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Chemokine Signatures of Pathogen-Specific T Cells II: Memory T Cells in Acute and Chronic Infection

Bennett Davenport, Jens Eberlein, Tom T. Nguyen, Francisco Victorino, Verena van der Heide, Maxim Kuleshov, Avi Ma’ayan, Ross Kedl and Dirk Homann
J Immunol October 15, 2020, 205 (8) 2188-2206; DOI: https://doi.org/10.4049/jimmunol.2000254
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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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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Tom T. Nguyen
*Barbara Davis Center for Childhood Diabetes, 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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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 TM are a prodigious source of chemokines.

  • The chemokine-expression patterns of TM largely resemble those of TE.

  • Unique TM chemokine signatures in acute/chronic infection are of diagnostic value.

Abstract

Pathogen-specific memory T cells (TM) contribute to enhanced immune protection under conditions of reinfection, and their effective recruitment into a recall response relies, in part, on cues imparted by chemokines that coordinate their spatiotemporal positioning. An integrated perspective, however, needs to consider TM as a potentially relevant chemokine source themselves. In this study, we employed a comprehensive transcriptional/translational profiling strategy to delineate the identities, expression patterns, and dynamic regulation of chemokines produced by murine pathogen-specific TM. CD8+TM, and to a lesser extent CD4+TM, are a prodigious source for six select chemokines (CCL1/3/4/5, CCL9/10, and XCL1) that collectively constitute a prominent and largely invariant signature across acute and chronic infections. Notably, constitutive CCL5 expression by CD8+TM serves as a unique functional imprint of prior antigenic experience; induced CCL1 production identifies highly polyfunctional CD8+ and CD4+TM subsets; long-term CD8+TM maintenance is associated with a pronounced increase of XCL1 production capacity; chemokines dominate the earliest stages of the CD8+TM recall response because of expeditious synthesis/secretion kinetics (CCL3/4/5) and low activation thresholds (CCL1/3/4/5/XCL1); and TM chemokine profiles modulated by persisting viral Ags exhibit both discrete functional deficits and a notable surplus. Nevertheless, recall responses and partial virus control in chronic infection appear little affected by the absence of major TM chemokines. Although specific contributions of TM-derived chemokines to enhanced immune protection therefore remain to be elucidated in other experimental scenarios, the ready visualization of TM chemokine-expression patterns permits a detailed stratification of TM functionalities that may be correlated with differentiation status, protective capacities, and potential fates.

Introduction

Pathogen-specific memory T cells (TM) are an integral component of the anamnestic immune response and can provide immune protection by curtailing secondary (II°) infections, limiting morbidity, and forestalling potential host death (1–5). These clinical outcomes are the net result of highly complex and coordinated interactions between multiple organ systems, tissues, cell types, and extracellular factors that are marshaled into action following pathogen detection, and the relevant contributions of specific TM to these processes are grounded in three fundamental determinants: their numbers, their location, and their differentiation status (i.e., the particular phenotypic, molecular, and epigenetic makeup that permits TM populations to respond with the elaboration of rapid effector activities as well as cooperative cellular interactions, local and systemic mobilization, II° effector T cell [TE] differentiation, and proliferative expansion). The choreography of these events is, in part, governed by chemokines, a large family of mostly secreted small molecules that regulates the spatiotemporal positioning of motile cells (6–8).

Pathogen-specific TM, by virtue of their distinct chemokine receptor expression patterns, are acutely attuned to varied chemokine cues, as demonstrated in numerous in vitro and in vivo studies (6–11); however, as has been known for over two decades (12), T cells are also a relevant source for certain chemokines themselves, notably for CCL3, CCL4, and CCL5, which, beyond their chemotactic functions, can also act as competitive inhibitors of HIV binding to its coreceptor CCR5 (13–15); at micromolar concentrations, CCL5 may exert receptor-independent cellular activation, apoptosis, and even antimicrobial activity, although some of the evidence is contradictory and the in vivo relevance unclear (16–21). Several other chemokines, including CCL1, CCL9/10, and XCL1, have further been reported as products of pathogen-specific TM (22–26), but to date, experimental evidence in support of pathogen-specific, TM-derived chemokines as nonredundant contributors to effective immune protection at the level of II° TE expansion, pathogen control, and/or host survival remains limited to CCL3 and possibly XCL1 in some, but not other, murine model systems (27, 28).

Chemokine synthesis and secretion by TM, similar to other effector functions, such as cytokine and TNFSF ligand production, typically require a brief period of TCR activation, a prerequisite that may provide a safeguard against inappropriate TM activation and immunopathology (29). It is therefore of interest that CCL5 is expressed in a constitutive fashion by human CD8+T cell subsets (22, 30, 31), in which it is confined to a unique subcellular compartment and released with near-instantaneous kinetics upon TCR engagement (30). Other studies, however, have demonstrated a preferential association of constitutively expressed CCL5 with cytolytic granules in HIV-specific CD8+T cell clones or primary (I°) human CD8+T cells (13, 31), but murine memory-phenotype CD8+T cells (CD8+TMP), which contain abundant Ccl5 in addition to Ccl3 and Ccl4 transcripts, apparently do not express the corresponding proteins, the synthesis of which requires TCR stimulation (32, 33). Our recent work on chemokine signatures of pathogen-specific CD8+TE may provide clues for a reconciliation of these discrepancies (34): at the peak of the effector response, CD8+TE express constitutive CCL5 in a subcellular compartment distinct from granzyme (Gzm)–containing cytolytic granules, but upon TCR stimulation, these structures partly coalesce just prior to secretion; in fact, release of prestored CCL5 proceeds so rapidly that temporarily, and within the confines of the immunological synapse, CCL5 concentrations well in excess of 1 μM may be achieved (34), a potential foundation for in vivo CCL5 action at supraphysiological levels (16, 17).

Importantly, in the same study, we provide detailed evidence that the induced production of all of the above (CCL1/3/4/5/9/10 and XCL1), but no other chemokines, constitutes a largely invariant functional signature of specific CD8+ and CD4+TE generated in response to acute viral and bacterial infections, as well as protective immunization (34). Building on this work, we have now extended our investigations to an interrogation of the identities, patterns, regulation, and relevance of chemokines expressed by pathogen-specific TM populations maintained after resolution of acute and under conditions of chronic infections. To be sure, because of the fundamentally different disease courses in these experimental scenarios, such TM populations present with both distinctive and shared properties (35). If those differences warrant a terminological distinction may remain a matter of debate, but we favor an emphasis on relevant commonalities, as argued by Jameson and Masopust (5). Hence, regardless of the acute or chronic course of an infection, we, in this study, refer to populations generated in the immediate wake of a pathogen challenge (∼1 wk) as TE, and to populations present at later stages (>1 mo) as TM.

Materials and Methods

Ethics statement

All procedures involving laboratory animals were conducted in accordance with 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 of the University of Colorado (permit numbers 70205604[05]1F, 70205607[05]4F, and B-70210[05]1E) and the Icahn School of Medicine at Mount Sinai (IACUC-2014-0170), and all efforts were made to minimize suffering of animals.

Mice, pathogens, and challenge/vaccination protocols

C57BL6/J (B6), congenic B6.CD90.1 (B6.PL-Thy1a/CyJ), congenic B6.CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ), and B6.CCL3−/− (B6.129P2-Ccl3tm1Unc/J) mice on a B6 background as well as BALB/c mice were purchased from The Jackson Laboratory; p14 TCR transgenic mice were obtained on a B6.CD90.1 background from Dr. M. Oldstone (CD8+T cells from these mice [p14 cells] are specific for the dominant lymphocytic choriomeningitis virus [LCMV]-GP33–41 determinant restricted by Db). B6.CCL5−/− mice (36) were obtained from Dr. M. von Herrath (these mice are identical to the commercially available B6.129P2-Ccl5tm1Hso/J strain), and B6.CCL1−/− mice were a gift from Dr. S. Manes (37). LCMV Armstrong (Arm; clone 53b) and clone 13 (cl13) 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 described (38). Recombinant Listeria monocytogenes expressing full-length OVA (rLM-OVA) (39) was grown and titrated as described (34, 40). For acute infections, 8–10-wk-old mice were inoculated with a single i.p. dose of 2 × 105 PFUs LCMV Arm or 2 × 103 CFU rLM-OVA i.v.; for persistent infections, mice were challenged with 2 × 106 PFU LCMV cl13 i.v. (control cohorts were acutely infected with 2 × 106 PFU LCMV Arm i.v.). Combined TLR/CD40 vaccinations were performed as described (34, 41) by immunizing mice i.p. with 500 μg OVA (Sigma-Aldrich) in combination with 50 μg anti-CD40 (FGK4.5; Bio X Cell) and 50 μg polyinosinic/polycytidylic acid [poly(I:C); Amersham/GE Healthcare]; all vaccinations were performed by mixing each component in PBS and injection in a volume of 200 μl.

Lymphocyte isolation, T cell purification, and stimulation cultures

Our procedures for isolation of lymphocytes from lymphatic and nonlymphoid tissues, including total body perfusion with PBS, are detailed elsewhere (42, 43). To generate p14 chimeras, naive CD90.1-congenic 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 (25). For microarray analyses, splenic p14 TM were positively selected using anti-CD90.1-PE Ab and PE-specific magnetic beads (STEMCELL Technologies) and further enriched to >99% purity by FACS sorting (BD Biosciences FACSAria) as described (25). Adoptive transfer/rechallenge experiments (Supplemental Fig. 3) were conducted with spleen cells obtained from LCMV-immune B6 mice and depleted of CD19+B cells or CD19+B and CD4+T cells using magnetic beads prior to i.v. transfer into B6 congenic recipients and LCMV challenge (25). For specific T cell stimulation, spleen cells were cultured for 5 h with MHC class I (MHC-I) (1 μg/ml)– or MHC-II (5 μg/ml)–restricted peptides in the presence (for flow cytometry [FC] analyses) or absence (for ELISA assays) of 1 μg/ml brefeldin A (BFA; Sigma-Aldrich) (43). In some cases, highly purified p14 TM were stimulated for 3 h with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) prior to processing for microarray hybridization.

In vivo T cell activation and chemokine blockade

In vivo activation of CD8+TM (Fig. 5D) was performed as described (44) (i.e., vaccine-immune mice were injected with 100 μg OVA257 peptide i.v., followed by 250 μg BFA/PBS i.p. 30 min later); spleens were harvested 2 h after peptide injection, processed, and analyzed by FC. For in vivo chemokine neutralization in the context of CD8+TM recall responses, we employed experimental designs, Ab dosages, and treatment schedules as detailed in the legend to Supplemental Fig. 3E and 3F using the following Abs for CCL5 blockade: anti-CCL5 clone R6G9 [mIgG1 (45)] or mIgG1 isotype clone MOPC-21 (Sigma-Aldrich); combined CCL3/4/5 blockade: anti-CCL3 clone 756605 (rIgG1), anti-CCL4 clone 46907 (rIgG2a), anti-CCL5 clone 53405 (rIgG2a) (R&D Systems), or rIgG control; and XCL1 blockade: polyclonal anti-XCL1 AF486 or goat IgG control AB-108-C (R&D Systems).

Microarray analyses

Microarray experiments were conducted with highly purified p14 TM populations (directly ex vivo or after 3 h in vitro stimulation with anti-CD3/anti-CD28) and Affymetrix M430.2 arrays as detailed (25, 34). Selected data are shown in Figs. 1A and 2C, Supplemental Figs. 1A and 2A and 2C, and the entire datasets, including those for ex vivo and anti-CD3/anti-CD28–stimulated p14 TE, can be retrieved from the Gene Expression Omnibus repository accession number GSE143632 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE143632); MAS5, RMA, and GC-RMA normalization were performed and yielded essentially similar results (data not shown).

Peptides and MHC tetramers

Peptides corresponding to the indicated pathogen 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. LCMV epitopes are the following: GP33–41 (Db/KAVYNFATC), GP276–286 (Db/SGVENPGGYCL), NP396–404 (Db/FQPQNGQFI), GP64–80 (IAb/GPDIYKGVYQFKSVEFD), and NP118–126 (Ld/RPQASGVYM); and rLM-OVA epitopes: OVA257–264 (Kb/SIINFEKL) and LLO190–201 (IAb/NEKYAQAYPNVS). Note that the LCMV-GP33 peptide also stimulates KbGP34+ CD8+TE/M not captured by DbGP33 tetramer stains; we therefore refer to the T cells activated in GP33 stimulation cultures as GP33/4-specific CD8+TE/M. DbNP396, DbGP33, DbGP276, LdNP118, KbOVA257, and IAbGP66 complexes were obtained from the National Institutes of Health Tetramer Core Facility as APC or PE conjugates and/or biotinylated monomers, and CD1/αGalCer tetramers were a gift from Dr. L. Gapin. Note that the IAbGP66 tetramer identifies the same population of CD4+TE/M responsive to stimulation with the longer GP64–80 peptide (46). Tetramer staining was performed as described (i.e., for 45 min at 4°C or room temperature for MHC-I tetramers and for 90 min at 37°C in the presence of sodium azide for MHC-II tetramers) (47).

Abs, staining protocols, and FC

All Abs and FC staining protocols used in the current study are described elsewhere. Specifically, this includes Abs/protocols for standard cell surface and intracellular staining as well as Ab conjugation (25), our detailed Ab characterization and staining strategies for detection practically all murine chemokines (22, 34), and the procedure for visualization of LCMV-NP expression in infected Io cells (42, 48); perforin stains were performed with Ab clone S16009B conjugated to PE (BioLegend). All samples were acquired on FACSCalibur, LSR II, or LSRFortessa (BD Biosciences) or Attune NxT (Thermo Fisher Scientific) flow cytometers and analyzed with DIVA (BD Biosciences) and/or FlowJo (Tree Star) software. To estimate the relative magnitude of the CD8+TM chemokine response in the context of other CD8+TM activities (Fig. 3D), we determined the percentage of individual NP396-specific CD8+TM (∼d42) subsets expressing constitutive (GzmA/B and perforin) or inducible (CCL1/3/4/5/9/10 and XCL1; IFN-γ, IL-2, IL-3, and GM-CSF; TNF-α, FasL, and CD40L; and degranulation) effector functionalities as shown/described in this study (Figs. 3A, 3B, 4A, 4B) or in Ref. 25; details about our calculations to estimate the relative magnitude and distribution of individual functional activities as displayed in Fig. 3D are provided (34).

ELISA

Quantitation of CCL3, CCL4, and CCL5 in sera and/or peptide-stimulated tissue culture supernatants (Figs. 2F, 5D) was performed using respective Quantikine ELISA Kits and protocols provided by the manufacturer (R&D Systems). To determine secretion kinetics of prestored CCL5 by specific CD8+TM (Fig. 5E), spleen cells from LCMV-immune mice were preincubated for 30 min at 37°C with 10 μg/ml cycloheximide (CHX; Sigma-Aldrich) to prevent translation and were stimulated with NP396 peptide, and CCL5 in the supernatant was quantified by ELISA (the amount of CCL5 secreted in the absence of peptide stimulation was subtracted from stimulated samples at all time points). To calculate CCL5 release on a per-cell basis, FC analyses were performed in parallel to calculate the exact numbers of DbNP396+ CD8+TM in the stimulation culture.

Statistical analyses

Data handling, analysis, and graphic representation was performed using Prism 4.0 and 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 Student t test and adopt the following convention: *p < 0.05, **p < 0.01, and ***p < 0.001. The 50% effective time (ET50) values (response kinetics; Fig. 5A–C) and EC50 values (activation thresholds; Fig. 5F–H) were calculated by plotting the fraction of specific TM demonstrating detectable chemokine/cytokine staining (FC) or the amount of secreted chemokines (ELISA) as a function of stimulation time (10−6 M peptide for 0–5 h) or peptide concentration (10−6–10−11 M peptide for 5 h), followed by nonlinear regression analysis using appropriate data format and analysis functions in the Prism software. Note that ET50 and EC50 values are independent of the fact that not all effector functions are induced in all TM of a given specificity.

Results

Chemokine signatures of virus-specific CD8+TM

To define the range of chemokines produced by virus-specific CD8+TM, we employed a comprehensive transcriptional/translational profiling strategy previously used for the characterization of CD8+TE generated in response to an acute infection with LCMV (34). In brief, so-called p14 chimeras were generated by transfusing congenic B6 mice with a trace population of naive TCR transgenic CD8+T cells specific for the dominant LCMV-GP33–41 determinant; following infection with LCMV Arm (2 × 105 PFU i.p.), p14 TE rapidly differentiate, expand, and contribute to efficient virus control before contracting and developing into p14 TM populations ∼6 wk later (25, 49, 50); highly purified p14 TM were then subjected to gene array analyses conducted directly ex vivo or after a 3-h in vitro TCR stimulation, as detailed in Materials and Methods and previous studies (25, 34).

In these analyses, 11 distinct chemokine mRNA species scored as present in p14 TM and, according to their expression patterns, could be clustered into three groups (Fig. 1A): 1) absence of ex vivo detectable mRNA but robust transcription after TCR stimulation (Ccl1 and Ccl17); 2) constitutive mRNA expression that significantly increased upon TCR engagement (Ccl3, Ccl4, Ccl9/10, Ccl27, Cxcl10, and Xcl1); and 3) chemokine mRNA species that remained unaffected or were downregulated by TCR activation [Ccl5, Ccl6, and Ccl25; this group also contains Cklf and Cklfsf3/6/7 (Supplemental Fig. 1A), members of the related chemokine-like factor superfamily, about which relatively little remains known to date (51)]. Altogether, the chemokine mRNA expression patterns of p14 TM are notably similar to the corresponding transcriptional profiles of p14 TE (34).

FIGURE 1.
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FIGURE 1.

Chemokine mRNA and protein expression by virus-specific CD8+TM. (A) p14 TM were obtained from spleens of LCMV-immune p14 chimeras (d46), 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 = 4 individual mice). The bar diagrams display MAS5-normalized values of chemokine mRNA expression of p14 TM–analyzed ex vivo (gray bars) or after TCR stimulation (black bars). Statistically significant differences are indicated by asterisks, and the broken line indicates the detection threshold set at an MAS5 value of 40; coverage: 39/40 chemokines (Ccl26 not on chip). (B) Left, p14 TM (d68) were analyzed for chemokine protein after 5 h culture in the absence (gray histograms) or presence of GP33 peptide (black tracings); all histograms are gated on p14 TM (the broken line histogram in the CCL5 panel indicates a negative control stain). Right, Summary of constitutive (no peptide) and induced (GP33 peptide stimulation) chemokine expression by p14 TM (SEM, n = 3 individual mice, data from one of three similar experiments, asterisks indicate significant expression differences between unstimulated and stimulated p14 TM). *p < 0.05, **p < 0.01, ***p < 0.001.

Complementary protein expression analyses were conducted with chemokine FC using a portfolio of extensively characterized Abs that permit detection of practically all murine chemokines at the single-cell level (22, 34). Upon TCR stimulation with GP33 peptide, nearly all p14 TM produced CCL3, CCL4, and CCL5; a large subset made XCL1; and smaller fractions expressed CCL1 and CCL9/10. Of note, neither Ccl6, Ccl17, Ccl25, Ccl27, Cxcl10, nor any other chemokine mRNA was translated by p14 TM (Fig. 1B and data not shown). Remarkably, most p14 TM expressed abundant CCL5 even in the absence of TCR activation (Fig. 1B), establishing an expression pattern that is unique not only for CD8+TM chemokines but for cytokines at large (25). With only six chemokine mRNA species serving as templates for induced, and in the case of Ccl5, also constitutive translation, the chemokine signatures of p14 TM are therefore equivalent to those of p14 TE (34).

Constitutive CCL5 expression by endogenously generated pathogen-specific CD8+TM

We next extended our investigations to endogenously generated LCMV-specific CD8+TM. Just like p14 TM, these populations, regardless of epitope specificity, constitutively expressed CCL5 but no other chemokine, cytokine, or TNFSF ligand (Fig. 2A, Ref. 25, and data not shown). Moreover, this expression pattern was largely identical in CD8+TM recovered from lymphatic and nonlymphoid tissues, was independent of mouse strain, and pertained to specific CD8+TM generated in response to infection with the bacterium L. monocytogenes (Fig. 2A, 2B). We note, however, that constitutive CCL5 expression by CD8+TM in peripheral lymph nodes was somewhat reduced in comparison with all other tissues (Fig. 2A, 2B). Because lymph nodes are enriched for CD62L-expressing central TM (TCM), we evaluated the possibility that TCM in the periphery express lower CCL5 levels than effector TM (TEM). Although this was indeed the case (Fig. 2A), the differences were small, and the progressive TEM→TCM conversion in aging pathogen-immune mice (25, 52) was associated with only a slight reduction of Ccl5 message and overall CCL5 content in splenic CD8+TM (Fig. 2C, 2D). Thus, constitutive CCL5 expression, in contrast to the rapid (GzmB) or gradual (GzmA) decline of constitutively expressed components within the cytolytic effector pathway, is a distinctive hallmark of most peripheral CD8+TM populations maintained in long-term memory (Fig. 2D and Ref. 25).

FIGURE 2.
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FIGURE 2.

Constitutive CCL5 expression by pathogen-specific TM. (A) Constitutive chemokine expression by LCMV-specific CD8+TM was analyzed in different tissues on d175 (top row, spleen) or d55 (middle and bottom rows) after acute LCMV infection. Bottom row, middle, PBMCs from rLM-OVA–immune mice were analyzed on d83. Two additional contour plots (right) gated on epitope-specific CD8+TM recovered from LCMV-immune B6 (DbNP396+, d175, spleen) or BALB/c (LdNP118+, d174, PBMC) mice demonstrate a slight, but significant, reduction of constitutive CCL5 expression levels by CD62Lhi CD8+TCM versus CD62Llo TEM subsets. (B) Summary of constitutive CCL5 expression by LCMV-specific CD8+TM recovered from lymphatic and nonlymphoid organs. (C) Temporal regulation of ex vivo detectable Ccl5 mRNA and CCL5 protein expression by aging p14 TM (the gray shaded area demarcates the transitional period from effector [d8] to early memory [d42] stage, and asterisks identify significant differences comparing young [∼d40–50] to older p14 TM). (D) Complementary ex vivo CCL5 expression data by endogenously generated DbNP396+ CD8+TM; the dot plot insert shows constitutive CCL5 versus GzmA expression in blood-borne d53 DbNP396+ CD8+TM. (E) Ex vivo CCL5 expression by CD8+T cells from age-matched naive versus LCMV-immune (day 45 [d45]) mice; the corresponding bar diagram quantifies the fraction (percentage) and expression levels (geometric mean fluorescence intensity [GMFI]) of CCL5+ CD8+TMP in naive versus young and aged LCMV-immune mice (statistical significance indicated by asterisks, no difference between d45 and d206 LCMV-immune mice). (F) Serum CCL5 levels as a function of time after LCMV infection of B6 mice (top) and age-matched uninfected B6 mice (bottom); the broken line indicates the detection threshold as determined in LCMV-immune B6.CCL5−/− mice. All summary data represent SEM with three to four individual mice analyzed per group or time point in multiple independent experiments [(A, B, and E) two to four experiments; (C, D, and F) one to two experiments]. *p < 0.05, **p < 0.01.

Our findings are in apparent contrast to the reported presence of Ccl5 mRNA but absence of protein in murine CD44hi CD8+TMP (32, 33). To reconcile these discrepancies, we evaluated constitutive CCL5 expression in major hematopoietic cell subsets isolated from lymphatic and nonlymphoid organs of naive mice (Fig. 2E, Supplemental Fig. 2). In this study, ex vivo detectable CCL5 was restricted in all tissues to two hematopoietic lineages, NK and CD3ε+T cells. Moreover, substantial CCL5 expression by NK cells as reported previously (22) contrasted with the weak CCL5 expression by CD8+TMP that was slightly more pronounced in the CD122+ subset (Supplemental Fig. 1B–E); additional minor CCL5+ T cell subsets included γδTCR+T cells as well as CD4+TMP and NKT cells that harbored constitutive CCL5 at marginal levels (Supplemental Fig. 1C, 1F, 1G). In no case did we find ex vivo CCL5 expression by B cells, monocytes, macrophages, dendritic cell (DC) subsets, or granulocytes (Supplemental Fig. 2B).

The low-level CCL5 content of CD8+TMP in naive mice provides an explanation for the seeming absence of CCL5 in these cells (32, 33) and stands in stark contrast to the robust CCL5 expression pattern of CD8+TMP in age-matched, LCMV-immune mice (Fig. 2E). Thus, a viral challenge can leave an imprint of prior infection by shifting the relative distribution of constitutive CCL5 expressors from both NK cells and CD8+TMP (each contributing ∼50% to the CCL5+ population in naive mice) to CD8+TMP as the predominant constituent of hematopoietic CCL5+ cells (>90%) (Supplemental Fig. 2H, 2I). These observations, together with the presence of CCL5 in the sera of naive mice at levels of ∼1 ng/ml (Fig. 2F), may warrant a reclassification of CCL5 as a dual function rather than inflammatory chemokine. Although CD8+TM would be an obvious source for the serum CCL5, levels were not increased in LCMV-immune mice (Fig. 2F), and the absence of spontaneous CCL5 release by CD8+TMP confirms earlier reports about the need for TCR stimulation to induce CCL5 secretion (32, 33).

Induced chemokine synthesis by pathogen-specific CD8+ and CD4+TM

To delineate the principal chemokine production capacity of endogenously generated pathogen-specific CD8+TM, we visualized induced chemokine expression by peptide restimulation of CD8+TM obtained from LCMV- and rLM-OVA–immune mice. Similar to p14 TM, nearly all specific CD8+TM readily made CCL3/4/5, large subsets produced XCL1, and 15–25% expressed CCL1 and CCL9/10; no other chemokines were synthesized in response to TCR activation (Fig. 3A, 3B and data not shown). Because these expression patterns were further consistent across different epitope specificities [and therefore also independent of immunodominant determinants and functional avidities (25, 34)], they apparently constitute a largely invariant functional property of pathogen-specific CD8+TM. Similar analyses conducted with LCMV- and rLM-OVA–specific CD4+TM also revealed subsets producing CCL3/4/5 (40–50%), CCL1 (20–30%), XCL1 (5–15%), or CCL9/10 (<5%) (Fig. 3A, 3B); these expression patterns essentially recapitulate the chemokine response of the respective pathogen-specific CD4+TE (34).

FIGURE 3.
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FIGURE 3.

Induced chemokine expression by pathogen-specific TM. (A) Induced chemokine expression by splenic GP33/4-specific CD8+ (top row) and GP64-specific CD4+TM (bottom row) evaluated on d55 after acute LCMV challenge of B6 mice. (B) Top, Summary of induced chemokine expression by LCMV epitope–specific (IFN-γ+) CD8+ and CD4+TM (SEM, three mice per group, d42). Bottom, Induced chemokine profiles of specific TM recovered from the spleens of rLM-OVA–immune mice (SEM, three mice per group, d83). Data are representative for two to three independent experiments conducted at various time points after LCMV or rLM-OVA infection, and asterisks indicate significant differences between respective pathogen-specific CD8+ and CD4+TM. (C) Induced chemokine production by NP396-specific CD8+TM (black) and GP64-specific CD4+TM (gray) as a function of time after LCMV challenge; details for data display and statistics as in legend to Fig. 2C (SEM of three to six mice per time point; all data were generated with splenic TM, with the exception of XCL1 production, for which blood-borne NP396-specific CD8+TM were analyzed, and were combined from four independent experiments). (D) Composition of the NP396-specific CD8+TM response (∼d42) as reflected by the relative magnitude of individual subsets expressing constitutive (GzmA/B and perforin) or inducible (all other including CCL5) effector activities (data for the pie chart represent averages of ≥3 mice analyzed in up to five separate experiments). *p < 0.05, **p < 0.01, ***p < 0.001.

The long-term maintenance of CD8+TM populations is associated with a gradual remodeling process that promotes their functional diversification, as reflected in an increasing capacity for IFN-γ, IL-2, CD40L, and FasL synthesis (25). We therefore monitored the temporal modulation of chemokine mRNA species in aging p14 TM and observed four distinct patterns of longitudinal mRNA expression: enduring absence (Ccl1), a continuous decrease (Ccl5 and Ccl9), a progressive increase (Xcl1), and an initial ∼5 mo decline followed by a subsequent rise (Ccl3 and Ccl4) (Supplemental Fig. 2A), a somewhat unusual dynamic comparable to that previously reported for Ifng, Tnf, Prf1, and Gzmb/k/m (25). The relevance of the latter kinetics, however, remains unclear because inducible CCL3/4 production was not altered in aging NP396- or GP33/4-specific CD8+TM (Fig. 3C, Supplemental Fig. 2B). Similarly, and notwithstanding the slight decline of constitutive CCL5 expression (Fig. 2C, 2D), the capacity for stimulated CCL5 synthesis remained a stable property of aging CD8+TM, as did the CCL1 production potential by a major subset (Fig. 3C, Supplemental Fig. 2B). In contrast, inducible CCL9/10 and XCL1 expression tracked with the modulation of the respective mRNA patterns in the memory phase (i.e., aging CD8+TM populations featured a decreasing potential for CCL9/10, but increasing proficiency for XCL1 production) (Supplemental Figs. 2A, 2B, 3C). In fact, within the temporal context of increasingly diversified CD8+TM activities (25), the rising XCL1-production competence constitutes the most pronounced functional gain for aging CD8+TM, prompting further investigations, as discussed below. Finally, the relative proportions of chemokine-producing CD4+TM subsets, despite the gradual decline of total specific CD4+TM numbers (47), did not significantly change over time (Fig. 3C).

CD8+TM-produced chemokines in context

The full spectrum of rapid effector functions elaborated by pathogen-specific CD8+TM remains, at present, unknown. Although the induction of cytokines (IFN-γ, IL-2, IL-3, and GM-CSF), TNFSF ligands (TNF-α, CD40L, and FasL) and degranulation are readily observed in short-term restimulation cultures (25, 43, 53); virus-specific, CD8+TM-producing IL-4, IL-5, IL-10, IL-13, or IL-17 are rare, if present at all, in our experimental systems. To provide a provisional estimate for the relative contribution of chemokine production to the gamut of established CD8+TM functionalities, we quantified the fractions of NP396-specific CD8+TM capable of individual chemokine, cytokine, and TNFSF ligand synthesis, constitutive GzmA/B and perforin expression, and degranulation (Fig. 3D); although at best a rough approximation, our calculations indicate that chemokine synthesis may account for >40% of CD8+TM activities and thus suggest a potential importance of CD8+TM-derived chemokines in the context of recall responses.

Induced CCL1 expression as a determinant for polyfunctional CD8+ and CD4+TM subsets

The extent to which individual effector molecules contribute to I° T cell–mediated pathogen control has been delineated in multiple experimental systems, yet comparatively little information is available about their precise role in immune protection afforded by recall responses. Rather, the concept of T cell polyfunctionality, typically measured in IFN-γ/TNF-α/IL-2 coexpression analyses, argues that the presence of TM with a diversified and robust functional repertoire correlates with better pathogen control irrespective of the precise mechanisms by which the analyzed T cell functions may in fact contribute to this task (53, 54). In this study, we demonstrate that induced CCL1 expression by virus-specific CD8+ and CD4+TM identifies subsets characterized by highly diversified effector functions and increased production of multiple effector molecules (because we were unable to concurrently visualize more than two to three chemokines, we relied on a series of successive and complementary stains to define complex functional TM profiles according to 11 parameters) (Fig. 4).

FIGURE 4.
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FIGURE 4.

Induced CCL1 expression as a determinant for highly polyfunctional CD8+ and CD4+TM subsets. (A) Patterns of induced cytokine and TNFSF ligand expression by GP33/4–specific CD8+TM (d67, spleen). Middle and bottom rows, Red dots designate specific CD8+T cells producing a third cytokine (identified in red font) in addition to IFN-γ/TNF-α (or CD40L/IL-2) expression displayed in conventional dot plots. (B) Top row, left, CCL3/4 coexpression by all GP33/4–specific CD8+TM; top row, right, specific CD8+TM subsets producing CCL1, CCL9/10, or XCL1 also express higher levels of CCL3 (the color-coded CCL3 geometric mean fluorescence intensity [GMFI] values refer to cells identified with the same color; statistical significance is indicted by asterisks). Middle row, Induced CCL1/XCL1 expression and corresponding CCL3 and IFN-γ expression levels in subpopulations identified by color-coded histograms and corresponding GMFI values. In addition, IL-2 production by CCL1− (black tracing) versus CCL1+ (red tracing) subsets is shown. Bottom row, left, Approximately eighty percent of CCL1+ cells fail to induce CCL9/10 expression, suggesting that XCL1 and CCL9/10 are expressed in a reciprocal fashion in CCL1+ subsets (compare with above CCL1/XCL1 plot). Lack of CCL9/10 production is associated with higher CCL3 (as well as IFN-γ and IL-2) expression. Bottom row, right, CCL4 GMFI values and IL-2/CD40L expression patterns by CCL1− (blue) versus CCL1+ (red) subsets. All values listed in (A) and (B) are the average of three mice analyzed. (C) Complementary analyses of cytokine and TNFSF ligand production by GP64-specific CD4+TM [data display as in (A); note that IL-2+ cells are CD40L+, IFN-γhi, and TNF-αhi]. (D) Top row, Coproduction of CCL3/4 by a major CD4+TM subset and enrichment of CCL1, CCL9/10, and XCL1 expression in the CCL3/4+ subpopulation. Middle row, Progressive enrichment of CD40L+ IL-2 producers in CCL1−/4− → CCL1−/4+ → CCL1+/4+ subsets (color-coded gating strategy). Bottom row, No clear association of induced CCL9/10 and XCL1 production with CCL1 expression. However, CCL1+ CD4+TM not only contain more IL-2 producers, the latter cells are also enriched for a pronounced IFN-γhi phenotype. Representative data from two independent experiments are shown, and the text boxes below (A)–(D) summarize the functional characteristics of LCMV-specific CD8+TM and CD4+TM subsets identified by induced CCL1 expression. *p < 0.05, **p < 0.01, ***p < 0.001.

In addition to IFN-γ, LCMV-specific CD8+TM subsets rapidly synthesize TNF-α, IL-2, GM-CSF, and CD40L (25, 43), and the level of IFN-γ expression in TNF-α+, IL-2+, GM-CSF+, and CD40L+ subsets was consistently and significantly higher than in CD8+TM subsets, producing only IFN-γ (p < 0.05, analysis of NP396-, GP33/4-, and GP276-specific CD8+TM; data not shown). Combinatorial analysis of these functions demonstrated that IL-2+ and CD40L+, but not GM-CSF+, CD8+TM belong to subsets that also produce IFN-γ and TNF-α at higher levels. Among the IL-2 and CD40L producers, however, only ∼25% exhibited coexpression, whereas another ∼25% were IL-2+/CD40L−, and ∼50% expressed CD40L in the absence of IL-2 (Fig. 4A). Similar to CD8+TE, CD8+TM coproduced CCL3/4 (Fig. 4B, upper left panel), the particularly tight association of which may, in part, be due to the formation of intracellular CCL3/4 hetero-oligomers (34). Further comparing the extent of induced CCL3 production in subsets expressing additional chemokines with those that do not, CCL3 was significantly increased in CCL1+ and XCL1+ and, to a lesser extent, CCL9/10+ populations (Fig. 4B, upper panels). In fact, the CCL1+ population is a subset of XCL1+ cells that produced significantly more CCL3 and IFN-γ compared with the XCL1+/CCL1− CD8+TM, which in turn, exhibited higher CCL3/IFN-γ levels than the XCL1−/CCL1− population. In addition, although IL-2 producers were found in both CCL1− and CCL1+ populations, the latter subset was significantly enriched for IL-2+ cells (∼45 versus 23%), which also produced IL-2 at higher levels (p = 0.0206) (Fig. 4B, middle panels). CCL1− and CCL1+ populations were also stratified according to CD40L expression: in this study, the CCL1+ subset was clearly enriched for both IL-2+/CD40L+ and IL-2−/CD40L+ subsets (Fig. 4B, lower panels). Finally, the majority (∼3/4) of CCL9/10 expressors were found among CCL1− cells and, expectedly, expressed significantly lower amounts of CCL3 (Fig. 4B, lower panels). Therefore, induced CCL1 expression defines a highly polyfunctional, virus-specific CD8+TM subset comprising ∼25% of epitope-specific cells that are characterized by a CCL3hi/CCL4hi/CCL5+/XCL1+/IFN-γhi/TNF-αhi phenotype, are enriched for IL-2hi and/or CD40L+ cells, and exhibit variable GM-CSF and/or CCL9/10 expression (CCL5 analyses are not displayed in Fig. 4B because all specific CD8+TM are capable of induced CCL5 production, shown in Fig. 3A–C).

Similar functional stratifications were also performed for LCMV-specific CD4+TM, taking into account that their relative IL-2+ and CD40L+ subsets are significantly larger than the corresponding CD8+TM subpopulations (43) (Fig. 4C, 4D). Collectively, these analyses demonstrate that induced CCL1 production is also useful to delineate a polyfunctional CD4+TM subset that accounts for ∼25% of the specific CD4+TM compartment and is defined by a CCL3hi/CCL4hi/IFN-γhi/TNF-αhi/IL-2+/CD40Lhi expression pattern with variable induction of XCL1, GM-CSF, and/or CCL9/10.

Orchestration of the CD8+TM chemokine response (I): kinetics of chemokine production

The presence of chemokine transcripts in TM has been suggested to confer a kinetic advantage that permits more rapid protein synthesis (32, 55), but this hypothesis has, to our knowledge, not yet been experimentally tested. The distinct expression patterns of ex vivo detectable chemokine mRNA species in LCMV-specific CD8+TM (absence of Ccl1 mRNA, abundant Ccl3/4/5 mRNA, and progressively increasing Xcl1 mRNA; Supplemental Fig. 2A) would therefore suggest that early synthesis of CCL3/4 is followed by that of XCL1 and, finally, CCL1 protein. Experiments performed with LCMV-immune p14 chimeras indeed confirmed this prediction: by calculating the time point at which p14 TM demonstrate detectable chemokine expression in half of the responder population (ET50), we found ET50 values of ∼1 h and 10 min for induced CCL3/4 and IFN-γ production, whereas XCL1 synthesis was significantly delayed (∼2.0 h, p < 0.0002), yet faster than the eventual induction of CCL1 (∼3.5 h, p < 0.0001) (Fig. 5A, 5B). In fact, a direct comparison with other effector functions demonstrated that induced chemokine production dominates the earliest (CCL3/4) and later (CCL1 and CCL9/10) stages of the CD8+TM response (Fig. 5B) and also illustrates that the presence/absence of pre-existing mRNA species is not the sole predictor for the temporal elaboration of effector functions: although absent at the mRNA level in resting p14 TM, Il2, Csf2/Gmcsf, and Cd40lg mRNA species were rapidly transcribed following TCR stimulation (Supplemental Fig. 2C), but translation of CD40L, IL-2, and GM-CSF proteins occurred with significantly different kinetics (Fig. 5B). Conversely, despite the presence of Ccl9 mRNA (Supplemental Fig. 2A), synthesis of the corresponding protein was a relatively late event (Fig. 5B). Because differential mRNA stability may shape the temporal order in which genes encoding inflammatory mediators are expressed (56), it is conceivable that this phenomenon also contributes to the observed differences in translation kinetics. Nevertheless, the significant increase of Xcl1 message in aging p14 TM (Supplemental Fig. 2A) allowed for an analysis of the kinetics with which an individual chemokine is synthesized as a function of changing mRNA levels. As shown in Fig. 5C, not only did more aged p14 TM (∼21 mo) produce XCL1, but they did so ∼20 min faster than younger p14 TM (∼9 wk). In contrast, the kinetics of CCL1/3/4 and IFN-γ production were not significantly different in young and old CD8+TM (data not shown).

FIGURE 5.
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FIGURE 5.

Kinetics and activation thresholds for induced chemokine production by specific CD8+TM. (A and B) Spleen cells from LCMV-immune p14 chimeras (∼10 wk) were stimulated for 0–5 h with GP33 peptide, and expression of indicated chemokines, cytokines, and TNFSF ligands was determined as a function of stimulation time. The dot plots (gated on p14 TM) show representative data for the temporal regulation of induced CCL1/3/4, XCL1, and IFN-γ synthesis, and the bar diagram ranks inducible chemokine (black) and other effector molecule (gray) production according to ET50 values (time required to induce effector functions in 50% of the population capable of producing a given effector molecule); significant differences are indicated by asterisks. Overall, induced CCL3/4 and IFN-γ synthesis proceeds with significantly faster kinetics as compared with other effector functions (p < 0.003), with the exception of TNF-α. (C) ET50 values for induced XCL1 expression by young (d64) versus aged (d637) p14 TM were determined as above. (D) Approximately two months after OVA/anti-CD40/poly(I:C) vaccination, B6 mice were injected with BFA only or OVA257 peptide and BFA as indicated, and activation status (CD69) and in vivo IFN-γ, TNF-α, and chemokine production by KbOVA257+ CD8+TM was determined as described in Materials and Methods; values are the percentage of KbOVA257+ CD8+TM in respective quadrants synthesizing indicated cytokines/chemokines. (E) Kinetics of prestored CCL5 release by individual NP396-specific CD8+TM (d45) were determined in stimulation cultures supplemented with CHX to prevent protein neosynthesis; for comparison, CCL5 secretion in the absence of CHX is featured for the 5-h time point (gray symbol). (F) Intracellular IFN-γ and CCL3/4 content of p14 TM (∼9 wk) determined after 5 h stimulation with graded doses of GP33 peptide (plots gated on p14 TM). The adjacent diagrams summarize the emergence of p14 TM expressing effector functions as a function of GP33 peptide concentration. (G) Summary of activation thresholds (EC50 values) determined for individual chemokines (black bars) and other effector functions (gray bars) elaborated by splenic p14 TM stimulated with graded doses of GP33 peptide (1 × 10−11–1 × 10−6M; data are combined results from three similar experiments evaluating three p14 chimeras each; to account for interexperimental variability, all samples were costained for IFN-γ, the average IFN-γ EC50 value was set at 1.0 [dotted line], and the EC50 values of other effector functions were calculated accordingly). Statistically significant differences comparing the IFN-γ activation threshold to CCL1, CCL4, CD40L, and IL-2 are indicated by asterisks. (H) CCL3/4/5 secreted by p14 CD8+TM as a function of GP33 peptide concentration was determined by ELISA (5 h stimulation), and EC50 values are summarized in the adjacent bar diagram. All data in (B), (C), and (E)–(H) are representative for individual experiments conducted two to four times (SEM, n = 3–4 mice per experiment). *p < 0.05, **p < 0.01.

To relate the preceding in vitro data to a specific in vivo CD8+TM response, B6 mice previously immunized with combined TLR/CD40 vaccination were subjected to a peptide challenge and coadministration of BFA to allow for the intracellular accumulation of chemokines and other effector molecules in vivo (34, 44). Within 2 h after challenge, ∼2/3 of the vaccine-specific CD8+TM became activated, as determined by CD69 expression, and the majority of these cells readily synthesized CCL3/4 and XCL1 (Fig. 5D), demonstrating that the in vivo CD8+TM response features rapid and abundant chemokine production. Finally, we determined the secretion kinetics and quantities of CCL5, constitutively expressed by LCMV-specific CD8+TM (day 45 [d45]) in an ELISA format. Interestingly, both the rapid speed of CCL5 release and the amount of prestored CCL5 secreted by individual CD8+TM (Fig. 5E) were equivalent to CD8+TE (34). Thus, the CD8+TM chemokine response proceeds through an ordered sequence of overlapping events that are, in part, dictated by the extent of pre-existing protein and/or mRNA species: CCL5 → CCL3/4 and IFN-γ → TNF-α → CD40L → XCL1 → IL-2 → GM-CSF → CCL1 → CCL9/10. Given the particularly fast release of CCL3/4/5, stimulated CD8+TM rather than DCs or other APCs (57) likely constitute the principal source for these chemokines in a recall response.

Orchestration of the CD8+TM chemokine response (II): activation thresholds for chemokine production and secretion

In addition to the temporal regulation of the T cell chemokine response, production of individual chemokines may be controlled by distinct activation thresholds. The functional avidities of specific T cells are often determined by measuring induced IFN-γ synthesis in response to graded concentrations of antigenic peptide (34, 58). Although the interpretation of such data provides insights into properties intrinsic to the interaction between TCRs and peptide/MHC complexes, it should be noted that the experimental readout is modulated by complex signaling events that precede the induction of a given T cell function. For example, functional avidities for induced IFN-γ production were reported to be lower than those required for IL-2 production by the same cells, suggesting that limiting Ag concentrations may permit the elaboration of direct T cell effector functions (IFN-γ), whereas induction of proliferative T cell responses, mediated by T cell–produced IL-2, preferentially occurs at higher Ag loads (53).

We therefore compared the functional avidities/activation thresholds for induced chemokine and cytokine/TNFSF expression by LCMV-specific CD8+TM (Fig. 5F–H). Although the activation thresholds for 10 distinct CD8+TM effector functions varied, at most, by a factor of only ∼3.0 (Fig. 5G), several findings are noteworthy: 1) the activation thresholds for CCL3/4 production were only slightly lower than for IFN-γ induction, yet we consistently observed the emergence of a small CCL3+ or CCL4+T cell subset in the absence of detectable IFN-γ at limiting peptide concentrations. In fact, at a peptide concentration of 10−10 M, the very-small subset of responding p14 TM consisted to >50% of CCL3/4+ cells that were IFN-γ−. In contrast, no IFN-γ induction was observed in the absence of CCL3/4 expression (Fig. 5F). Parallel analyses of aged p14 TM (∼21 mo) yielded identical results and indicate that subtle differences pertaining to the thresholds for defined effector functions are preserved in long-term memory (data not shown). 2) Although production of CCL1 and XCL1 was initiated with delayed kinetics (Fig. 5B), the activation thresholds for these chemokines were relatively low and comparable to CCL3/4 (Fig. 5G). And 3) the highest activation thresholds were recorded for CD40L and, in agreement with earlier observations (53), IL-2 (Fig. 5G). To directly compare the thresholds for CCL3/4 synthesis with CCL5 release and production, we determined the respective functional avidities by ELISA (Fig. 5H). The activation thresholds for CCL3 and CCL4 secretion were expectedly comparable but, importantly, significantly higher than that for induced CCL5 release. In summary, at limiting Ag concentration, the IFN-γ response is preceded by secretion of the chemokines CCL5 and, to a certain extent, CCL1/3/4 and XCL1. These chemokines may stabilize interactions between T cells and target cells or APCs (59), may provide costimulatory signals (60), exert direct effector functions (27), or mediate additional functions currently under investigation.

II° CD8+ and CD4+TE/M immunity: expression profiles and functional roles of T cell chemokines

Given the prominence of the TM chemokine response, we next assessed the potential modulation of chemokine-expression profiles in the context of a recall response. To this end, we employed an adoptive transfer system in which T cell–enriched populations from LCMV-immune mice are transferred into naive congenic recipients that are subsequently challenged with LCMV; in this study, II° TE responses derived from the transferred population can be concurrently visualized with the I° TE response generated by the host (25). At the level of II° CD8+TE responses, we observed a distinct reduction of functional diversities (i.e., in comparison with I° CD8+TE, the former cells produced less IFN-γ, TNF-α, and CD40L as well as CCL1 and XCL1); at the same time, constitutive CCL5 and induced CCL3/4/5/9/10 expression were undistinguishable (Supplemental Fig. 3A, 3B). In contrast, II° CD4+TE presented with a comparatively enhanced functional diversity, as reflected in substantially larger fractions of TNF-α+, CCL1+, CCL3+, CCL4+, CCL5+, and XCL1+ subsets, a chemokine profile that in fact resembles the response of I° CD8+TE (Supplemental Fig. 3B). We also extended these analyses to the formation of II° memory, a stage at which II° CD8+TM typically display a less-mature phenotype than I° CD8+TM (Ref. 25 and Supplemental Fig. 3C). In agreement with this notion, II° CD8+TM produced more CCL9/10 and less XCL1 (Supplemental Fig. 3D), the only two chemokines subject to CD8+TM maturation-associated modulations (cf. Fig. 3C); induced CCL1/3/4/5 expression, however, was largely similar between I° and II° CD8+TM. II° CD4+TM, in contrast, mostly preserved the functional gains accrued in the II° effector phase and exhibited enhanced CCL1/3/4/5 and XCL1 production capacity in comparison with I° CD4+TM (Supplemental Fig. 3D).

To determine if the major TM-derived chemokines contribute to the regulation of II° CD8+TE responses, we conducted adoptive transfer/rechallenge experiments with CD8+T cell–enriched donor populations under conditions of systemic chemokine neutralization. We first focused on the potential role of CCL5 and observed that its blockade resulted only in a very minor impairment of II° CD8+TE responses (Supplemental Fig. 3E). A similarly negligible impact on the concurrent I° CD8+TE expansions (Supplemental Fig. 3E) is in general agreement with the unperturbed I° CD8+TE responses generated by B6.CCL5−/− mice in the wake of an acute LCMV challenge (34, 61). Our conclusion that CCL5 is dispensable for CD8+TM recall responses is further supported by experiments that demonstrated commensurate II° CD8+TE reactivities of wild-type and CCL5-deficient CD8+TM (data not shown and Ref. 61). Somewhat surprisingly, even the combined neutralization of CCL3/4/5 or XCL1 failed to alter II° CD8+TE responses (Supplemental Fig. 3F). Thus, at least in the context of an acute LCMV rechallenge, none of the major CD8+TM chemokines appear to contribute to the regulation of II° CD8+TE responses.

Chemokine profiles of specific T cells in chronic LCMV infection (I): effector stage

Challenge of naive mice with the LCMV variant cl13 (2 × 106 PFU i.v.) results in a chronic infection characterized by prolonged virus persistence and T cell exhaustion, as reflected in a progressive functional deterioration of their IL-2, TNF-α, and IFN-γ production capacity (48, 62, 63). We previously noted that an escalation of acute pathogen infection dosage depressed CCL1/3/4/5 and XCL1 synthesis potential of specific CD8+, but not CD4+TE (34) and therefore speculated that a similar CD8+T cell incapacitation would also occur under conditions of chronic viral infection. Accordingly, we inoculated mice with LCMV cl13 to initiate a persistent infection and performed a first set of analyses at the height of the TE stage (d8; control cohorts were infected with 2 × 106 PFU LCMV Arm i.v., which does not result in viral persistence or T cell exhaustion). In agreement with its previously described tropism (64, 65), LCMV cl13 preferentially infected fibroblastic reticular cells (FRCs) and APC subsets but largely spared lymphocytes (Fig. 6A and data not shown); furthermore, CD8+TE expansions after cl13 infection were diminished in the expected epitope-dependent fashion (i.e., NP396 > GP33/34 > GP276), and the functional impairment of CD4+TE appeared particularly pronounced (compare numbers of IAbGP66+ and corresponding IFN-γ–producing, GP64-specific CD4+TE) (Fig. 6B, 6C; note that DbGP33 tetramer stains and GP33 peptide stimulation cannot be directly compared because the latter method also activates KbGP34+ CD8+TE). In regard to constitutive chemokine expression by specific CD8+TE, CCL5 was the only ex vivo detectable chemokine after high-dose Arm i.v. infection (Fig. 6D), presenting with a chemokine profile practically identical to that observed after lower dose i.p. Arm infection (34). In contrast, CD8+TE generated in response to cl13 infection expressed, in addition to CCL5, low levels of all other T cell chemokines indicative of recent TCR activation in the presence of enhanced viral replication (Fig. 6D). The latter pattern (i.e., low-level ex vivo detectable chemokine expression) was also observed for specific CD4+TE, yet its presence after both cl13 and Arm infection indicates that the CD4+TE chemokine response is more impervious to chronic viral challenge. Indeed, this contention is supported by a comparison of induced chemokine production profiles in which CD8+TE in cl13-infected mice displayed impaired CCL1/3/4 and XCL1 (and elevated CCL9/10) expression in an epitope-dependent manner (NP396 > GP33/34 and GP276), whereas the CD4+TE chemokine response was not compromised and, if anything, presented with greater CCL9/10 and XCL1 expression (Fig. 6E).

FIGURE 6.
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FIGURE 6.

Chemokine profiles of specific T cells in chronic LCMV infection (I): effector stage. B6 mice were infected with 2 × 106 PFU LCMV Arm or cl13 i.v. as indicated and subsequently analyzed on d8. (A) LCMV–nucleoprotein (NP) expression in myeloid and lymphoid cell subsets (the APC dot plots are gated on CD3ε−CD19−NK1.1− cells and feature LCMV-NP+ subsets in red; the histograms are gated on indicated lymphoid cell subsets and compare LCMV-NP expression in Arm [gray] versus cl13 [black tracing] infection). (B and C) Enumeration of specific TE frequencies and numbers by tetramer and IFN-γ stains; the arrows/values indicate the factor by which respective TE frequencies or numbers differ between Arm- and cl13-infected mice. (D) Ex vivo chemokine expression by specific CD8+TE (top) and CD4+TE (bottom) in Arm and cl13 infection. (E) Quantification of induced chemokine production by epitope-specific CD8+ and CD4+TE; asterisks indicate significant differences between Arm- and cl13-infected mice (all summary data are SEM with n = 4 mice per group and are representative for results obtained in two to three independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001.

Chemokine profiles of specific T cells in chronic LCMV infection (II): memory stage

Extending our investigations to the d30 time point, at which chronic LCMV infection is fully established, we found preferential infection of APC subsets and, in particular, lymphoid reticular cells identified as CD45−/podoplanin+ cells (66) in cl13-challenged, but not Arm-challenged, mice (Fig. 7A), a numerical reduction of specific CD8+TM compartments according to epitope specificity (NP396 > GP33 > GP276) but also a trend toward increased specific CD4+TM numbers in comparison with the corresponding CD4+TM pool in Arm-immune mice (Fig. 7B, 7C). Although the effects of cl13 infection on compromised CD8+TE expansions are well known, a direct enumeration of specific TM numbers by ex vivo tetramer staining versus induced IFN-γ production can help to clarify certain peculiarities pertaining to impaired functionalities (Fig. 7D): reduction of the splenic NP396-specific CD8+TM pool by a factor of ∼6 is captured in absolute numbers by both analysis modalities, implying that despite the considerable pressure of the chronic infection (67), the IFN-γ production capacity of surviving NP396-specific CD8+TM is reasonably well preserved. In contrast, a minor numerical decrease of DbGP276+ CD8+TM is associated with 2.5-fold fewer IFN-γ–producing, GP276-specific CD8+TM, demonstrating a considerable functional incapacitation (Fig. 7D). Similar comparisons of GP64-specific CD4+TM further complicate this picture because in both LCMV cl13 and Arm infection, ∼3.5-fold fewer IFN-γ–producing than tetramer+ CD4+TM were recovered from the spleen (Fig. 7D). Thus, in the dynamic interplay between specific TM and persisting virus that may constrain (CD8+TM) or expand (CD4+TM) reactive T cell pools, concurrent functional impairments, at least at the level of IFN-γ production, may become uncoupled such that lesser numerical reduction can be associated with greater functional handicaps.

FIGURE 7.
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FIGURE 7.

Chemokine profiles of specific T cells in chronic LCMV infection (II): memory stage. Analyses of LCMV Arm- or cl13-infected B6 mice (2 × 106 PFU i.v.) as conducted on d30. (A) Left, LCMV–nucleoprotein (NP) expression (red) by APC and other cell subsets in cl13, but not Arm, infection (plots gated on CD3ε−CD19−NK1.1− cells). Right, LCMV-NP detection in fibroblastic reticular (CD45−podoplanin+) in cl13 but not Arm infection. (B and C) Enumeration of specific TM frequencies and numbers by tetramer and IFN-γ stains; the arrows/values indicate the factor by which respective TE frequencies or numbers differ between Arm- and cl13-infected mice (black font, significant differences; gray font, NS). (D) Comparison of epitope-specific TM numbers in the spleen as based on tetramer (ex vivo) versus IFN-γ (stimulated) stains; the arrows/values indicate significant differences emerging as a consequence of the two distinct analysis modalities used for TM enumeration. (E) Cytokine and TNFSF ligand production by GP33/4-specific CD8+TM. (F) Ex vivo (top) and induced (bottom) chemokine expression by GP276-specific CD8+TM in Arm and cl13 infection (all plots gated on CD8+T cells). All summary data are SEM, with n = 4 mice per group, and represent results from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

We therefore sought to broaden the functional characterization of specific TM populations in cl13 infection. At the level of inducible cytokines and TNFSF ligands, GP33/4-specific CD8+TM, and to a similar, although lesser, extent also GP64-specific CD4+TM, produced less TNF-α and CD40L, whereas no significant differences were noted for residual IL-2, GM-CSF, IL-3, or IL-10 expression (Fig. 7E and data not shown). Importantly, although constitutive chemokine expression by specific CD8+TM in both infection models was restricted to CCL5, induced chemokine production in cl13-infected mice revealed the emergence of CCL3+, CCL4+, and XCL1+ subsets that lacked IFN-γ expression; at the same time, very few CD8+TM synthesized CCL1 or CCL9/10 (Fig. 7F). We therefore extended and summarized these observations in two complementary ways: 1) traditional data display quantifying chemokine-producing cells as a fraction of IFN-γ+ TM subsets demonstrated impaired chemokine responses (CCL1/3/4/5 and XCL1) in cl13 infection that were more pronounced in NP396- as compared with GP33/4- and GP276-specific CD8+TM; differences were less evident in the CD4+TM compartment, although similar to the TE stage, GP64-specific tended to make more CCL9/10 and XCL1, whereas their CCL3/4 expression was somewhat diminished (Fig. 8A). And 2) by including IFN-γ–negative CCL3+, CCL4+, and XCL1+ subsets in our analyses and relating the functional distributions (IFN-γ−/chemokine+, IFN-γ+/chemokine+, and IFN-γ+/chemokine−) to the number of tetramer+ cells quantified in parallel assays, we found that the seeming numerical reduction of functional GP276-specific CD8+TM subsets as determined solely by IFN-γ production largely disappears (Figs. 7D, 8B, left). Thus, in the context of these complementary analyses, CD8+TM in chronic LCMV infection are found to incur both functional deficits (reduced chemokine production by the IFN-γ+ fraction) and functional gains (chemokine production in the absence of IFN-γ expression). We also note that the limited expression of CCL1, including the lasting absence of a CCL1+/IFN-γ− subset (Fig. 8B), may make this chemokine a particularly useful marker to ascertain the overall functional capacities of CD8+TM in chronic infection. Finally, analyses conducted on d107 after cl13 infection (i.e., at a time point when infectious virus is eliminated from many tissues, and a phenotypic/functional recovery of CD8+TM is underway) (63, 68) revealed a slightly smaller functional GP276-specific CD8+TM pool with a relative reduction of the compartments expressing only CCL3/4 or XCL1 in favor of subsets coproducing IFN-γ (Fig. 8B, right). At the same time, the NP396-specific CD8+TM pool, likely as a consequence of its protracted recovery, exhibited particularly prominent fractions of IFN-γ−/chemokine+ subsets (Fig. 8B, right).

FIGURE 8.
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FIGURE 8.

Total TM chemokine responses and viral persistence under conditions of systemic chemokine deficiency. (A) Quantification of chemokine production by specific CD8+ and CD4+TM on d30 after Arm or cl13 infection (significant differences are indicated by asterisks). As before, these analyses depict chemokine-producing TM subsets as a fraction of the respective IFN-γ+ epitope–specific TM populations. (B) Quantification of chemokine-producing CD8+TM subsets on d30 (left; Arm and cl13) and d107 (right; cl13 only). In this study, the percentage of DbGP276+ and DbNP396+ CD8+TM as revealed by tetramer staining in parallel experiments (data not shown) was set at 100%; the relative fraction of IFN-γ− and chemokine-producing CD8+TM populations was calculated accordingly and was further stratified into IFN-γ+/chemokine− (light gray), IFN-γ+/chemokine+ (medium gray), and IFN-γ−/chemokine+ (dark gray) subsets [the summary data in (A) and (B) are SEM with n = 4 mice per group analyzed in two independent experiments]. (C) Quantification of LCMV serum titers in cl13-infected B6, B6.CCL1−/−, CCL3−/−, and CCL5−/− mice (n = 5 mice per group and time point; data summarized from individual time course experiments). *p < 0.05, **p < 0.01, ***p < 0.001.

Chronic LCMV infection under condition of systemic chemokine deficiency

Finally, to explore potential roles for major T cell–derived chemokines in control of a chronic LCMV infection, we challenged B6, B6.CCL1−/−, B6.CCL3−/−, and B6.CCL5−/− mice with cl13 and quantified viral titers in the serum in biweekly intervals over a period of ∼10 wk. In B6 mice, high titers of infectious virus in the serum are maintained for ∼1 mo before onset of a slow decline and eventual virus clearance from this compartment by ∼3 mo (48, 63). In the present experiments, the kinetics of virus control were identical in B6, B6.CCL1−/−, and B6.CCL3−/− mice, whereas B6.CCL5−/− mice, as reported in an earlier study (61), presented with a modest impairment of late LCMV control (Fig. 8C). Furthermore, virus titers in various tissues as well as specific TM frequencies, numbers, phenotypes, and functionalities were not significantly different in B6, B6.CCL1−/−, and B6.CCL3−/− mice (Supplemental Fig. 4 and data not shown). Although these experiments cannot directly address the role of TM-derived CCL1 or CCL3, the absence of a phenotype under conditions of systemic chemokine deficiency strongly suggests that TM-produced CCL1 or CCL3 are unlikely to contribute to the partial control of a chronic LCMV infection.

Discussion

We previously profiled the chemokine response of pathogen- and vaccine-specific TE and defined a series of distinctive properties pertaining to its constituents (CCL1/3/4/5/9/10 and XCL1, but no other chemokines), apparent magnitude, quantitative differences between CD8+ and CD4+TE, consistency of expression patterns across different challenge models, and to unique aspects of individual chemokine synthesis, coexpression, cooperative regulation, and/or secretion (34). We now demonstrate that most of these properties are carried forward into the memory stage, identify discrete traits subject to further temporal modulation, and delineate notable adjustments of the TM chemokine response under conditions of pathogen persistence. Collectively, these characteristics establish TM-derived chemokines as particularly prominent, robust, and rapidly mobilized components of the recall response; in addition, we demonstrate that the analytical visualization of TM chemokine-expression patterns permits a more detailed stratification of TM functionalities that may be correlated with differentiation status, protective capacities, and potential fates.

Similar to pathogen-specific CD8+TE (34), specific CD8+TM evaluated 6–12 wk after acute challenge with LCMV or L. monocytogenes present with a spectrum of inducible chemokines that is both circumscribed and distributed across larger and smaller subsets (CCL3/4/5, 85–95%; XCL1, 60–70%; and CCL1 and CCL9/10, 15–30%). The same select chemokines are also produced by pathogen-specific CD4+TM, although in a resemblance to the corresponding effector populations (34), induced chemokine synthesis is restricted to somewhat smaller subsets (CCL3/4/5, 40–50%; XCL1, 5–15%; CCL1, 20–30%; and CCL9/10, <5%). These CD8+ and CD4+TM chemokine signatures are largely preserved throughout long-term memory but with two significant exceptions: a precipitous decline of Ccl9 mRNA in aging CD8+TM diminishes their corresponding protein production capacity, and a progressive increase of Xcl1 message permits larger CD8+TM subsets to synthesize XCL1 with faster kinetics. Escalating XCL1 production potential, part of an intriguing functional diversification associated with long-term CD8+TM maintenance (25), is especially pronounced in blood-borne CD8+TM and arguably constitutes one of the largest functional gains bestowed on aging CD8+TM populations. At the same time, however, the repertoire of CD8+TM-produced chemokines is not broadened with age and remains strictly confined to the same six chemokines distinctive for effector and early memory stage.

Our conclusion about the defined, restricted, and mostly invariant nature of CD8+ and CD4+TM chemokine responses shared across different infectious disease models is contingent on the sensitivity, specificity, and breadth of our assay systems. As we have detailed in our earlier reports (22, 34), the analytical power of gene array and FC technology is delimited by a variety of factors that, together with the specifics of the experimental context, inform the degree of certainty with which statements about the presence/absence of individual gene products can be made. Accordingly, although we cannot categorically rule out the possibility that minor quantities of other chemokines are produced by pathogen-specific TM under some experimental or naturally occurring conditions, we are confident that the visualization of induced CCL1/3/4/5/9/10 and XCL1 production accurately captures their distinctive chemokine production potential. Related caveats also pertain to our attempt to position the prima facie already considerable magnitude of the CD8+TM chemokine response in a larger frame of reference: to estimate the relative contribution of chemokine production to the overall size of the inducible CD8+TM functionome, we quantified a broad although likely incomplete spectrum of specific CD8+TM activities and estimate that >40% of CD8+TM effector functions are dedicated to chemokine production. Although clearly only a provisional approximate, it nonetheless suggests a remarkable prominence of chemokine synthesis as part of the immediate pathogen-specific CD8+TM response.

By extending our investigations to the visualization of complex chemokine coexpression patterns, we further expand the notion of T cell polyfunctionality and, at the same time, propose a potential analytical simplification as based on inducible CCL1 expression. T cell polyfunctionality as a diagnostically relevant concept was first introduced by R. Seder’s group (69), who identified a simple metric (composed of the number of IFN-γ+/TNF-α+/IL-2+-specific CD4+TM [triple producers] as well as the amount of cytokines produced by these cells [measured by the mean fluorescence intensity of cytokine stains]) that can predict the extent of immune protection in a model for Leishmania major infection. Although it is important to note that T cell polyfunctionality is a correlate for immune protection (i.e., it does not make claims about the precise mechanisms by which specific T cell activities contribute to immune protection), the concept has been successfully applied to other pathophysiological scenarios and CD8+T cells and may further incorporate cytotoxic capacities (54, 70). Our stratification of LCMV-specific CD8+TM responses according to five cytokines/TNFSF ligands and six chemokines now reveals that visualization of induced CCL1 marks a highly polyfunctional CD8+TM subset that produces larger quantities of IFN-γ, TNF-α, CCL3, and CCL4, readily expresses CCL5 and XCL1, and is enriched for IL-2hi and/or CD40L+ cells; a corresponding dissection of LCMV-specific CD4+TM populations demonstrates a similar utility of CCL1 expression analyses because this subset presents with a distinct IFN-γhi/TNF-αhi/CCL3hi/CCL4hi/IL-2+/CD40Lhi phenotype. Furthermore, and in contrast to all other chemokines, CCL1 synthesis is strictly dependent on de novo mRNA transcription, and its induction in specific CD8+TM is compromised under conditions of persistent viral infection. Although the simple visualization of CCL1-expressing TM subsets does not allow for an integration of T cell activities into a polyfunctionality index (54), it may nevertheless serve as a singular metric for the quantification of highly polyfunctional T cells (acute infection) or T cells with residual functional reserves (chronic infection). Mechanistically, however, CCL1 appears to be dispensable, as shown by the normal virus control kinetics achieved by CCL1-deficient mice in acute (34) and chronic LCMV infection.

Perhaps the most striking aspect of the CD8+TM chemokine response is the constitutive expression of CCL5 by specific CD8+TM in lymphatic and nonlymphoid organs alike throughout long-term memory and even in chronic LCMV infection. Unique among CD8+TM chemokines, cytokines, and TNFSF ligands, the CCL5 expression pattern is reminiscent of that reported for constituents of the perforin/Gzm pathway. However, following acute infection, GzmB expression is quickly downregulated to low or undetectable levels in LCMV-, L. monocytogenes–, and other pathogen-specific CD8+TM recovered from lymphatic tissues (25, 52, 71–73), and constitutive GzmA expression by LCMV-specific CD8+TM is subject to a protracted downmodulation (25) (although perforin is expressed by practically all CD8+TM in the early memory stage, its subsequent temporal regulation remains, at present, unknown). Together, these dynamics provide further evidence for the differential regulation of cytotoxic effector mechanisms (74–76) and establish enduring constitutive CCL5 expression by pathogen-specific CD8+TM in the absence of inflammatory stimuli as a distinctive functional signature.

Several additional aspects pertaining to the constitutive CCL5 expression by CD8+TM are noteworthy: 1) acute pathogen infections leave a conspicuous and lasting imprint that is readily discernible even without an analytical focus on specific CD8+TM: in naive mice, ∼25% of CD8+TMP express low levels of constitutive CCL5; in contrast, ∼70% of CD8+TMP in LCMV-immune mice present with a robust CCL5+ phenotype. In fact, in such mice, CD8+TMP rather than innate immune cells constitute the predominant hematopoietic source of prestored CCL5 (CD8+TMP, ∼90%; NK cells, and ∼10%; other [mostly CD4+TMP], <5%). 2) As shown earlier, constitutive CCL5 expression is a dynamic process that continuously replenishes intracellular CCL5 storage pools (31, 34); given the associated bioenergetics demands, the long-term preservation of a CCL5+ CD8+TM phenotype may therefore provide crucial kinetic advantages for the recall response. 3) Under steady-state conditions, however, prestored CCL5 is not released from CD8+TM, as evidenced by comparable CCL5 serum levels in LCMV-immune and naive mice; rather, CCL5 secretion requires TCR stimulation, as reported previously (32, 33). And 4) yet very similar to CD8+TE (34), the initial burst of TCR-triggered CCL5 secretion by CD8+TM occurs independently of transcriptional/translational regulation and proceeds with extraordinary speed; we estimate that within 30 min after TCR activation, individual CD8+TM release as much as 1.0 fg of prestored CCL5.

The latter kinetics also impinge on the precise coordination of the CD8+TM chemokine response; because of a very low activation threshold and its rapid release, TCR-induced CCL5 secretion constitutes the first step in a cascade of CD8+TM activities that otherwise require efficient protein synthesis. In this study, a combination of pre-existing mRNA levels, functional avidities (EC50 values), and protein production/secretion kinetics (ET50 values) particular for individual effector functions shapes the coordinated elaboration of the CD8+TM response. Accordingly, and in direct comparison with cytokine (IFN-γ, IL-2, and GM-CSF) and TNFSF ligand (TNF-α and CD40L) synthesis, chemokines play a preeminent role because the production of CCL3/4 and IFN-γ dominates the early stages of the CD8+TM response and, together with CCL1 and XCL1, is preferentially induced under conditions of limited Ag availability. These conclusions are further reinforced by in vivo experiments in which just 2 h after peptide injection, >55% of specific CD8+TM produce CCL3/4 and XCL1, whereas ∼45% express IFN-γ and only ∼25% TNF-α.

Collectively, this work identifies a previously unappreciated and striking prominence of chemokine production as part of the CD8+TM recall response. Nevertheless, the evidence for a decisive role of TM-produced chemokines in the regulation of II° responses remains limited. Our own experiments, in agreement with a previous report (61), demonstrate that CCL5 is largely dispensable for the proliferative expansion of II° CD8+TE and even combined CCL3/4/5 or XCL1 blockade does not compromise the LCMV-specific recall response. To our knowledge, there are two reports documenting a pertinent role for CD8+TM-derived chemokines under conditions of an II° pathogen challenge. In the first, II° L. monocytogenes–specific CD8+TE were shown to provide immune protection through CCL3 secretion and induction of TNF-α and reactive oxygen species in phagocytes (27). The evidence in the second report is more indirect. In their elegant study, Alexandre et al. (28) conditionally depleted DCs bearing XCR1 (the sole XCL1 receptor) in pathogen-immune mice and found that II° CD8+TE expansions generated in response to L. monocytogenes, vesicular stomatitis virus, and vaccinia virus, but not CMV, were compromised. Complementary experiments revealed that early IFN-γ production by II° CD8+TE was contingent on an NK cell/IFN-γ–dependent induction of IL-12 and CXCL9 in XCR1+ DCs, but although IL-12, CXCR3, or NK cell neutralization/depletion all depressed the emergence of the early IFN-γ+ II° CD8+TE phenotype, neither IL-12, CXCL9, nor NK cells were required for efficient II° CD8+TE expansions (28). Other than NK cells, the only major hematopoietic XCL1 source are CD8+TE/M (22, 23, 34), raising the possibility that in some model systems, II° CD8+TE-derived XCL1 is the defining factor for productive engagement of XCR1+ DCs and the regulation of II° CD8+TE proliferative responses.

In regard to the functional properties of II° CD8+TE and, in particular, TM generated in the wake of a recall response, earlier work has described discrete alterations in comparison with the respective I° CD8+TE/M populations, such as reduced IL-2 production or enhanced GzmB expression, killing capacities, and TNF-α induction (72, 73). Together with corresponding phenotypic alterations, these observations are consistent with a greater degree of II° CD8+TE differentiation and delayed II° CD8+TM maturation (25). In agreement with this interpretation, we demonstrate reduced IL-2, TNF-α, CD40L, CCL1, and XCL1 production by II° CD8+TE, as well as a somewhat greater induction of TNF-α, CCL3/4, and CCL9/10 but decreased XCL1 expression by II° CD8+TM. Interestingly, II° CD4+TE avoid a corresponding attrition of functional diversity and display a substantial increase of CCL1/3/4/5 and XCL1 production that also extends into the II° CD4+TM stage. Given the distinct CCL3/4 coexpression pattern of a major I° CD4+TM subset, it might be tempting to postulate the existence of yet another Th cell subset, but we consider this option a distraction (5) and have focused ongoing investigations on the question if the functional skewing observed in II° CD4+TE responses involves a conversion of I° CD4+TM previously incapable of CCL1/3/4 production or the preferential recruitment of CCL1/3/4 expressors into the recall response (data not shown).

Chronic viral infections are associated with molecular, phenotypic, and functional alterations of the specific T cell compartments that are aptly described by the term T cell exhaustion because cardinal TE activities are compromised to varying degrees (77). For our interrogation of the TE/M chemokine signatures in chronic LCMV infection, not previously undertaken in a systematic fashion (61, 78), we therefore had to account for the fact that TE/M dysfunctions also affect inducible IFN-γ production, the extent of which is furthermore regulated in an epitope-specific fashion for CD8+TM (63, 67). Because concurrent analyses of MHC tetramer binding and peptide-induced functionalities are technically not feasible for murine T cells, we conducted careful complementary analyses to assess the extent of TE/M abundance and dysfunction. Although these experiments expectedly confirmed a hierarchical numerical deficit of CD8+TE/M as a function of epitope specificity in LCMV cl13– versus high-dose Arm–infected control mice, we also noted a modest increase of both tetramer+ CD4+T cell frequencies/numbers and IFN-γ producers in the memory, but not effector, stage. This unexpected expansion is reminiscent of the phenomenon of memory inflation described for some epitope-specific CD8+TM populations in persistent infections with CMV, HSV-1, polyomavirus, or parvovirus (79–81) and may be related to a more restricted exposure of CD4+TM to persisting LCMV because of its preferential localization to FRCs (64). Although FRCs may express MHC-II under certain inflammatory conditions (82), LCMV-infected FRCs do not (64), thus limiting the productive engagement of specific CD4+TE/M populations to MHC-II–expressing, LCMV-infected APCs (42). Accordingly, and despite the fact that IFN-γ production by CD4+TEM in both LCMV cl13 and high-dose Arm infection is partially reduced, we predicted and indeed observed an overall lesser functional impairment of CD4+TE/M at the chemokine production level.

In contrast, although specific CD8+TE generated immediately after a cl13 infection preserve a constitutive CCL5+ phenotype, their chemokine production capacity is modestly altered (reduced CCL1/3/4 and XCL1 and increased CCL9/10), and the extent of impaired CCL1 induction tracks with CD8+TE epitope specificity (i.e., NP396 > GP33/4 > GP276). The compromised chemokine profiles, as a property of IFN-γ–producing subsets, are maintained, if not somewhat exacerbated, by the time chronic LCMV infection is fully established but, importantly, are complemented by the emergence of CCL3+, CCL4+, and XCL1+ subsets that do not make IFN-γ. In fact, inclusion of these subsets into a calculation of GP276-specific CD8+TM frequencies confirms an abundance that is roughly equivalent to the corresponding functional CD8+TM subset in acute Arm infection. At later stages of cl13 infection (>3 mo), a gradual functional resurgence of CD8+TM occurs (68) and is reflected at the chemokine level by a contraction of the CCL3/4/XCL1-only compartment in favor of their coexpression with IFN-γ. Of note and in further support of CCL1 as a marker for CD8+TM with residual functionalities, CCL1-production is not observed in the absence of IFN-γ coexpression in either early or late phases of cl13 infection. We therefore suggest that future functional profiling of T cells in chronic viral infection and related scenarios of prolonged Ag persistence such as cancer and autoimmunity should include, ideally in a highly multiplexed fashion, a visualization of the T cell chemokine response to account for the full presence and diversity of reactive T cell populations.

Finally, and in an echo of a seeming disconnect between the prominence of TE and TM chemokine responses and their limited relevance for the regulation of I° (34) and II° TE expansions, systemic CCL1 or CCL3 deficiency also has no bearing on virus control or T cell exhaustion in chronic LCMV infection; a modest impact observed under conditions of CCL5 deficiency corroborates an earlier report (61) but leaves unclear the specific contribution of T cell–produced CCL5. As argued previously (34), an apparent absence of pronounced phenotypes in mice deficient for major T cell–produced chemokines may be grounded in redundancies of the chemokine system and thus emerge only in the context of compound chemokine mutations or may be observed in other experimental or natural scenarios characterized by a distinctive dynamic interplay between specific T cells and persisting Ags.

In summary, the present work defines six chemokines (CCL1/3/4/5/9/10 and XCL1) as a major component integral to the functional repertoire of pathogen-specific T cells in long-term memory and chronic viral infection. Unique expression patterns, such as constitutive CCL5 and inducible CCL1 expression, may serve targeted diagnostic purposes, and the relative dominance exerted by T cell–derived chemokines in the early recall response establishes TM populations as a principal source for CCL1/3/4/5 and XCL1 in particular. How such prodigious chemokine production, which necessitates the mobilization of not inconsiderable bioenergetic resources, exactly contributes to enhanced immune protection, however, remains to be investigated in greater detail.

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. L. Gapin for CD1/αGalCer tetramers, Drs. P. Marrack and J. Kappler and the entire Kappler/Marrack laboratory for critical discussion, and Dr. F. Mortari (R&D Systems) for the generous gift of most of the chemokine Abs used in this study.

Footnotes

  • This work was supported by National Institutes of Health (NIH) Grants AG026518 and AI093637, 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 Training Grants T32 AI07405, T32 AI052066, and T32 DK007792 (to B.D.). This work was also supported by JDRF Grant CDA 2-2007-240 (to D.H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

  • The microarray data presented in this article have been submitted to Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE143632.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    Arm
    Armstrong
    BFA
    brefeldin A
    CD8+TMP
    memory-phenotype CD8+T cell
    CHX
    cycloheximide
    cl13
    clone 13
    ET50
    50% effective time
    FC
    flow cytometry
    FRC
    fibroblastic reticular cell
    Gzm
    granzyme
    I°
    primary
    II°
    secondary
    LCMV
    lymphocytic choriomeningitis virus
    MHC-I
    MHC class I
    rLM-OVA
    recombinant Listeria monocytogenes expressing full-length OVA
    TCM
    central TM
    TE
    effector T cell
    TEM
    effector TM
    TM
    memory T cell.

  • 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
  • Table of Contents
  • Table of Contents (PDF)
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Chemokine Signatures of Pathogen-Specific T Cells II: Memory T Cells in Acute and Chronic Infection
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Chemokine Signatures of Pathogen-Specific T Cells II: Memory T Cells in Acute and Chronic Infection
Bennett Davenport, Jens Eberlein, Tom T. Nguyen, Francisco Victorino, Verena van der Heide, Maxim Kuleshov, Avi Ma’ayan, Ross Kedl, Dirk Homann
The Journal of Immunology October 15, 2020, 205 (8) 2188-2206; DOI: 10.4049/jimmunol.2000254

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Chemokine Signatures of Pathogen-Specific T Cells II: Memory T Cells in Acute and Chronic Infection
Bennett Davenport, Jens Eberlein, Tom T. Nguyen, Francisco Victorino, Verena van der Heide, Maxim Kuleshov, Avi Ma’ayan, Ross Kedl, Dirk Homann
The Journal of Immunology October 15, 2020, 205 (8) 2188-2206; DOI: 10.4049/jimmunol.2000254
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