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Chemokine Signatures of Pathogen-Specific T Cells I: Effector T Cells

Jens Eberlein, Bennett Davenport, Tom T. Nguyen, Francisco Victorino, Kevin Jhun, Verena van der Heide, Maxim Kuleshov, Avi Ma’ayan, Ross Kedl and Dirk Homann
J Immunol October 15, 2020, 205 (8) 2169-2187; DOI: https://doi.org/10.4049/jimmunol.2000253
Jens Eberlein
*Barbara Davis Center for Childhood Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
†Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
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Bennett Davenport
*Barbara Davis Center for Childhood Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
†Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
‡Department of Anesthesiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
§Diabetes, Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029;
¶Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY;
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Tom T. Nguyen
*Barbara Davis Center for Childhood Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
†Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
‡Department of Anesthesiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
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Francisco Victorino
*Barbara Davis Center for Childhood Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
†Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
‡Department of Anesthesiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
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Kevin Jhun
§Diabetes, Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029;
¶Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY;
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Verena van der Heide
§Diabetes, Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029;
¶Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY;
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Maxim Kuleshov
‖Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and
#Mount Sinai Center for Bioinformatics, Icahn School of Medicine at Mount Sinai, New York, NY 10029
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Avi Ma’ayan
‖Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and
#Mount Sinai Center for Bioinformatics, Icahn School of Medicine at Mount Sinai, New York, NY 10029
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Ross Kedl
†Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
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Dirk Homann
*Barbara Davis Center for Childhood Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
†Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
‡Department of Anesthesiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
§Diabetes, Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029;
¶Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY;
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Key Points

  • Pathogen-specific TE are a prodigious source of chemokines.

  • The complete TE chemokine spectrum is CCL3, CCL4, CCL5 > XCL1 ≥ CCL1 > CCL9/10 >> CXCL2.

  • TE exhibit unique and shared chemokine synthesis/expression/secretion patterns.

Abstract

The choreography of complex immune responses, including the priming, differentiation, and modulation of specific effector T cell populations generated in the immediate wake of an acute pathogen challenge, is in part controlled by chemokines, a large family of mostly secreted molecules involved in chemotaxis and other patho/physiological processes. T cells are both responsive to various chemokine cues and a relevant source for certain chemokines themselves; yet, the actual range, regulation, and role of effector T cell–derived chemokines remains incompletely understood. In this study, using different in vivo mouse models of viral and bacterial infection as well as protective vaccination, we have defined the entire spectrum of chemokines produced by pathogen-specific CD8+ and CD4+T effector cells and delineated several unique properties pertaining to the temporospatial organization of chemokine expression patterns, synthesis and secretion kinetics, and cooperative regulation. Collectively, our results position the “T cell chemokine response” as a notably prominent, largely invariant, yet distinctive force at the forefront of pathogen-specific effector T cell activities and establish novel practical and conceptual approaches that may serve as a foundation for future investigations into the role of T cell–produced chemokines in infectious and other diseases.

This article is featured in Top Reads, p.1979

Introduction

The immune system is a distributed network of organs, tissues, cells, and extracellular factors. Functional integration of these components faces a particular challenge because the principal sentinels, regulators, and effectors of immune function are often highly mobile single cells. The controlled spatiotemporal positioning of these cells is achieved by adhesion molecules, such as integrins and selectins, as well as chemokines and their receptors that function as a “molecular address” system in the coordination of cellular traffic in specific tissue microenvironments (1–4). The defining function of chemokines (chemoattractant cytokines), demonstrated in numerous in vitro experiments, is their capacity to induce the directed migration of locomotive cells by establishing a spatial gradient. However, chemokines exhibit a host of additional functions, including control of lymphopoiesis and lymphoid organogenesis, alterations of leukocyte adhesive properties by modulation of integrins as well as regulation of lymphocyte differentiation, proliferation, survival, cytokine release, and degranulation (1, 3, 5–7). Given this functional diversity, chemokines have been implicated in a wide variety of pathological states, such as infectious disease and cancer, autoimmunity, allergy, and transplant rejection (7–12).

The family of chemokines comprises a large number of mainly secreted molecules that share a defining tetracysteine motif and can be classified according to structural criteria, functional properties (homeostatic versus inflammatory) and genomic organization (13–15). Among the many different cell types capable of chemokine production, pathogen-specific T cells were identified as a relevant source over two decades ago (16). However, whereas the T cell–produced chemokines CCL3/4/5 have received considerable attention as competitive inhibitors of HIV binding to its coreceptor CCR5 (17–19), an inclusive perspective on specific T cell–produced chemokines has not been established, a likely consequence of both an experimental and conceptual emphasis on chemokine action on T cells rather than chemokine production by T cells (20–23).

In the more circumscribed context of pathogen-specific effector T cell (TE) immunity (i.e., T cell responses generated in the immediate wake of an acute pathogen challenge) and the topic of the present investigations, murine models of infectious disease have by and large confirmed the prodigious CCL3/4/5 production capacity of TE populations. For example, Dorner et al. (24) demonstrated that CCL3/4/5 as well as XCL1 are readily synthesized by CD8+ but not CD4+TE specific for the bacterium Listeria monocytogenes, are coexpressed with IFN-γ and thus may constitute a family of “type 1 cytokines.” Moreover, CCL3-deficient but not wild-type (wt) L. monocytogenes–specific CD8+TE, after transfer into naive wt recipients, failed to protect against a lethal L. monocytogenes infection, to this date one of the most striking phenotypes reported for a T cell–specific chemokine deficiency (25). Abundant CCL3/4/5 is also made by CD8+TE and to a lesser extent by CD4+TE, generated in response to acute infection with lymphocytic choriomeningitis virus (LCMV) (26). In the related LCMV model of lethal choriomeningitis, CCL3/4/5 secretion by CD8+TE has been associated with the recruitment of pathogenic myelomonocytic cells into the CNS and lethal choriomeningitis (27), but the precise role of these chemokines remains to be determined, given that mice deficient for CCL3 or CCR5 (the only receptor for CCL4 that also binds CCL3/5) are not protected from fatal disease (28). Even during the initial stages of T cell priming, CCL3/4 production by activated CD4+ or CD8+ T cells (induced by peptide immunization or vaccinia virus infection, respectively) contributes to the effective spatiotemporal organization of T and dendritic cell (DC) interactions (29, 30). A similar role has most recently also been demonstrated for CD8+T cell–derived XCL1 (30) and, following an earlier report that CD8+T cell–secreted XCL1 is required for optimal proliferative expansion of allogeneic CD8+TE (31), mice lacking XCR1 (the sole XCL1 receptor) were shown to generate reduced L. monocytogenes–specific CD8+TE responses associated with delayed bacterial control (32). Collectively, these observations demonstrate that pathogen-specific CD8+ and CD4+T cells, beyond their responsiveness to numerous various chemokine cues, are themselves a relevant source for select chemokines that exert nonredundant effects on the development of effective TE responses and, in some cases, efficient pathogen control.

The complete range of chemokines produced by pathogen-specific CD8+ and CD4+TE, however, has not yet been defined, and the respective expression patterns of T cell–derived chemokines, their coregulation as well as their synthesis, and secretion kinetics remain incompletely understood. In this study, we have addressed these issues in a series of complementary investigations that chiefly rely on the use of stringently characterized chemokine-specific Abs that permit the flow cytometry (FC)– based detection of practically all (37 out of 38) murine chemokines at the single-cell level (33). Our results demonstrate that production of chemokines by pathogen-specific CD8+ and CD4+TE constitutes a restricted (CCL1, CCL3, CCL4, CCL5, CCL9/10 and XCL1), remarkably prominent, uniquely regulated, integral, and consistent component of the TE response across different infectious disease models and protective vaccination; together, these properties position mature TE-derived chemokines at the forefront of coordinated host pathogen defenses.

Materials and Methods

Ethics statement

All procedures involving laboratory animals were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health; the protocols were approved by the Institutional Animal Care and Use Committees (IACUC) of the University of Colorado (permit numbers 70205604[05]1F, 70205607[05]4F, and B-70210[05]1E) and Icahn School of Medicine at Mount Sinai (IACUC-2014-0170), and all efforts were made to minimize suffering of animals.

Mice

C57BL6/J (B6), congenic B6.CD90.1 (B6.PL-Thy1a/CyJ), and B6.CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mice, B6.CCL3−/− (B6.129P2-Ccl3tm1Unc/J), B6.CCL5−/−(B6.129P2-Ccl5tm1Hso/J), B6.CCR5−/− (B6.129P2-Ccr5tm1Kuz/J), B6.Jnk1−/− (B6.129S1-Mapk8tm1Flv/J), and B6.Jnk2−/− (B6.129S2-Mapk9tm1Flv/J) mice on a B6 background, as well as BALB/c and CCR3−/− (C.129S4-Ccr3tm1Cge/J) mice on a BALB/c background were purchased from The Jackson Laboratory; CCR1−/− mice (B6.129S4-Ccr1tm1Gao) (34) were obtained from Taconic Biosciences; CCL3/CCR5–deficient mice were derived from intercrosses of B6.CCL3−/− × B6.CCR5−/− F1 offspring; p14 TCRtg mice on a B6.CD90.1 background were provided by Dr. M. Oldstone [CD8+T cells from these mice (“p14 cells”) are specific for the dominant LCMV-GP33-41 determinant restricted by Db (35)]; and B6.CCL1−/− mice were a gift from Dr. S. Manes (36). To generate p14 chimeras, naive p14 T cells were enriched by negative selection and ∼5 × 104 cells were transferred i.v. into sex-matched B6 recipients that were challenged 24 h later with LCMV (37); to assure reliable detection of phenotypically defined CD8+TE subsets (38) and cytolytic effector molecules (39), additional p14 chimeras were generated with lower numbers (∼5 × 103 and ∼1 × 103, respectively) of p14 cells.

Pathogen infections and vaccination

LCMV Armstrong (clone 53b) and vesicular stomatitis virus (VSV) Indiana were obtained from Dr. M. Oldstone; stocks were prepared by a single passage on BHK-21 cells, and plaque assays for determination of virus titers were performed as previously described (40). Recombinant L. monocytogenes expressing full-length OVA (rLM-OVA) (41) was grown and titrated as described (42). In brief, aliquots of ∼1 × 108 mouse-passaged rLM-OVA were frozen at −80°C. To estimate titers prior to in vivo challenge, thawed aliquots were used to inoculate 5–10 ml of fresh tryptic soy broth media, grown at 37°C in a shaker for 2–3 h to log phase followed by determination of OD600 values. Eight- to ten-week-old mice were infected with a single i.p. dose of 2 × 105 PFUs LCMV Armstrong, 1 × 106 PFU VSV i.v., or 3 × 102–3 × 104 CFU rLM-OVA i.v. as indicated; combined TLR/CD40 vaccinations were performed essentially as described previously (43) (i.e., mice were immunized i.p. with 500 μg of OVA [Sigma-Aldrich] or 100 μg of 2W1S peptide [Pi Proteomics] in combination with 50 μg of anti-CD40 [FGK4.5, Bio X Cell] and 50 μg of poly(I:C) (Amersham/GE Healthcare); all vaccinations were performed by mixing each component in PBS and injecting in a volume of 200 μl. In some cases (Fig. 9), mice were challenged intracerebrally (i.c.) with 1 × 103 PFU LCMV Armstrong (27); because of the lethal disease course in wt mice, we employed an IACUC-approved scoring matrix to measure morbidity, and terminally ill mice were euthanized and scored as deceased.

Lymphocyte isolation, T cell purification, and stimulation cultures

Lymphocytes were obtained from spleen and blood using standard procedures (44, 45). Splenic CD90.1+ p14 TE from LCMV-infected p14 chimeras were positively selected using anti-CD90.1-PE Ab and PE-specific magnetic beads (STEMCELL Technologies); additional purification (>99%) was achieved by FACS sorting (FACSAria; BD Biosciences). Primary cells were cultured for 0.5–5.0 h in complete RPMI (RPMI 1640 supplemented with 7% FCS, 1% l-glutamine, 1% penicillin/streptomycin; Life Technologies) and, where indicated, stimulated with specific peptides (1 μg/ml for MHC class I– and 5 μg/ml for MHC class II–restricted peptides); plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml); PMA/ionomycin (5–20 and 500 ng/ml, respectively); or LPS (500 ng/ml; Sigma-Aldrich) in the presence or absence of 1 μg/ml brefeldin A (BFA; Sigma-Aldrich). For transcriptional and/or translational blockade, cells were preincubated for 30 min at 37°C with 5 μg/ml actinomycin D (ActD; Sigma-Aldrich) and/or 10 μg/ml cycloheximide (CHX; Sigma-Aldrich) prior to addition of peptide and/or BFA. In vitro and in vivo T cell proliferation was monitored by CFSE dilution as previously described (44, 46).

Microarray hybridization and analysis

For microarray analyses (Fig. 1A, Supplemental Fig. 2A–C), p14 TE were purified (>99%) from individual p14 chimeras on day 8 after LCMV challenge by sequential magnetic- and fluorescence-activated cell sorting as detailed above; DNA-digested total RNA was extracted either directly postsort (ex vivo) or after 3 h stimulation with anti-CD3/anti-CD28 (see above) using a MinElute kit (QIAGEN), and RNA integrity was confirmed by PicoChip RNA technology (Agilent Technologies) according to the manufacturer’s instructions. Amplification and labeling of mRNA (Ovation Biotin RNA Amplification and Labeling System; NuGEN), hybridization to Affymetrix M430.2 arrays, and quality control were performed by the Affymetrix Core Facility of the University of Colorado Cancer Center according to standard protocols; further experimental and analytical details are provided in Ref. 37, and the data can be retrieved from the Gene Expression Omnibus https://www.ncbi.nlm.nih.gov/geo) repository under accession number GSE143632. Note that the files deposited therein also contain data for ex vivo p14 TE previously uploaded in the context of a related study (GSE38462) as well as data on ex vivo and anti-CD3/anti-CD28–stimulated p14 memory T cells (TM) discussed in the accompanying report by Davenport et al. (47) (although all ex vivo and stimulated p14 TE and TM data were generated in the same set of experiments). Robust multi-array analysis (RMA), GC-RMA, and Affymetrix Micro Array Suite 5.0 (MAS5) normalization were performed and yielded essentially similar results (data not shown). In addition, ex vivo–purified and anti-CD3/anti-CD28–stimulated p14 TE were analyzed by macroarrays (OMM022 chemokine array; SuperArray Bioscience) according to protocols provided by the manufacturer and yielded results comparable to Affymetrix analyses (data not shown).

Peptides and MHC tetramers

Peptides corresponding to the indicated pathogens, OVA, or I-Eα epitopes were obtained from Peptidogenic, the National Jewish Health Molecular Core Facility, or GenScript at purities of >95%; their MHC restriction and amino acid sequences are indicated as follows: LCMV epitopes were GP33–41 (Db/KAVYNFATC), GP67–77 (Db/IYKGVYQFKSV), GP92–101 (Db/CSANNAHHYI), GP118–125 (Kb/ISHNFCNL), GP276–286 (Db/SGVENPGGYCL), NP396–404 (Db/FQPQNGQFI), NP166–175 (Db/SLLNNQFGTM), NP205–212 (Kb/YTVKYPNL), NP118–126 (Ld/RPQASGVYM), GP64–80 (IAb/GPDIYKGVYQFKSVEFD), NP309–328 (IAb/SGEGWPYIACRTSIVGRAWE); VSV epitopes were N52–59 (Kb/RGYVYQGL), GP415–433 (IAb/SSKAQVFEHPHIQDAASQL); rLM-OVA epitopes were OVA257–264 (Kb/SIINFEKL), LLO190–201 (IAb/NEKYAQAYPNVS); and the I-Eα–derived epitope was 2W1S (IAb/EAWGALANWAVDSA). DbNP396, DbGP276, DbGP33, LdNP118, IAbGP66, and IAbhuCLIP87 complexes were obtained from the National Institutes of Health tetramer core facility as allophycocyanin or PE conjugates and/or biotinylated monomers; KbOVA257, IAb2W1S, and IAbGP61–80 tetramers were prepared in the laboratory as previously described (46, 48, 49). Note that the shorter sequences (GP64–80 and GP66–77) within the dominant IAb-restricted LCMV GP61–80 epitope are recognized by the same population of LCMV-specific CD4+T cells (50). Tetramer staining was performed as previously described (46, 51).

Abs, staining procedures, and FC

All FC Abs were obtained as purified, biotinylated, and/or fluorochrome-conjugated reagents from R&D Systems, BD Biosciences, eBioscience, BioLegend or Invitrogen; our protocols for cell surface and intracellular FC staining, including the stringent characterization and usage of chemokine-specific mAb and polyclonal Abs (pabs), are detailed elsewhere (33, 45, 46); the use of a new CXCL3 pab included in this study (AF5568; R&D Systems) is demonstrated in Supplemental Fig. 1B. For concurrent use of two chemokine-specific goat pabs, we performed preconjugations with Zenon AF488 and AF647 kits according to the manufacturer’s instructions (Invitrogen). Note that the pabs anti-CCL1, anti-CCL4, and anti-XCL1 do not exhibit cross-reactivity with any other chemokines; anti-CCL3 is weakly cross-reactive with CX3CL1 (not expressed by any hematopoietic cells); and anti-CCL5 demonstrates very minor cross-reactivity with CCL3 (33). Analyses of CCL6 and CCL9/10 expression are complicated by the fact that anti-CCL6 and anti-CCL9/10 pabs exhibit significant cross-reactivity with the respective noncognate (but no other) chemokine (33). However, because T cells fail to produce CCL6 as determined with the non–cross-reactive 262016 mAb (R&D Systems) (33), chemokine expression by T cells stained with the anti-CCL9/10 pab can be attributed exclusively to the presence of CCL9/10. Additional chemokine Abs employed in this study include anti-CCL3-PE mAb (clone 39624; R&D Systems) and anti-CCL5 mAb R6G9 (mIgG1), generation of which has been described elsewhere (52). For detection of murine granzymes, we used granzyme A (GzmA) clone 3G8.5 (mIgG2b) conjugated to FITC or PE [similar results were obtained with a rabbit anti-serum provided by Dr. M. Simon (53); Santa Cruz Biotechnology] and granzyme B (GzmB) clones GB12 (mIgG1) conjugated to PE or allophycocyanin (Invitrogen) or GB11 (mIgG1) conjugated to AF647 (BioLegend). All samples were acquired on FACSCalibur or LSR II flow cytometers (BD Biosciences) and analyzed with CellQuest, FACSDiva (BD Biosciences), and/or FlowJo (Tree Star) software. Comprehensive functional CD8+TE profiling (Fig. 2F) was performed by quantification of NP396-specific CD8+TE subsets constitutively expressing cytolytic effector molecules (GzmA, GzmB, and perforin visualized directly ex vivo in DbNP396+CD8+TE) or featuring inducible effector activities after 5-h NP396 peptide stimulation (CCL1/3/4/5/9/10, XCL1; IFN-γ, IL-2, IL-3, GM-CSF; TNF-α, FasL, CD40L; degranulation/CD107a surface translocation); primary data are found in Figs. 2C, 2E, 4D, and Supplemental Fig. 2E. Inducible IL-3 and FasL expression, as well as degranulation, were quantified as previously described (37), and perforin stains were performed with Ab clone S16009B (BioLegend; data not shown). To provide an overall context for the relative abundance of individual functional properties, fractions of specific CD8+TE demonstrating individual ex vivo or inducible effector activities (expressed as the percentage of DbNP396+CD8+TE or IFN-γ+ NP396-specific CD8+TE, respectively) were added, and their distribution across the total response (sum of all discrete analytical readouts determined in this study) was calculated accordingly.

In vivo killing and CD8+TE activation assays

In vivo killing assays (Supplemental Fig. 4E) were performed as described (54). In brief, frequencies of DbNP396+ CD8+TE or p14 TE in control and experimental groups of day 8 LCMV-infected mice were determined prior to assay execution to assure the presence of equal specific CD8+TE numbers; then, differentially CFSE-labeled and peptide-coated (NP396 or GP33 peptide) versus uncoated CD45.1 spleen cells were transferred i.v., followed by longitudinal blood sampling (10–240 min) and assessment of killing kinetics by calculating the specific loss of peptide-coated targets as a function of time after transfer; for CCL5 neutralization, mice were treated i.v. with 100 μg of anti-CCL5 (clone R6G9) or mIgG1 isotype control (clone MOPC-21; Sigma-Aldrich) ∼10 min prior to injection of target cells. In vivo CD8+TE activation assays (Fig. 8C) were conducted according to modified protocols originally developed by Haluszczak et al. (55). In this study, wt and chemokine-deficient mice 8 d after LCMV infection were injected with 250 μg of BFA i.p., followed 30 min later by i.v. injection of saline (negative control) or 100 μg of GP33 peptide; spleens were harvested 1 h later, processed, and immediately stained with anti-CD8α Ab and DbGP33 tetramers (surface) and chemokine Abs (intracellular).

Conjugation assays

For conjugation assays, bead-purified p14 TE (CD90.1) obtained from LCMV-infected p14 chimeras (day 8) were combined at a ratio of 1:1 with EL4 thymoma cells (CD90.2, magnetically depleted of a small CCR3/5–expressing subset [∼8%] and pulsed for 1 h with 1 μg/ml GP33 peptide or left uncoated, followed by two washes to remove excess peptide) in prewarmed media in a V-bottom microtiter plate, pelleted by brief centrifugation, and cultured for 20–60 min. At indicated time points, cells were immediately fixed by the addition of an equal volume of 4% PFA buffer, stained for CD90.2 and cell surface CCL5, and analyzed by FC (Fig. 7B) or confocal microscopy (Fig. 7C).

Chemokine and cytokine ELISAs

Quantitation of CCL3, CCL4, CCL5, and IFN-γ in tissue culture supernatants or serum was performed using respective Quantikine ELISA Kits and protocols provided by the manufacturer (R&D Systems) (Fig. 7D, Supplemental Figs. 3C, 4B). For evaluation of CCL3/4/5 chemokine complex formation, supernatants of NP396 peptide–stimulated spleen cells (day 8 after LCMV, 5-h stimulation, no BFA) were diluted and incubated for 2.5 h at room temperature in plates precoated with 5.0 μg/ml polyclonal goat IgG, anti-CCL3, anti-CCL4, or anti-CCL5, and absorbed supernatants were immediately analyzed for CCL3/4/5 content by standard ELISA. To determine chemokine production on a per cell basis (Fig. 7D), FC analyses were performed in parallel to calculate the numbers of DbNP396+ CD8+TE in the stimulation culture.

Confocal microscopy

PBMC or splenocyte suspensions were prepared 8 d after LCMV infection of B6 mice. For ex vivo colocalization studies (Fig. 5), CD4−CD19−NK1.1− PBMCs were sorted into GzmA− and GzmA+ populations using a MoFlo cell sorter (Beckman Coulter); for colocalization studies of CCL3/4/5 and GzmB in 5 h NP396 peptide–stimulated splenocytes (Supplemental Fig. 4A), cells were stained for surface and intracellular markers followed by sorting on IFN-γ+B220−CD4− cells using a FACSAria Cell Sorter (BD Biosciences); for an assessment of conjugate formation (Fig. 7C), we employed the conjugation assay described above. Cells were resuspended in 22% BSA and spun onto glass slides (Gold Seal Microscope Slides, Ultra StickTM, catalog number 3039) for 5 min at 800 rpm using a cytospin (Cytospin3; Shandon) and mounted using one drop of ProLong Gold reagent (Invitrogen) with or without DAPI, and a cover slip was placed on top (number 1 1/2; Corning). After drying overnight, slides were sealed with nail polish and stored in the dark at 4°C until acquisition. Slides were analyzed with a Leica TCS SP5 confocal laser scanning microscope equipped with an inverted Leica DMI6000 microscope, a high performance TCS workstation, a 488/543/633 excitation beam splitter, a UV laser (405 nm, diode 50 mW), an argon laser (458/476/488/496/514 nm, 100 mW, attenuated to 20%), a green helium/neon laser (543 nm, 1 mW) and a red helium/neon laser (633 nm, 10 mW) for excitation of DAPI, FITC/AF488, Cy3, PE, and Cy5/allophycocyanin/AF647, respectively. The 2048 × 2048 and 1024 × 1024 pixel images were acquired sequentially with a 63×/numerical aperture 1.4 oil immersion lens at 1.9× and 5.95× zoom, respectively, resulting in respective effective pixel sizes of 63.2 and 80.24 nm. Prism spectral detectors were manually tuned to separate labels (DAPI, 415–487 nm; FITC/AF488, 497–579 nm; Cy3, 551–641 nm; PE, 585–699 nm; Cy5, 640–778 nm). The pinhole size was set at one airy unit to give an effective optical section thickness of ∼0.5 μm. Gray-scale images were digitized at eight bits per channel and pseudo-colored as indicted in the figure legends using the Leica Sp5 Software or exported as TIFF files for processing in Adobe Photoshop CS (version 8.0).

Statistical analyses

Data handling, analysis, and graphic representation was performed using Prism 4.0 or 6.0c (GraphPad Software, San Diego, CA). All data summarized in bar and line diagrams are expressed as mean ± 1 SE. Asterisks indicate statistical differences calculated by unpaired or paired two-tailed Student t test and adopt the following convention: *p < 0.05, **p < 0.01, and ***p < 0.001. EC50 values (activation thresholds, Fig. 2E) were calculated by plotting the fraction of specific (IFN-γ+) T cells as a function of peptide concentration (1 × 10−6 − 1 × 10−11 M peptide for 5 h) followed by nonlinear regression analysis using the appropriate data format and analysis functions in the Prism software.

Results

Broad survey of T cell–produced chemokines

To delineate the principal spectrum of chemokines synthesized by activated T cells, spleen cells obtained from unmanipulated wt mice were stimulated for 5 h with PMA/ionomycin and interrogated by FC for production of IFN-γ and 37 individual chemokines using a stringently validated panel of chemokine-specific Abs that reliably detect chemokine expression in appropriate positive control samples (33). In stimulated T cells, robust chemokine induction was observed for CCL3, CCL4, and XCL1 and to a lesser extent also CCL5; 1–2% of T cells synthesized CXCL2; and very small subsets produced CCL1 or CCL9/10 (Supplemental Fig. 1A–C). CCL3, CCL4, CCL5, and XCL1 production was particularly prominent in the IFN-γ+ T cell subset (40–80% coexpression), and despite their low frequency, CCL1- and CCL9/10–expressing T cells were also enriched in the IFN-γ+ population (3–4% coexpression); in contrast, no such enrichment was observed for CXCL2 (Supplemental Fig. 1A, 1C). Although T cells have previously been described as a source for these chemokines in various experimental scenarios, we note that the comprehensive nature of our screen, within the limits of specific experimental constraints and the sensitivity afforded by chemokine FC (33), can apparently rule out 30 other chemokines as potential products of highly activated T cells.

Defining the complete spectrum of chemokines produced by virus-specific CD8+TE

To refine these analyses within the context of infectious diseases and to define the complete range of chemokines produced by pathogen-specific CD8+TE, we first employed the established p14 chimera system to quantify chemokine mRNA and protein expression by virus-specific CD8+TE with a combination of gene arrays and FC-based assays (33, 37). In brief, p14 chimeras were generated by transducing congenic B6 mice with a trace population of naive TCR-transgenic CD8+T cells (p14 TN) specific for the dominant LCMV–GP33–41 determinant; after challenge with LCMV, p14 TE populations rapidly differentiate, expand and contribute to efficient virus control before contracting and developing into p14 TM ∼6 wk later (35, 37, 51). At the peak of the effector phase (day 8), p14 TE were purified, and RNA was extracted either immediately or after a 3-h in vitro TCR stimulation and processed for gene array hybridization. Overall, 10 chemokine mRNA species were detectable in p14 TE evaluated ex vivo and/or after TCR stimulation, and their expression patterns could be allocated to three groups (Fig. 1A): 1) absence of ex vivo detectable mRNA but robust transcription after TCR stimulation (Ccl1 and Xcl1), 2) constitutive mRNA expression that significantly increased upon TCR engagement (Ccl3, Ccl4, Ccl9/10, and Cxcl10), and 3) chemokine mRNA species that were slightly downregulated by TCR activation (Ccl5, Ccl6, Ccl25, and Ccl27). A list of all murine chemokine genes and gene array IDs is found in Supplemental Fig. 2A. We also quantified chemokine mRNA expression for the known members of the related chemokine-like factor superfamily (CKLFSF) (56) (Supplemental Fig. 2B). Four out of ten Cklfsf mRNA species were detected in p14 TE but none were increased or decreased upon TCR stimulation. Information about the biological function of CKLFSF members remains limited and is centered around the pleiotropic effects of CKLF1, which may be produced by human T cells after prolonged in vitro stimulation (57, 58). At the present stage, we have refrained from a further analysis of this gene family.

FIGURE 1.
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FIGURE 1.

Chemokine mRNA and protein expression by virus-specific CD8+TE. (A) p14 TE (day 8) were obtained from spleens of LCMV-infected p14 chimeras, enriched to >99% purity, and processed for RNA extraction (either immediately or after 3-h anti-CD3/anti-CD28 stimulation) and gene array analysis (n = 3 individual mice). The bar diagrams display MAS5-normalized values of chemokine mRNA expression of p14 TE analyzed ex vivo (gray bars) or after TCR stimulation (black bars); statistically significant differences are indicated by asterisks. The broken line indicates the detection threshold set at a MAS5 value of 40; coverage: 39/40 chemokines (Ccl26 not on chip). (B) p14 TE (day 8) were analyzed for chemokine protein expression ex vivo or after 5-h stimulation with GP33 peptide. Histograms are gated on splenic p14 cells (gray histograms: control stains; black tracings: indicated chemokine stains; red dots identify panels demonstrating detectable chemokine expression; representative data from three experiments with [n ≥ 3 individual mice per experiment]). (C) Summary of p14 TE- and TM-expressed chemokines and chemokine receptors; gray font indicates presence of mRNA in the absence of constitutive or induced protein expression. (D) Genomic organization of murine chemokine genes transcribed and translated by T cells (modified after Ref. 14). The genes for 4–6 chemokines produced by T cells (Ccl3/4/5 and Ccl9/10) are found in the MIP region on mouse chromosome 11; the Ccl1 gene is located in the MCP region but rather distantly related to other members of the MCP group, and the nonclustered Xcl1 gene is found on chromosome 1. Arrows indicate chemokine genes and their transcriptional orientation; colors identify homeostatic (green), inflammatory (red), and dual function (yellow) chemokine genes; gray arrows indicate pseudogenes. Based on our results reported here and in Ref. 33, we propose to classify the CCL3/4/5 and 9/10 as dual function chemokines rather than simply inflammatory (CCL3/4/5) or homeostatic (CCL9/10). *p < 0.05, **p < 0.01, ***p < 0.001.

Traditional gene array analyses are fraught with several limitations, including measurement of mRNA levels across entire albeit purified cell populations rather than individual cells, difficulties in directly comparing mRNA expression between different mRNA species, and the impossibility of predicting whether mRNA is in fact translated. We therefore deployed chemokine FC to the interrogation of p14 TE and found that specific TCR stimulation with GP33 peptide induced CCL3, CCL4, and CCL5 expression in practically all p14 TE, whereas CCL1, CCL9/10, and XCL1 production was restricted to p14 TE subsets; neither Ccl6, Ccl25, Ccl27, Cxcl2, nor Cxcl10 or any other chemokine RNA was translated. Of note, and to our knowledge not previously reported, CCL5 was also detectable directly ex vivo and is thus the only chemokine expressed in the absence of TCR activation (Fig. 1B). The robust induction of CCL1 and CCL9/10 in p14 TE contrasts with their more limited expression in our initial T cell survey (Supplemental Fig. 1A–C). To resolve these discrepancies, we quantified CCL1 and CCL9/10 production by CD8+T cells from LCMV-immune mice in response to stimulation with peptide anti-CD3/anti-CD28 or PMA/ionomycin. Interestingly, >20% of IFN-γ+ CD8+T cells also synthesized CCL1 or CCL9/10 after activation with peptide or anti-CD3/anti-CD28, but fewer than 5% produced these chemokines in response to PMA/ionomycin stimulation (Supplemental Fig. 1D). The reason for the only sparse CCL1 and CCL9/10 induction after PMA/ionomycin treatment remains unclear but emphasizes important limitations associated with this widely used T cell–stimulation protocol. In summary, six mRNA species found ex vivo and/or after brief TCR engagement in virus-specific p14 TE serve as templates for induced protein synthesis and, in the case of Ccl5, also for effective constitutive translation (Fig. 1B, 1C). The genes of four of these six chemokines (Ccl3/4/5 and 9/10) are clustered in the “MIP region” on murine chromosome 11 with an additional gene (Ccl1) immediately adjacent in the “MCP region;” the Xcl1 gene is unclustered and located on chromosome 1 (Fig. 1D).

Finally, transcriptional profiling of p14 TE–expressed chemokine receptors revealed a prominent presence of Ccr2, Cxcr3, Cxcr4, Cx3cr1, and Ccrl1 (all of which were significantly downregulated upon activation); Ccr5 and Cxcr6 (slightly increased after stimulation), and low levels of Ccr7 that remained unaffected by TCR engagement (Fig. 1C, data not shown). It therefore appears that CCR5 is the only receptor that may sensitize p14 TE to potential auto- or paracrine actions of T cell–produced chemokines themselves (i.e., CCL3/4/5).

Constitutive and induced chemokine expression profiles of endogenously generated LCMV-specific CD8+ and CD4+TE

Extending our findings from the p14 chimera system to endogenously generated TE, direct ex vivo analyses of the dominant DbNP396+ CD8+TE population in LCMV-infected B6 mice demonstrated patterns comparable to p14 TE in that constitutive chemokine expression was largely limited to CCL5. The small subsets of specific CD8+TE showing weak CCL1/3/4 (but no CCL9/10 or XCL1) staining suggest that their expression, in contrast to IFN-γ and other cytokines (59), can be maintained somewhat longer after cessation of TCR activation (Fig. 2A). Constitutive CCL5 expression, in contrast, is a general feature of LCMV-specific CD8+TE, as based on analyses of additional epitope-specific CD8+TE and the inclusion of LCMV-infected Ccl5-deficient mice as a negative control (we also observed a slight reduction of ex vivo–detectable CCL5 in Ccl5-heterozygous mice, indicative of a modest gene-dose effect) (Fig. 2A), and appears to be in fact unique for cytokines at large because no other effector molecules readily detected in restimulated CD8+TE (IFN-γ, TNF-α, IL-2, GM-CSF, CD40L) are expressed in a constitutive fashion (Supplemental Fig. 2C, 2D). Rather, CCL5 expression resembles that of constituents of the granzyme/perforin pathway (60–63) (Supplemental Fig. 2C, 2E). Similar to CD8+TE, LCMV-specific CD4+TE cells also contained ex vivo detectable CCL5 albeit only in a subset (∼60%) and at lower levels (Fig. 2B).

FIGURE 2.
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FIGURE 2.

Constitutive and induced chemokine expression by LCMV-specific CD8+ and CD4+TE. (A) Top row, Endogenously generated LCMV-specific CD8+TE (day 8) were analyzed directly ex vivo by chemokine FC (plots gated on CD8+T cells); values indicate SEM of chemokine+ subsets among NP396-specific CD8+TE. Middle and bottom rows, Constitutive CCL5 expression by LCMV-specific CD8+TE subsets generated by LCMV-infected B6, B6.CCL5+/−, and B6.CCL5−/− mice. (B) Ex vivo–detectable CCL5 expression by LCMV-specific CD4+TE. The adjacent bar diagram compares the fractions (percentage) and CCL5 expression levels in geometric mean of fluorescence intensity (GMFI) of CCL5+-specific CD8+ and CD4+T cells; statistical differences are indicated by asterisks. For the purpose of this direct comparison, MHC class I and II tetramer stains were performed under the same experimental conditions (90-min incubation at 37°C). (C) Induced chemokine production by NP396-specific CD8+ (top row) and GP64-specific CD4+ (bottom row) T cells as determined after 5-h in vitro peptide stimulation culture. (D) Left, Induced CCL1 expression following NP396 peptide stimulation of day 8 spleen cells from LCMV-infected B6 and B6.CCL1−/− mice. Right, Chemokine coexpression by NP396-specific CD8+TE (plots gated on IFN-γ+ CD8+TE). (E) Summary of induced chemokine expression by LCMV-specific TE subsets stratified according to epitope specificity; their restriction elements, relative size (immunodominance), and functional avidities (peptide concentration required to induce IFN-γ production in 50% of a given epitope-specific population) are indicated. Significant differences between chemokine-expressing CD8+ and CD4+TE subsets are indicated by asterisks. (F) The composition of the NP396-specific CD8+TE response (day 8) was assessed by quantification of subsets expressing individual constitutive (GzmA/B and perforin) or inducible (all other, including CCL5) effector activities, and the pie chart depicts the sum and relative distribution thereof. Unless noted otherwise, data (SEM) were generated with splenic T cell populations and are representative of two to five experiments comprising groups of three to five individual mice. *p < 0.05, **p < 0.01, ***p < 0.001.

Upon in vitro restimulation, endogenously generated CD8+TE rapidly synthesized the same six chemokines induced in p14 TE but no other chemokines (Fig. 2C, data not shown). Accordingly, CCL1, CCL9/10, and XCL1 production by specific CD8+TE was mostly restricted to a subset of IFN-γ+ cells, whereas CCL3/4/5 were produced by virtually all epitope-specific CD8+TE. The patterns of induced chemokine synthesis further indicated the existence of particularly potent CD8+TE populations, as demonstrated by the coexpression of high CCL3/4/5 levels and the relative restriction of CCL1 production to a subset of XCL1+ CD8+TE (Fig. 2D). To ascertain whether “polyfunctionality” at the level of inducible chemokine production is a property of defined CD8+TE subsets, we differentiated specific CD8+TE according to CD127 and KLRG1 expression, a distinction that captures CD127−KLRG1+ short-lived effector cells and CD127+KLRG1− memory precursor effector cells (MPECs) (38). As shown in Supplemental Fig. 2F, 2G, MPECs in comparison with short-lived effector cells were enriched for inducible CCL1, CCL9/10, and, in particular, XCL1 expression. This is broadly consistent with the chemokine profiles of specific CD8+TM that overall exhibit greater CCL9/10 and XCL1 production capacity (37, 47). In regard to LCMV-specific CD4+TE, their chemokine signatures were qualitatively similar, but induced CCL3/4/5 production was confined to a subset (∼60%), and very few cells produced CCL9/10 or XCL1 (Fig. 2C). Fig. 2E summarizes the above observations by displaying the fraction of endogenously generated chemokine+ LCMV-specific TE stratified according to MHC restriction elements. Because the individual epitope-specific TE populations not only differed according to immunodominance but also activation threshold (Fig. 2E), our findings establish that induced chemokine production is independent of mouse strain, immunodominant determinants, and functional avidities but quantitatively different in specific CD8+ and CD4+TE.

To provide a rough estimate for the relative contribution of chemokine production to the totality of quantifiable CD8+TE functionalities, we determined the respective fractions of NP396-specific CD8+TE capable of individual chemokine, cytokine, and TNFSF ligand synthesis, constitutive GzmA/B and perforin expression, and degranulation (see Materials and Methods for details); according to this estimate, >40% of the CD8+TE response is in fact dedicated to chemokine production (Fig. 2F).

Similar chemokine expression profiles of LCMV-, VSV-, L. monocytogenes–, and vaccine-specific CD8+ and CD4+TE

The regulation of pathogen-specific CD8+ and CD4+T immunity generated in response to VSV or L. monocytogenes shares many cardinal properties with the LCMV system (41, 44, 46, 64–67), yet the distinct biology of these pathogens may have an impact on aspects of the T cell chemokine response. In contrast to the noncytopathic arenavirus and natural murine pathogen LCMV, VSV is an abortively replicating cytopathic virus that causes a polio- or rabies-like neurotropic infection in immunodeficient mice (68). Similar to other pathogenic bacteria, such as Mycobacteria, Salmonella, Rickettsia, and Chlamydia, L. monocytogenes is a facultative intracellular bacterium, and the model of murine listeriosis constitutes one of the best-characterized experimental systems for bacterial infection (69). Acute infection with L. monocytogenes (rLM-OVA for induction of a traceable specific CD8+TE population) or VSV generated specific CD8+TE with constitutive CCL5 and inducible CCL3/4/5/9/10 and XCL1 expression akin to those found in LCMV-specific CD8+TE; similarly, L. monocytogenes– and VSV-specific CD4+TE presented with chemokine production profile resembling LCMV-specific CD4+TE (Fig. 3A, 3B, Supplemental Fig. 3A). The considerable uniformity of chemokine signatures by T cells specific for three disparate pathogens therefore identifies fundamental functional attributes of the pathogen-specific T cell response at large (Figs. 2E, 3C).

FIGURE 3.
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FIGURE 3.

Constitutive and induced chemokine expression by L. monocytogenes–, VSV-, and vaccine-specific CD8+ and CD4+TE. (A) Induced chemokine expression by specific CD8+ and CD4+TE (day 8) following challenge with rLM-OVA (data displayed as in Fig. 2C). (B) Constitutive CCL5 expression by rLM-OVA257–specific CD8+TE (day 8). (C) Summary of induced chemokine production by rLM-OVA– and VSV-specific CD8+ and CD4+TE (restriction elements are indicated); asterisks denote significant differences between CD8+ and CD4+TE specific for the same pathogen. (D) Ex vivo CCL5 expression by vaccine-specific CD8+ and CD4+TE (day 7 after vaccination as explained below); dot plots are gated on blood-borne CD8+ or CD4+T cells as indicated. The two-tone dot plot is gated on both total CD4+T cells (gray) and I-Ab2WS1+ CD4+TE (black). (E and F) Induced chemokine profiles of specific CD8+ and CD4+TE generated by combined TLR/CD40 vaccination. Mice were challenged with OVA/anti-CD40/poly(I:C) (CD8+ vaccination) or 2WS1 peptide/anti-CD40/poly(I:C) (CD4+ vaccination) as detailed in Materials and Methods and analyzed 7 d later; asterisks indicate differences between CD8+ and CD4+TE. All data (SEM) were generated with splenic T cell populations unless noted otherwise and are representative of two to five experiments comprising groups of three to five individual mice. *p < 0.05, **p < 0.01, ***p < 0.001.

Nevertheless, we noted some quantitative differences associated with the use of different infection protocols, and to ascertain whether the degree of infection-associated inflammation could modulate TE chemokine production profiles in a given model system, we infected B6 mice with escalating doses of rLM-OVA (3 × 102–3 × 04 CFU). As expected, an increase of bacterial dose heightened early inflammation as determined by serum IFN-γ levels, but the numbers of OVA257-specific CD8+TE as well as their ex vivo CCL5 expression levels peaked postinfection with 3 × 103 CFU rLM-OVA and declined at higher infection doses; in contrast, LLO190-specific CD4+TE numbers steadily rose with escalating challenge doses (Supplemental Fig. 3B). Although the fraction of IL-2–, TNF-α–, and CD40L-producing specific CD8+ and CD4+TE remained impervious to bacterial challenge dose, induced chemokine synthesis by OVA257-specific CD8+TE was compromised by infection with higher rLM-OVA titers, especially the production of CCL1 and XCL1 and to a lesser extent also CCL3/4/5; the chemokine expression profiles of LLO190-specific CD4+TE, however, remained largely unaffected by the different challenge protocols (Supplemental Fig. 3C, 3D). These observations suggest that chemokine production by CD8+ but not CD4+TE may be partially impaired under conditions of chronic infection, and we have further pursued this question in the accompanying report by Davenport et al. (47).

We also extended our delineation of chemokine expression profiles to vaccine-specific CD8+ and CD4+TE. Using a strategy for induction of protective T cell immunity by combined TLR/CD40 vaccination [i.e., the immunization with whole proteins or peptides in conjunction with poly(I:C) and anti-CD40 administration (43, 70, 71)], we found that vaccine-induced CD8+ and CD4+TE were remarkably similar to the respective pathogen-specific TE populations at the level of constitutive (CCL5) and induced chemokine production capacity (Fig. 3D–F). Both effective vaccines and different infectious pathogens therefore elicit essentially the same TE chemokine response that is quantitatively adjusted according to the particular conditions of T cell priming.

Finally, small subsets of both LCMV- and L. monocytogenes–specific CD4+TE have been described to exhibit a TH2 phenotype (72, 73). Although the existence of specific IL-4–producing CD4+TE in these model systems has been contested by others (67, 74), the description of CXCL2 production as a characteristic for in vitro–generated TH2 cells (24, 75) permits an analysis of TH2 functionality at the chemokine level. Indeed, primary murine T cells expressed CXCL2 after polyclonal activation preferentially under exclusion of IFN-γ (Supplemental Fig. 1A), and a very small subset of LCMV- but not rLM-OVA–specific CD4+TE produced CXCL2 (Supplemental Fig. 3E). However, given the clearly predominant TH1 phenotype of LCMV- and L. monocytogenes–specific CD4+T cells (46, 67, 74), we have not pursued a further characterization of TH2 chemokines in these model systems.

Delayed acquisition of CCL5 production capacity by CD8+TE

The acquisition of defined effector functions constitutes a hallmark of primary TE differentiation, and the detailed work by A. Krensky’s (76–78) group has identified an unusual property of T cell–produced CCL5, namely its comparatively late synthesis only after 3–5 d of TCR stimulation as a consequence of regulation through the transcription factor KLF13. To elucidate these dynamics for pathogen-specific T cells, we compared the regulation of CCL5 expression with that of principal effector molecules (GzmB and IFN-γ) during the transition from naive to early effector stage of developing p14 TE. Assayed over a 72-h period in vitro, the rapid and progressive induction of IFN-γ and slightly delayed GzmB synthesis as a function of cell division contrasted with a lack of constitutive and only minimal inducible CCL5 expression (Fig. 4A, 4B). Similarly, within the first 60 h after in vivo challenge, p14 TE remained CCL5-negative and constitutive CCL5 expression by ∼50% of p14 TE or endogenously generated CD8+TE became discernible only on day 5 after LCMV infection; by day 8, however, practically all LCMV-specific CD8+TE had acquired a CCL5+/GzmB+ phenotype (Fig. 4C, 4D). The protracted dynamics of CCL5 expression are indeed unique, as they differed not only from GzmB and IFN-γ but also GzmA and all other inducible CD8+TE functionalities evaluated (CCL1/3/4, XCL1, IL-2, GM-SF, TNF-α, and CD40L; data not shown); accordingly, constitutive CCL5 protein expression may serve as a novel functional marker for mature Ag-experienced CD8+TE.

FIGURE 4.
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FIGURE 4.

Expression and acquisition kinetics of CD8+TE effector molecules. (A) Constitutive (gray histograms) and induced (black tracings) CCL5, GzmB, and IFN-γ expression levels by naive CD44lo p14 cells (p14 TN; note that the functionality of p14 TN is restricted in this study to limited IFN-γ production). (B) CCL5, GzmB, and IFN-γ expression as a function of early in vitro p14 TE proliferation. Dot plots are gated on p14 T cells analyzed directly after 72-h stimulation culture (no restimulation) or following GP33 peptide restimulation in the presence of BFA during the final 5 h of culture as indicated. The diagrams on the right summarize the individual expression patterns as a function of p14 CFSE dilution (generation #0, no division). (C) Acquisition of constitutive CCL5 expression by p14 CD8+TE in vivo was analyzed 60 h after adoptive transfer of CFSE-labeled p14 cells and LCMV challenge (top dot plot) or in p14 chimeras on day 5 postinfection (bottom plot gated on blood-borne CCL5−/− [gray] and wt [black] p14 TE); the adjacent diagrams depict the emergence of constitutive CCL5 expression by developing CD8+TE in the p14 chimera system (middle) and LCMV-infected B6 mice (right diagram, fraction of CCL5+ T cells among total [gray] and DbNP396+ [black] CD8+T cells). (D) Left, GzmA and GzmB expression by specific CD8+TE (day 8) analyzed ex vivo (top) or after 5-h restimulation culture (bottom); all dot plots were gated on splenic CD8+T cells. Right, Constitutive CCL5, GzmA, and GzmB expression by LCMV-specific CD8+TE (the small subset of CCL5- and GzmA/B–negative CD8+T cell subset corresponds to the CD44lo naive CD8+T cell fraction, data not shown). All data are representative of two to three experiments comprising groups of three or more individual mice (unless noted otherwise, all data obtained with splenic cell populations).

Constitutive coexpression and subcellular localization of CCL5 and granzymes in antiviral CD8+TE

The precise subcellular localization of CCL5 remains a matter of controversy. In humans, a reported preferential association with the content of cytolytic granules (GzmA, perforin, granulysin) (17, 79) contrasts with the identification of a unique subcellular CCL5 compartment (80), and the frequent use of T cell clones or blasts, the differential regulation of cytolytic effector gene and protein expression in primary murine CTL (63, 81–83), and the previously reported absence of constitutive CCL5 expression by mouse CD8+ memory phenotype T cells, in particular (84, 85), further complicate resolution of this issue. Our direct ex vivo FC analyses of LCMV-specific CD8+TE now demonstrate a clear association of CCL5 and GzmB expression, whereas GzmA, as reported previously by us [and also similar to influenza-specific CD8+TE (37, 63)], was expressed by only ∼60% of the CD8+TE population (Fig. 4D). These observations indicate that CCL5 colocalization studies in murine CD8+TE should be extended beyond the visualization of GzmA. Accordingly, confocal microscopy revealed the existence of multiple discrete vesicles that contained either GzmA, GzmB, or both (Fig. 5, rows 1, 2); in contrast, CCL5+ vesicles appeared mostly devoid of GzmA/B (Fig. 5, rows 3–5) and thus presented with an expression pattern reminiscent of the subcellular CCL5 localization in primary human CD8+ memory phenotype T cells (80). Nevertheless, we did observe polarization and coalescence of GzmA/B+ and CCL5+ vesicles in some cells, perhaps a result of recent CD8+TE activation and an indication that these effector molecules are likely cosecreted (Fig. 5, row 6, 7). The overall distribution of granzymes and CCL5 across individual vesicles in CD8+TE is therefore somewhat heterogeneous, a conclusion also reported for the subcellular expression patterns of CCL5, perforin, and granulysin in human CD8+T cells (79).

FIGURE 5.
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FIGURE 5.

Subcellular localization of GzmA, GzmB, and CCL5 in CD8+TE. Blood-borne CD8+TE (day 8) were stained with anti-GzmA–FITC, anti-GzmB–allophycocyanin, and anti-CCL5/anti-goat–Cy3, and sorted GzmA+ subsets were analyzed by confocal microscopy as detailed in Materials and Methods (GzmA− subsets were used as a negative staining control). (Rows 1 and 2) Subcellular GzmA and GzmB localization in two different cells; (rows 2–5) same cell analyzed for GzmA, GzmB, and/or CCL5 colocalization; (row 6) GzmB and CCL5 expression in the sorted GzmA− subset; (row 7) example for partial polarization and coalescence of intracellular GzmA/B and CCL5 stores in another CD8+TE (representative images are taken from one of three experiments conducted with two to three mice each). Original magnification ×63 with 1.9 or 5.95 digital zoom.

The precise molecular mechanisms underpinning constitutive CCL5 expression by CD8+TE, unique among the cytokines/chemokines, remain unclear. Given the general similarity between CD8+T and NK cells (86) and, more specifically, the chemokine production profiles largely shared between CD8+TE and NK cells (33), we considered the proposal that constitutive CCL5 expression by human NK cells is dependent on the JNK pathway (87). As shown in Supplemental Fig. 3F, 3G, however, Jnk1−/− and Jnk2−/− mice, both of which readily control an acute LCMV infection (88), did not present with any abnormalities at the level of constitutively expressed CCL5 in NK cells or virus-specific CD8+ or CD4+TE.

Kinetics of chemokine synthesis by virus-specific CD8+TE

The elaboration of diverse T cell effector functions is a coordinated event that integrates spatial and temporal constraints with potentially different activation requirements. To determine the velocity of chemokine production by specific CD8+TE, p14 TE were restimulated for 0–5 h with GP33 peptide in the presence or absence of transcriptional (ActD), translational (CHX), and/or protein secretion inhibitors (BFA) and analyzed for intracellular chemokine content by FC using IFN-γ production as a reference. Induced IFN-γ, CCL3, and CCL4 expression became detectable after as little as 30 min of stimulation and reached a maximum after 4–5 h. The synthesis of these proteins was sufficiently robust to allow detection of intracellular IFN-γ and CCL3/4, even in the absence of BFA (Fig. 6A–C, panels 1, 2). Given the presence of ex vivo–detectable mRNA species for CCL3/4 and IFN-γ (Fig.1A, Supplemental Fig. 2C), protein synthesis, although reduced, was still observed under conditions of transcriptional blockade in the presence but not absence of BFA (Fig. 6A–C, panels 3, 4). The fact that protein synthesis increased over time in a homogeneous fashion in all p14 TE (data not shown) suggests that the constitutive IFN-γ and CCL3/4 message is evenly distributed among individual cells rather than preferentially allocated to a particular p14 subset. As expected, inhibition of translation or combined transcriptional/translational blockade completely prevented the accumulation of IFN-γ and CCL3/4 proteins (Fig. 6A–C, panels 5–8).

FIGURE 6.
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FIGURE 6.

Regulation and kinetics of chemokine production by p14 CD8+TE. (A–D) Spleen cells from LCMV-infected p14 chimeras (day 8) were cultured for indicated time periods in the presence (closed symbols) or absence (open symbols) of GP33 peptide and indicated transcriptional (ActD), translational (CHX), and/or protein transport (BFA) inhibitors. Graphs depict the geometric mean of fluorescence intensity (GMFI) of IFN-γ, CCL3, CCL4, and CCL5 expression by p14 TE as a function of culture time and inhibitor presence or absence (panels depicting traditional stimulation conditions [i.e., peptide plus BFA] are shaded in gray). To better compare the kinetic regulation of cytokine and chemokine production, the respective GMFI values were normalized (IFN-γ: the GMFI of rat isotype control stains was subtracted from all corresponding IFN-γ GMFI values and the resulting values of BFA/GP33 cultures for the t = 5.0 h time point [A.1] were set at 100%; CCL3 and CCL4: similar normalization performed by subtraction GMFI values of goat IgG stains from corresponding CCL3 or CCL4 GMFI values; CCL5: ex vivo goat IgG control stain GMFI was subtracted from all CCL5 GMFI values, and the resulting normalized ex vivo CCL5 values [B.1] were set at 100%). The sigmoidal curve fit is based on optimal fits determined by nonlinear regression analyses of samples containing additional time points (n = 3 mice per group); data were from three similar experiments.

The kinetics of intracellular CCL5 accumulation were predictably more complex because TCR-induced release of prestored CCL5 and initiation of protein neosynthesis occurred in parallel. In fact, a modest loss of CCL5 observed 30 min after TCR stimulation was quickly compensated by a pronounced increase of intracellular CCL5 in cultures containing BFA, a pattern that contrasted with rapid if only partial CCL5 depletion in the absence of BFA (Fig. 6D, panels 1, 2). It is therefore worth mentioning that the release of newly synthesized CCL5, in contrast to the release of prestored CCL5 (80), was largely inhibited by BFA. Furthermore, the kinetics of intracellular CCL5 accumulation were comparable in the presence and absence of transcriptional inhibition (Fig. 6D, panels 1, 3), consistent with our observation that TCR stimulation does not increase the level of CCL5 mRNA (Fig. 1A), and an early increase of intracellular CCL5 in cultures without TCR stimulation and translational blockade emphasizes that maintenance of constitutive CCL5 expression by T cells is an active process; the eventual decline of CCL5 expression at later time points is likely due to degradation because we did not observe CCL5 secretion by unstimulated T cells (Fig. 6D, panels 2, 4, data not shown). Interestingly, upon TCR stimulation in the presence of translational or combined transcriptional/translational blockade, ∼2/3 of prestored CCL5 were released within 30–60 min; additional depletion of CCL5 stores occurred with slower kinetics and was inhibited by BFA (Figs. 6D, panels 5–8, 7A).

FIGURE 7.
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FIGURE 7.

Rapid surface translocation and secretion of prestored CCL5 by antiviral CD8+TE. (A) Spleen cells from day 8 p14 chimeras were preincubated with CHX prior to initiation of TCR stimulation by addition of GP33 peptide and subsequent analysis of p14 GzmB and CCL5 content 0–5 h later. GzmB and CCL5 expression levels in geometric mean of fluorescence intensity (GMFI) were normalized such that t = 0 h levels correspond to 100% and the GMFI of respective control stains are set at to 0%. Left, Kinetics of prestored CCL5 release in the presence versus absence of BFA. Right, Immediate depletion of CCL5 stores versus delayed GzmB release. At t = 0.5 h, ∼2/3 of CCL5 but <10% of GzmB stores are emptied (n = 3 mice) for one to three independent experiments. (B) Conjugate formation between purified splenic p14 TE (CD90.1) and GP33 peptide-coated or uncoated EL4 cells (CD90.2) as well as CCL5 surface expression (sCCL5) were assessed 20 min after initiation of coculture as detailed in Materials and Methods. Four populations were distinguished according to CD90.1/2 expression levels and forward scatter (FSC) properties (cell size) as follows: (B1) EL4 cells; (B2) EL4:p14 TE conjugates; (B3) p14 TE expressing low levels of CD90.2, likely acquired by trogocytosis; and (B4) p14 TE. Note the weak but distinctive sCCL5 staining detectable among specific (black tracings) but not unspecific (gray histograms) EL4:p14 TE conjugates (population 2). (C) Conjugation assays were performed as above and analyzed by confocal microscopy to visualize sCCL5 (green) and CD90.2 (red) expression (panels C1 and C3 and panels C2 and C4 are identical with CD90.2 signals removed from panels 1 and 2 to better visualize sCCL5 expression). Note the “blebbing” of the EL4 cell in panel C5 consistent with the induction of apoptosis; panel C6 features a magnification of the p14 TE in panel 5 to demonstrate IS (white arrow) and antipolar (gray arrow) localization of sCCL5. (D) Spleen cells from LCMV-infected B6 mice (day 8) were preincubated with CHX and stimulated with NP396 peptide (no BFA), and CCL5 in the supernatant was quantified by ELISA. To calculate the amount of prestored CCL5 secreted by individual NP396-specific CD8+TE, complementary FACS analyses were performed to determine the absolute numbers of cultured specific CD8+TE. Further, the amount of CCL5 secreted in the absence of TCR stimulation was subtracted from stimulated samples at all time points. Note that after 30 min, ∼60% of total CCL5 is already secreted; at 1 h, it is ∼90%. Data are representative for two to four experiments conducted with three individual mice each. *p < 0.05, **p < 0.01.

Rapid CCL5 surface translocation and secretion by virus-specific CD8+TE

To interrogate the remarkably fast CCL5 release kinetics by CD8+TE in more detail, we compared the concurrent depletion of prestored CCL5 and GzmB from TCR-stimulated p14 TE in the presence of CHX. In this study, the near-instantaneous release of CCL5 contrasted with a ∼30-min lag period before intracellular GzmB began to decline; yet, the subsequent loss of GzmB proceeded so rapidly that the relative extent of CCL5 and GzmB depletion was comparable by 1 h after initiation of T cell stimulation (Fig. 7A). Overall, the kinetic differences between CCL5 and GzmB depletion, as well as the differential sensitivity of constitutive versus induced CCL5 expression/release to BFA, corresponds well with the heterogeneous distribution of CCL5 and GzmB across different subcellular compartments as shown in Fig. 5.

To better visualize the earliest events of TCR-induced CCL5 release, we performed an in vitro conjugation assay using purified p14 TE (CD90.1) and congenic (CD90.2) GP33 peptide-coated versus uncoated EL4 target cells (Fig. 7B, 7C). Within 20 min after TCR engagement and thus before initiation of CCL5 neosynthesis, CCL5 was translocated to the cell surface, deposited preferentially at the interface between p14 TE:EL4 conjugates, and the engagement of EL4 cells was readily demonstrated by the focused redistribution of CD90.2 around the immunological synapse (IS) (89); formation of “unspecific” conjugates (i.e., in the absence of GP33 peptide) was not associated with cell surface exposure of CCL5 or a clustering of EL4-expressed CD90.2 (Fig. 7B, 7C). Our data therefore support the notion that mobilization of intracellular CCL5 stores is primarily directed toward the IS, similar to the polarization reported for polyclonally activated human CD8+T cell blasts and clones (80, 90); yet, they apparently differ from results of another study in which the de novo synthesis of CCL5 by in vitro–generated murine CD4+T cell blasts resulted in multidirectional release of this chemokine (i.e., an early [2-h] association of intracellular chemokine stores with the IS followed by a later [4-h] distribution) in multiple compartments throughout the cytoplasm (91). In agreement with the latter report, however, we found that p14 TE on occasion presented low amounts of antipolar surface CCL5 (Fig. 7C, panel 6), and it is tempting to speculate that the heterogenous subcellular distribution of prestored CCL5 is related to the reported association with distinct trafficking proteins that mediate a multidirectional versus focused chemokine release (90, 91).

The rapid accumulation of CCL5 within the IS, a defined space with an estimated volume of 0.5–5.0 × 10−16 l (91, 92), suggests that local CCL5 concentrations may temporarily reach “supraphysiological” levels. The latter term describes multiple observations that in vitro exposure of cells to oligomeric CCL5 in excess of ∼1 μM promotes receptor-independent binding to surface glycosaminoglycans and generalized activation that, depending on the cell type under study, results in cellular proliferation and differentiation as well as enhanced survival, CTL activity, and cytokine and chemokine release (93–99). Whether these effects have an in vivo correlate has remained doubtful, and to provide a more quantitative estimate, we combined FC and ELISA assays conducted in the presence of CHX to calculate the amount of prestored CCL5 that is secreted by an individual LCMV-specific CD8+TE. The rapid increase of CCL5 in the ELISA culture supernatant (Fig. 7D) mirrors the loss of intracellular CCL5 in Fig. 7A and corresponds to ∼0.5 fg per CD8+TE released in the first 30 min after TCR triggering, an amount that could in principle result in a CCL5 concentration of >100 μM within the confines of the IS. Even if these calculations constitute a gross overestimate due to incomplete CCL5 release, multiple CD8+TE:target cell contact sites (100), limited spatial constraints, and/or rapid diffusion, it would appear likely that CCL5 concentrations of >1 μM could be achieved in a spatially and temporally confined fashion in vivo and therefore might contribute to target cell activation in a receptor-independent fashion.

Induced CCL3/4/5 colocalization and cosecretion by virus-specific CD8+TE

The coproduction of CCL3/4/5 by stimulated CD8+TE, as evidenced by the “diagonal” event distribution in FC plots (Fig. 2D), suggests a tight association and potential colocalization of these chemokines following CD8+TE activation. When analyzed by confocal microscopy under these conditions, CCL3/4/5 as well as GzmB and IFN-γ indeed tended to cluster in a single defined location close to the plasma membrane and in immediate proximity of the IS (Supplemental Fig. 4A, data not shown). The colocalization of induced CCL3/4/5 expression, in particular, might provide a basis for the joint release of these chemokines bound to sulfated proteoglycans as described for HIV-specific CD8+T cell clones (17). Moreover, human PBMC stimulated with PMA secrete CCL3/4 as heterodimeric complexes (101), and up to half of the CCL3/4 content in medium conditioned with lymph node cells from recently immunized mice is in a state of hetero-oligomerization (29). To determine the extent of heterologous chemokine complex formation in the context of a virus-specific T cell response, splenic CD8+TE were restimulated for 5 h, the supernatants were collected and preabsorbed with anti-CCL3, anti-CCL4, anti-CCL5, or control Abs prior to quantitation of CCL3/4/5 by ELISA (Supplemental Fig. 4B). Although we noted some variability in these cross-absorption experiments, we have previously confirmed the specificity of Abs used for preabsorption (33) and therefore can conclude that the biologically active form of CD8+TE–secreted CCL3/4/5 consists in part of hetero-oligomeric complexes. Beyond the apparently intimate coordination of CCL3/4/5 activities, this finding also emphasizes important limitations for the interpretation of any experiments that employ Ab-mediated in vivo neutralization of these chemokines.

Specific antiviral T cell immunity in the absence of systemic chemokine deficiencies

Despite their prominence among T cell–produced effector molecules, CCL3/4/5 are apparently dispensable for the control of an acute LCMV infection (102–104). Accordingly, we found that LCMV-challenged B6.CCL3−/− and B6.CCL5−/− mice generated a diversified virus-specific T cell response and controlled the infection with kinetics comparable to B6 wt mice (Supplemental Fig. 4C, 4D; similarly, B6.CCL1−/− mice mounted normal TE responses and readily controlled LCMV; Fig. 2D, data not shown). Because CCL5 may exert direct apoptotic functions (105) and in vitro degranulation and killing by CCL5−/− CD8+T cells in the context of a chronic LCMV infection is reportedly impaired (104), we also examined the in vivo–killing kinetics by LCMV-specific CD8+TE in the absence of CCL5. As shown in Supplemental Fig. 4E, however, in vivo target cell killing proceeded with the same rapid kinetics in wt and CCL5-deficient mice, and the lack of a role for CCL5 in this assay was further confirmed by treatment with a CCL5-neutralizing Ab.

However, a careful analysis of TE chemokine expression profiles in B6.CCL3−/− and B6.CCL5−/− mice demonstrated some unanticipated quantitative differences. In comparison with B6 mice, B6.CCL3−/− but not B6.CCL5−/− mice generated a slightly reduced antiviral CD8+ but increased CD4+TE response (Supplemental Fig. 4C, 4D). Furthermore, in B6.CCL3−/− mice, CCL4 and CCL5 production by specific CD8+ and CD4+TE was significantly diminished in comparison with B6 mice, and a somewhat lesser reduction of induced CCL3 and CCL4 expression was also observed for B6.CCL5−/− mice (Fig. 8A, 8B, Supplemental Fig. 4F). These differences were even more pronounced when chemokine production by specific CD8+TE was quantified in vivo following a 1-h peptide inoculation (Fig. 8C, data not shown). Lastly, both B6.CCL3−/− and B6.CCL5−/− mice also exhibited a modest but significant impairment of CCL9/10 production capacity by GP33-specific CD8+TE interrogated in vitro (Supplemental Fig. 4F). Altogether, functional impairments extending beyond specific chemokine gene deficiencies may be related to the cooperative regulation of chemokine expression/secretion and/or to artifacts arising in the mutant mice due to the proximity of respective gene loci on chromosome 11 (Fig. 1D); they will also need to be considered for any interpretation of relevant observations made with CCL3- or CCL5-deficient mice.

FIGURE 8.
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FIGURE 8.

Impact of CCL3 or CCL5 deficiency on related chemokine production capacity by antiviral TE. (A) Induced CCL3/4/5 production by GP33-specific CD8+ and GP64-specific CD4+TE analyzed on day 8 after LCMV challenge of B6, B6.CCL3−/−, and B6.CCL5−/− mice (all plots gated on CD8+ or CD4+T cells). (B) CCL3/4/5 content of stimulated GP33-specific CD8+ and GP64-specific CD4+TE in wt and chemokine-deficient mice (n = 3 per group) for one of three similar experiments; asterisks indicate significant differences between B6 and mutant mice by one-way ANOVA. (C) One hour in vivo CD8+TE activation assays were performed on day 8 after LCMV infection of B6 and B6.CCL5−/− mice by i.v. injection of GP33 peptide, as detailed in Materials and Methods (saline injection was negative control). Note the reduced CCL3 induction in CCL5-deficient DbGP33+CD8+TE (n = 3 mice per group) for one of two experiments; all plots were gated on splenic CD8+T cells; GP33 peptide also activates KbGP34+CD8+TE, accounting for the DbGP33 tetramer-negative population in the LR plot quadrants). *p < 0.05, **p < 0.01, ***p < 0.001.

Last, fatal lymphocytic choriomeningitis following i.c. LCMV infection of immunocompetent mice is contingent on a potent virus-specific CD8+TE population that may recruit pathogenic myelomonocytic cells into the CNS through secretion of CCL3/4/5 (27, 106, 107). Prior work with CCL3- and CCR5-deficient mice, however, has demonstrated a normal lethal phenotype after i.c. LCMV challenge (102, 103), leaving the possibility that CCL5 may uniquely contribute to the fatal disease course. In this study, we used a set of chemokine- and chemokine receptor-deficient mice to assess whether the lack of any CD8TE-produced chemokines delayed or prevented lethal choriomeningitis. Specifically, we employed CCL1−/−, CCL3−/−, CCR5−/− (CCL3/4/5 receptor), CCL5−/−, CCR1−/− (CCL3/5/9/10 receptor), and CCR3−/− (CCL5/9/10 receptor) mice and found that all of them succumbed to lethal disease with kinetics comparable to B6 or BALB/c control mice (Fig. 9). Even CCL3-deficient mice lacking CCR5 and thus exhibiting a reduced CCL4/5 production capacity (Fig. 8A, 8B) as well as decreased or absent responsiveness to CCL5 or CCL4, respectively, readily died after i.c. LCMV infection (Fig. 9). Although we cannot rule out that more complex compound chemokine/receptor deficiencies may alter the course of lethal disease and a potential contribution of the XCL1:XCR1 axis remains to be investigated, the fatal course of i.c. LCMV infection appears largely independent of chemokines produced by virus-specific CD8+TE.

FIGURE 9.
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FIGURE 9.

No role for antiviral TE-produced chemokines in the development of lethal choriomeningitis. The wt, chemokine- and/or chemokine receptor-deficient mice were infected with LCMV i.c., and survival was monitored (as per IACUC guidelines, we employed a scoring matrix to measure morbidity, and terminally ill mice were euthanized and scored as deceased). Multiple independent experiments were performed with matched experimental and control mice each, and the data displays feature the cumulative total (n) of individual mice analyzed in four separate experiments. The lower-right insert displays TE-produced chemokines and their respective receptors with specific chemokines/chemokine receptors interrogated in the present analysis highlighted in black.

Discussion

Pathogen-specific TE are cardinal components of the adaptive immune response to viral and bacterial infections. Despite a wealth of knowledge about the contribution of T cell–derived cytokines, TNFSF ligands, and cytolytic effector mechanisms to initial pathogen control, the full spectrum of potentially relevant T cell activities as well as their roles in shaping effective TE responses and providing immune protection remain incompletely defined. Our delineation of the entire range of chemokines produced by specific CD8+ and CD4+TE in the wake of different pathogen infections or immunizations constitutes an important addition to the analytically accessible repertoire of T cell functions for several important reasons: its near exclusive focus on six chemokines (CCL1, CCL3, CCL4, CCL5, CCL9/10, XCL1), a relative consistency of expression patterns across different in vivo challenge protocols, the sheer magnitude of the specific CD8+TE chemokine response, and quantitative differences between CD8+ and CD4+TE populations. In addition, T cells appear to be the major hematopoietic source for CCL1; CCL3/4/5 production/secretion is coregulated; and the unique temporospatial organization of CCL5 synthesis, storage, and secretion positions its targeted release at the forefront of the mature CD8+TE response.

Using a combination of transcriptomic profiling and chemokine FC, we have defined a hierarchy of inducible chemokine expression by LCMV-specific CD8+TE that pertains to all (CCL3/4) or nearly all (CCL5) CD8+TE as well as greater and smaller subsets thereof (XCL1 > CCL1 ≥ CCL9/10) (constitutive CCL5 expression is discussed below). At the same time, no other chemokine proteins are synthesized by LCMV-specific CD8+TE, a contextual contention that is based on our use of a rigorously vetted collection of highly sensitive chemokine-specific Abs deployed under optimal staining conditions (33). Although a lack of mRNA translation is a common feature of eukaryotic organisms (108), the absence of induced CXCL10 protein expression, in particular given the significant induction of Cxcl10 mRNA following TCR stimulation, would appear to contradict reports that have documented CD8+T cell–produced CXCL10 (e.g., Ref. 109). To our knowledge, however, direct visualization of CD8+T cell–expressed CXCL10 has not been demonstrated (including in our own exploration of multiple other experimental scenarios), and CXCL10 detection in supernatants from T cell stimulation cultures can arise from small populations of contaminating myeloid cells that readily produce CXCL10 in response to T cell–secreted IFN-γ (data not shown). Although our conclusion that neither CXCL10 nor 30 other chemokines are produced by activated CD8+TE is delimited by assay sensitivities and precise experimental context, the similarly restricted chemokine expression profiles of specific CD8+TE across different epitope-specific populations with distinct avidities and immunodominant determinants, mouse strains and infection, or vaccination modalities indicates that our analyses most likely capture the relevant components of the CD8+TE chemokine response in their entirety. Thus, the inducible production of CCL1, CCL3, CCL4, CCL5, CCL9/10, and XCL1 is a shared signature of protective CD8+TE populations generated in response to primary viral, bacterial, and vaccine challenges. Moreover, the development of a vigorous chemokine response by vaccine-elicited CD8+TE, which in contrast to pathogen-specific CD8+TE are not reliant on aerobic glycolysis to support their clonal expansion, reinforces the notion that the acquisition of robust effector functions is equally uncoupled from a Warburg metabolism (110). It is also noteworthy that CCL9/10, regarded a homeostatic chemokine, is part of the inflammatory CD8+TE response and therefore may be reclassified as a “dual function” chemokine; conversely, the constitutive expression of the inflammatory chemokines CCL3/4/5 by resting NK cells, as shown in this study and/or in Ref. 33, adds a homeostatic component that also may warrant the assignment of dual function to those chemokines.

One of the more striking aspects of the CD8+TE chemokine response is its apparent magnitude. By stratifying CD8+TE functionalities according to 17 individual parameters, we estimate that the synthesis and secretion of chemokines accounts for >40% of commonly quantified CD8+TE activities. The remarkable abundance and distinct profile of chemokines produced by CD8+TE therefore establish these cells as major focal points for the recruitment of other immune cells, the spatiotemporal organization of cellular interactions, and the overall coordination of complex effector immune responses. This conclusion, however, stands in marked contrast to the mostly modest phenotypes reported for specific T cell responses and/or pathogen control in mice lacking T cell–produced chemokines. Our own work confirms the generation of broadly normal LCMV-specific TE responses and virus clearance kinetics in CCL3- and CCL5-deficient mice and extends these observations to CCL1 deficiency; although these experiments do not focus on TE as a specific source for selected chemokines, the lack of a pronounced phenotype under conditions of systemic chemokine deficiency strongly suggests a negligible function for the respective TE-derived chemokines. We further demonstrate in a stringent disease model in which LCMV-specific CD8+TE activities are essential for the recruitment of pathogenic myelomonocytic cells (27) that CCL1/3/4/5 and 9/10 are apparently dispensable for the development of lethal immunopathology. Although these findings may in part be grounded in biological redundancies within the chemokine system, we note that lack of cardinal TE molecules, such as IFN-γ, TNF-α, IL-2, FasL, and GzmA and/or GzmB, often produces only subtle defects at the level of LCMV-specific T cell immunity and associated virus control (111–114). Rather, nonredundant contributions of specific T cell–produced chemokines in effective control of primary pathogen infections are likely to emerge in the context of compound immune deficiencies and within specific constraints of precisely delineated experimental scenarios for which the current study provides a comprehensive practical and conceptual foundation.

Another notable finding pertains to the chemokine response of CD4+TE as well as its shared and distinctive aspects in comparison with CD8+TE populations, which typically present with substantially greater primary expansions (46). The basic CD4+TE chemokine profile, largely preserved in different infection and immunization settings, is composed of the same chemokines made by CD8+TE (CCL1/3/4/5/9/10 and XCL1) but displays discrete quantitative differences: although CCL3/4/5 production is also the most prominent part of the CD4+TE response, only 30–60% of pathogen-specific CD4+TE readily synthesize these chemokines, and the strict coexpression of CCL3/4 (data not shown) points toward a specialized CD4+TE subset dedicated to the chemokine-dependent recruitment of CCR1/3/5-bearing immune cells. The fraction of CCL1-producing CD4+TE is comparable with or somewhat larger than that of the corresponding CD8+TE compartment, and only small subsets of CD4+TE (≤5%) make CCL9/10 or XCL1. Interestingly, these pathogen-specific CD4+TE chemokine profiles correspond remarkably well to a transcriptomic screen conducted for the presence of 28 chemokine mRNA species in in vitro–polarized polyclonal TH1 cells (115); the only other chemokine message detected in a complementary screen of TH2 cells was CXCL2 (115), also found at the protein level in polarized TH2 cells (24) and readily captured in our initial survey of primary T cell–produced chemokines. As expected for our TH1-dominated infection models, CXCL2-producing specific CD4+TE were either absent (rLM-OVA) or present at very low frequencies (∼0.2%; LCMV).

Several of the TE-produced chemokines characterized in this study exhibit additional unique properties. T cells are considered an important source for CCL1, as evidenced, for example, by Ccl1 mRNA transcription in activated CD4+ and CD8+T cell clones (116) or the secretion of CCL1 protein (in conjunction with the other CD4+TE chemokines CCL3/4/5/9/10 and XCL1) by diabetogenic CD4+T cell clones (117). Innate immune cells, such as mast cells (118) and L. monocytogenes–infected DCs (119), may constitute additional hematopoietic sources for this chemokine, but in our own work, we did not observe CCL1 expression by L. monocytogenes–infected DCs, activated B cells, myeloid cells, or NK cells (33); the lack of NK cell–produced CCL1 is particularly noteworthy because these cells readily produce all of the other CD8+TE chemokines (33). Thus, whereas innate immune cell populations capable of CCL1 synthesis remain to be characterized in greater detail, TE would appear to be a major and distinctive, if not exclusive, hematopoietic provenance of CCL1. Furthermore, its transcription/translation is strictly activation dependent (i.e., Ccl1 mRNA abundance displays the greatest differential of all T cell chemokines between ex vivo– and anti-CD3/anti-CD28–stimulated CD8+TE), and for reasons that remain unclear, CD8+TE activation with PMA/ionomycin fails to elicit CCL1 protein expression with same efficacy as anti-CD3/anti-CD28 or peptide stimulation (a similar disconnect was also observed for CCL9/10 induction). Perhaps most intriguingly, our chemokine coexpression analyses revealed that the CCL1+ CD8+TE subset exhibits pronounced functional diversity because this population coproduces XCL1 in addition to CCL3/4/5 and IFN-γ, and CD8+TE with enhanced potential for CD8+TM development (MPECs) foreshadowed CD8+TM chemokine profiles (37, 47) as reflected in their greater CCL1, CCL9/10, and especially XCL1 production capacity. Beyond the visualization of chemokine coexpression patterns by FC, our results also demonstrate that induced CCL3/4/5 production by primary virus-specific CD8+TE is coregulated, as shown by their shared compartmentalized subcellular localization and secretion in part as macromolecular complexes. Although this observation is in keeping with the general capacity for complex formation by disparate chemokines (99), our analyses of chemokine-deficient mice provide additional clues for the potentially cooperative nature of TE chemokine synthesis/secretion: CCL3-deficient CD8+TE and to a lesser extent also CD4+TE display a reduced capacity for CCL4, CCL5, and CCL9/10 production; similarly, CCL5-deficient TE present with a somewhat impaired CCL3/4/9/10 response. We note, however, that we cannot rule out the possibility that these defects do not at least in part arise from the close proximity of the respective chemokine gene loci to the mutant genes in CCL3- or CCL5-deficient mice.

Arguably the most distinctive feature of the primary pathogen-specific TE chemokine response pertains to the regulation of CCL5 production, expression, and secretion not, to our knowledge, previously detailed in murine model systems. Specifically, these properties comprise a delayed CCL5 production capacity of developing TE populations, the constitutive CCL5 (i.e., directly ex vivo quantifiable) expression in subcellular compartments largely distinct from cytolytic granules, and the extraordinarily fast kinetics of focused CCL5 release. In contrast to all other TE activities, CCL5 expression by T cells is delayed for 3–5 d after priming as a function of regulatory control exerted by KLF13 (76–78). Our in vitro experiments with LCMV-specific CD8+TE confirm this notion (ready induction of GzmB and IFN-γ but only minimal CCL5 expression within 72 h of stimulation) and, to our knowledge for the first time, demonstrate these kinetics in the context of a primary CD8+TE response in vivo: virtually undetectable for the first ∼3 d after LCMV challenge, constitutive CCL5 expression is found in ∼50% of specific CD8+TE on day 5 before emerging as a property of practically all antiviral CD8+TE by day 7–8. Thus, constitutive CCL5 expression is a distinctive hallmark for mature pathogen- and vaccine-specific CD8+TE (as well as a subset of CD4+TE) that may also serve as a diagnostic readout for the better staging of initial TE differentiation. Although we did not have the opportunity to study the impact of KLF13 deficiency in our model systems, we considered another potential mechanism that may contribute to the constitutive CCL5+ phenotype. Human NK cells were reported to regulate constitutive CCL5 expression through the JNK/MAPK pathway (87), and in mice, NK cells are the only hematopoietic population other than T cells that presents with substantial ex vivo–detectable CCL5 content (Ref. 33, data not shown). However, as based on the undiminished constitutive CCL5 expression by NK cells or specific CD8+ and CD4+TE under conditions of JNK1 or JNK2 deficiency, the JNK/MAPK pathway does not appear to contribute to the CCL5+ phenotype in mice.

The ready visualization of both constitutive and induced CCL5 expression by pathogen-specific CD8+TE also may resolve seeming discrepancies pertaining to its exact subcellular distribution in human and/or murine T cell clones, blasts, or primary CD8+T cell subsets (17, 79, 80, 84, 85). Evaluated directly ex vivo, the CCL5 content of CD8+TE is preferentially distributed across multiple vesicles discrete from GzmA- and/or GzmB-containing cytolytic granules; yet, an occasional polarization and coalescence of GzmA/B+ and CCL5+ vesicles, likely indicative of most recent T cell activation, is substantially increased following deliberate TCR stimulation, further incorporates newly synthesized CCL3/4, and thus provides a foundation for the focused release of CCL3/4/5 in part as macromolecular complexes. In fact, CCL3/4 translation by CD8+TE, just like that of IFN-γ, is initiated from abundantly present mRNA templates within just 30 min after TCR triggering and is subsequently amplified by the robust induction of additional mRNA transcription. In contrast, the release of prestored CCL5 after TCR engagement is near instantaneous, even precedes the full mobilization of cytolytic granules, and is primarily directed toward the IS formed between CD8+TE and sensitized target cells. The combination of remarkably fast and focused CCL5 accumulation in a tight interaction space may temporarily create conditions associated with a spike of local CCL5 concentrations in excess of 1.0 μM [i.e., a microenvironment that can promote conjugate stabilization, achieved, for example, already with 130 nM CCL5 added to in vitro cultures (120)] and may contribute to receptor-independent target cell activation (95). Interestingly, although the initial burst of CCL5 secretion is followed by additional protein production, translation is restricted to the use of pre-existing mRNA species because, in contrast to all other CD8+TE chemokines, cytokines, and TNFSF ligands, no further transcription is induced for at least 3 h of TCR activation, and secretion of newly synthesized, as opposed to prestored CCL5, is sensitive to inhibition by BFA; thus, CD8+TE activation promotes two successive waves of CCL5 release characterized by their distinctive temporospatial organization of CCL5 synthesis, storage, and secretion.

Again, however, it remains unclear to what extent the specific TE CCL5 response and its unique characteristics may provide relevant and nonredundant contributions to the control of infectious diseases, especially in experimental or natural scenarios beyond HIV infection. For one thing, the historically preferred experimental usage of chemokine receptor-deficient mice complicates any interpretation pertaining to the precise role of CCL5 because of its promiscuous receptor usage (CCR1/3/5) as well as receptor-independent modes of action (121). The use of CCL5 neutralization or CCL5-deficient mice can address these issues, and although to date employed less frequently in infectious disease studies, the targeting of CCL5 collectively shows a mostly modest impairment of pathogen-specific TE immunity that results, depending on experimental systems, in ameliorated immunopathology or exacerbated disease due to compromised pathogen control (reviewed in Ref. 121); whether any of these phenotypes are contingent on the specific lack CCL5 produced by TE rather than other hematopoietic sources remains an open question.

In summary, we demonstrate that the prodigious production of chemokines, purveyors of cues essential to the coordination of complex immune responses, constitutes a circumscribed yet diverse, prominent, and largely consistent component integral to the functionality of pathogen- and vaccine-specific TE. Further characterized by several unique aspects pertaining to the synthesis, coexpression and regulation as well as secretion of certain chemokines, the TE chemokine response is readily visualized, quantified, and dissected by analytical FC. As such, we propose that T cell profiling according to six distinct chemokines will considerably expand the repertoire of functional T cell assays and, importantly, may provide potentially important insights into specific T cell immunity under various experimental and naturally occurring conditions. We have pursued some of that work in an accompanying report that delineates the chemokine signatures of pathogen-specific TM under condition of effective Ag clearance as well as prolonged persistence (47).

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. S. Manes for the gift of B6.CCL1−/− mice; Dr. L. Lenz for rLM-OVA; Dr. T. Lane for the CCL5 Ab clone R6G9; Dr. M. Simon for GzmA anti-serum; Drs. L. Sheridan, L. Edwards, and J. Humann for experimental assistance; Drs. P. Marrack and J. Kappler and the entire the K/M lab for critical discussion; and Dr. F. Mortari (R&D Systems) for the generous gift of the majority of chemokine Abs used in this study.

Footnotes

  • This work was supported by National Institutes of Health (NIH) Grants AG026518 and AI093637, the JDRF (CDA 2-2007-240), a Barbara Davis Center Pilot and Feasibility grant, NIH Diabetes Endocrinology Research Center Grant P30-DK057516 (to D.H.), NIH Grants U54-HL127624 and U24-CA224260 (to A.M.), and NIH T32 Training Grants AI07405, AI052066, and DK007792 (to B.D.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ActD
    actinomycin D
    BFA
    brefeldin A
    CHX
    cycloheximide
    DC
    dendritic cell
    FC
    flow cytometry
    GzmA
    granzyme A
    GzmB
    granzyme B
    IACUC
    Institutional Animal Care and Use Committee
    i.c.
    intracerebral(ly)
    IS
    immunological synapse
    LCMV
    lymphocytic choriomeningitis virus
    MPEC
    memory precursor effector cell
    pab
    polyclonal Ab
    rLM-OVA
    recombinant L. monocytogenes expressing full-length OVA
    TE
    effector T cell
    TM
    memory T cell
    VSV
    vesicular stomatitis virus
    wt
    wild-type.

  • Received March 9, 2020.
  • Accepted August 7, 2020.
  • Copyright © 2020 by The American Association of Immunologists, Inc.

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The Journal of Immunology: 205 (8)
The Journal of Immunology
Vol. 205, Issue 8
15 Oct 2020
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Chemokine Signatures of Pathogen-Specific T Cells I: Effector T Cells
Jens Eberlein, Bennett Davenport, Tom T. Nguyen, Francisco Victorino, Kevin Jhun, Verena van der Heide, Maxim Kuleshov, Avi Ma’ayan, Ross Kedl, Dirk Homann
The Journal of Immunology October 15, 2020, 205 (8) 2169-2187; DOI: 10.4049/jimmunol.2000253

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Chemokine Signatures of Pathogen-Specific T Cells I: Effector T Cells
Jens Eberlein, Bennett Davenport, Tom T. Nguyen, Francisco Victorino, Kevin Jhun, Verena van der Heide, Maxim Kuleshov, Avi Ma’ayan, Ross Kedl, Dirk Homann
The Journal of Immunology October 15, 2020, 205 (8) 2169-2187; DOI: 10.4049/jimmunol.2000253
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