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

Chemokine signatures of virus-specific CD8⁺T_M

To define the range of chemokines produced by virus-specific CD8⁺T_M, we employed a comprehensive transcriptional/translational profiling strategy previously used for the characterization of CD8⁺T_E 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-GP_33–41 determinant; following infection with LCMV Arm (2 × 10⁵ PFU i.p.), p14 T_E rapidly differentiate, expand, and contribute to efficient virus control before contracting and developing into p14 T_M populations ∼6 wk later (25, 49, 50); highly purified p14 T_M 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 T_M 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 T_M are notably similar to the corresponding transcriptional profiles of p14 T_E (34).

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FIGURE 1.

Chemokine mRNA and protein expression by virus-specific CD8⁺T_M. (A) p14 T_M 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 T_M–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 T_M (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 T_M (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 T_M (SEM, n = 3 individual mice, data from one of three similar experiments, asterisks indicate significant expression differences between unstimulated and stimulated p14 T_M). *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 T_M 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 T_M (Fig. 1B and data not shown). Remarkably, most p14 T_M expressed abundant CCL5 even in the absence of TCR activation (Fig. 1B), establishing an expression pattern that is unique not only for CD8⁺T_M 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 T_M are therefore equivalent to those of p14 T_E (34).

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

We next extended our investigations to endogenously generated LCMV-specific CD8⁺T_M. Just like p14 T_M, 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⁺T_M recovered from lymphatic and nonlymphoid tissues, was independent of mouse strain, and pertained to specific CD8⁺T_M generated in response to infection with the bacterium L. monocytogenes (Fig. 2A, 2B). We note, however, that constitutive CCL5 expression by CD8⁺T_M 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 T_M (T_CM), we evaluated the possibility that T_CM in the periphery express lower CCL5 levels than effector T_M (T_EM). Although this was indeed the case (Fig. 2A), the differences were small, and the progressive T_EM→T_CM 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⁺T_M (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⁺T_M populations maintained in long-term memory (Fig. 2D and Ref. 25).

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FIGURE 2.

Constitutive CCL5 expression by pathogen-specific T_M. (A) Constitutive chemokine expression by LCMV-specific CD8⁺T_M 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⁺T_M recovered from LCMV-immune B6 (D^bNP₃₉₆⁺, d175, spleen) or BALB/c (L^dNP₁₁₈⁺, d174, PBMC) mice demonstrate a slight, but significant, reduction of constitutive CCL5 expression levels by CD62L^hi CD8⁺T_CM versus CD62L^lo T_EM subsets. (B) Summary of constitutive CCL5 expression by LCMV-specific CD8⁺T_M recovered from lymphatic and nonlymphoid organs. (C) Temporal regulation of ex vivo detectable Ccl5 mRNA and CCL5 protein expression by aging p14 T_M (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 T_M). (D) Complementary ex vivo CCL5 expression data by endogenously generated D^bNP₃₉₆⁺ CD8⁺T_M; the dot plot insert shows constitutive CCL5 versus GzmA expression in blood-borne d53 D^bNP₃₉₆⁺ CD8⁺T_M. (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⁺T_MP 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 CD44^hi CD8⁺T_MP (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⁺T_MP 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⁺T_MP 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⁺T_MP 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⁺T_MP 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⁺T_MP (each contributing ∼50% to the CCL5⁺ population in naive mice) to CD8⁺T_MP 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⁺T_M 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⁺T_MP confirms earlier reports about the need for TCR stimulation to induce CCL5 secretion (32, 33).

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

To delineate the principal chemokine production capacity of endogenously generated pathogen-specific CD8⁺T_M, we visualized induced chemokine expression by peptide restimulation of CD8⁺T_M obtained from LCMV- and rLM-OVA–immune mice. Similar to p14 T_M, nearly all specific CD8⁺T_M 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⁺T_M. Similar analyses conducted with LCMV- and rLM-OVA–specific CD4⁺T_M 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⁺T_E (34).

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FIGURE 3.

Induced chemokine expression by pathogen-specific T_M. (A) Induced chemokine expression by splenic GP_33/4-specific CD8⁺ (top row) and GP₆₄-specific CD4⁺T_M (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⁺T_M (SEM, three mice per group, d42). Bottom, Induced chemokine profiles of specific T_M 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⁺T_M. (C) Induced chemokine production by NP₃₉₆-specific CD8⁺T_M (black) and GP₆₄-specific CD4⁺T_M (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 T_M, with the exception of XCL1 production, for which blood-borne NP₃₉₆-specific CD8⁺T_M were analyzed, and were combined from four independent experiments). (D) Composition of the NP₃₉₆-specific CD8⁺T_M 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⁺T_M 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 T_M 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 GP_33/4-specific CD8⁺T_M (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⁺T_M, 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⁺T_M 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⁺T_M activities (25), the rising XCL1-production competence constitutes the most pronounced functional gain for aging CD8⁺T_M, prompting further investigations, as discussed below. Finally, the relative proportions of chemokine-producing CD4⁺T_M subsets, despite the gradual decline of total specific CD4⁺T_M numbers (47), did not significantly change over time (Fig. 3C).

CD8⁺T_M-produced chemokines in context

The full spectrum of rapid effector functions elaborated by pathogen-specific CD8⁺T_M 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⁺T_M-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⁺T_M functionalities, we quantified the fractions of NP₃₉₆-specific CD8⁺T_M 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⁺T_M activities and thus suggest a potential importance of CD8⁺T_M-derived chemokines in the context of recall responses.

Induced CCL1 expression as a determinant for polyfunctional CD8⁺ and CD4⁺T_M 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 T_M 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⁺T_M 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 T_M profiles according to 11 parameters) (Fig. 4).

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FIGURE 4.

Induced CCL1 expression as a determinant for highly polyfunctional CD8⁺ and CD4⁺T_M subsets. (A) Patterns of induced cytokine and TNFSF ligand expression by GP_33/4–specific CD8⁺T_M (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 GP_33/4–specific CD8⁺T_M; top row, right, specific CD8⁺T_M 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⁺T_M [data display as in (A); note that IL-2⁺ cells are CD40L⁺, IFN-γ^hi, and TNF-α^hi]. (D) Top row, Coproduction of CCL3/4 by a major CD4⁺T_M 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⁺T_M not only contain more IL-2 producers, the latter cells are also enriched for a pronounced IFN-γ^hi phenotype. Representative data from two independent experiments are shown, and the text boxes below (A)–(D) summarize the functional characteristics of LCMV-specific CD8⁺T_M and CD4⁺T_M subsets identified by induced CCL1 expression. *p < 0.05, **p < 0.01, ***p < 0.001.

In addition to IFN-γ, LCMV-specific CD8⁺T_M 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⁺T_M subsets, producing only IFN-γ (p < 0.05, analysis of NP₃₉₆-, GP_33/4-, and GP₂₇₆-specific CD8⁺T_M; data not shown). Combinatorial analysis of these functions demonstrated that IL-2⁺ and CD40L⁺, but not GM-CSF⁺, CD8⁺T_M 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⁺T_E, CD8⁺T_M 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⁺T_M, 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⁺T_M subset comprising ∼25% of epitope-specific cells that are characterized by a CCL3^hi/CCL4^hi/CCL5⁺/XCL1⁺/IFN-γ^hi/TNF-α^hi phenotype, are enriched for IL-2^hi 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⁺T_M are capable of induced CCL5 production, shown in Fig. 3A–C).

Similar functional stratifications were also performed for LCMV-specific CD4⁺T_M, taking into account that their relative IL-2⁺ and CD40L⁺ subsets are significantly larger than the corresponding CD8⁺T_M subpopulations (43) (Fig. 4C, 4D). Collectively, these analyses demonstrate that induced CCL1 production is also useful to delineate a polyfunctional CD4⁺T_M subset that accounts for ∼25% of the specific CD4⁺T_M compartment and is defined by a CCL3^hi/CCL4^hi/IFN-γ^hi/TNF-α^hi/IL-2⁺/CD40L^hi expression pattern with variable induction of XCL1, GM-CSF, and/or CCL9/10.

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

The presence of chemokine transcripts in T_M 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⁺T_M (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 T_M 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⁺T_M 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 T_M, 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 T_M (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 T_M (∼21 mo) produce XCL1, but they did so ∼20 min faster than younger p14 T_M (∼9 wk). In contrast, the kinetics of CCL1/3/4 and IFN-γ production were not significantly different in young and old CD8⁺T_M (data not shown).

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FIGURE 5.

Kinetics and activation thresholds for induced chemokine production by specific CD8⁺T_M. (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 T_M) 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 T_M 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 K^bOVA₂₅₇⁺ CD8⁺T_M was determined as described in Materials and Methods; values are the percentage of K^bOVA₂₅₇⁺ CD8⁺T_M in respective quadrants synthesizing indicated cytokines/chemokines. (E) Kinetics of prestored CCL5 release by individual NP₃₉₆-specific CD8⁺T_M (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 T_M (∼9 wk) determined after 5 h stimulation with graded doses of GP₃₃ peptide (plots gated on p14 T_M). The adjacent diagrams summarize the emergence of p14 T_M 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 T_M 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⁺T_M 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⁺T_M 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⁺T_M 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⁺T_M response features rapid and abundant chemokine production. Finally, we determined the secretion kinetics and quantities of CCL5, constitutively expressed by LCMV-specific CD8⁺T_M (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⁺T_M (Fig. 5E) were equivalent to CD8⁺T_E (34). Thus, the CD8⁺T_M 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⁺T_M rather than DCs or other APCs (57) likely constitute the principal source for these chemokines in a recall response.

Orchestration of the CD8⁺T_M 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⁺T_M (Fig. 5F–H). Although the activation thresholds for 10 distinct CD8⁺T_M 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 T_M 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 T_M (∼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⁺T_E/M immunity: expression profiles and functional roles of T cell chemokines

Given the prominence of the T_M 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° T_E responses derived from the transferred population can be concurrently visualized with the I° T_E response generated by the host (25). At the level of II° CD8⁺T_E responses, we observed a distinct reduction of functional diversities (i.e., in comparison with I° CD8⁺T_E, 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⁺T_E 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⁺T_E (Supplemental Fig. 3B). We also extended these analyses to the formation of II° memory, a stage at which II° CD8⁺T_M typically display a less-mature phenotype than I° CD8⁺T_M (Ref. 25 and Supplemental Fig. 3C). In agreement with this notion, II° CD8⁺T_M produced more CCL9/10 and less XCL1 (Supplemental Fig. 3D), the only two chemokines subject to CD8⁺T_M maturation-associated modulations (cf. Fig. 3C); induced CCL1/3/4/5 expression, however, was largely similar between I° and II° CD8⁺T_M. II° CD4⁺T_M, 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⁺T_M (Supplemental Fig. 3D).

To determine if the major T_M-derived chemokines contribute to the regulation of II° CD8⁺T_E 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⁺T_E responses (Supplemental Fig. 3E). A similarly negligible impact on the concurrent I° CD8⁺T_E expansions (Supplemental Fig. 3E) is in general agreement with the unperturbed I° CD8⁺T_E responses generated by B6.CCL5^−/− mice in the wake of an acute LCMV challenge (34, 61). Our conclusion that CCL5 is dispensable for CD8⁺T_M recall responses is further supported by experiments that demonstrated commensurate II° CD8⁺T_E reactivities of wild-type and CCL5-deficient CD8⁺T_M (data not shown and Ref. 61). Somewhat surprisingly, even the combined neutralization of CCL3/4/5 or XCL1 failed to alter II° CD8⁺T_E responses (Supplemental Fig. 3F). Thus, at least in the context of an acute LCMV rechallenge, none of the major CD8⁺T_M chemokines appear to contribute to the regulation of II° CD8⁺T_E 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⁺T_E (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 T_E 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⁺T_E expansions after cl13 infection were diminished in the expected epitope-dependent fashion (i.e., NP₃₉₆ > GP_33/34 > GP₂₇₆), and the functional impairment of CD4⁺T_E appeared particularly pronounced (compare numbers of IA^bGP₆₆⁺ and corresponding IFN-γ–producing, GP₆₄-specific CD4⁺T_E) (Fig. 6B, 6C; note that D^bGP₃₃ tetramer stains and GP₃₃ peptide stimulation cannot be directly compared because the latter method also activates K^bGP₃₄⁺ CD8⁺T_E). In regard to constitutive chemokine expression by specific CD8⁺T_E, 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⁺T_E 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⁺T_E, yet its presence after both cl13 and Arm infection indicates that the CD4⁺T_E 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⁺T_E in cl13-infected mice displayed impaired CCL1/3/4 and XCL1 (and elevated CCL9/10) expression in an epitope-dependent manner (NP₃₉₆ > GP_33/34 and GP₂₇₆), whereas the CD4⁺T_E chemokine response was not compromised and, if anything, presented with greater CCL9/10 and XCL1 expression (Fig. 6E).

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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 T_E frequencies and numbers by tetramer and IFN-γ stains; the arrows/values indicate the factor by which respective T_E frequencies or numbers differ between Arm- and cl13-infected mice. (D) Ex vivo chemokine expression by specific CD8⁺T_E (top) and CD4⁺T_E (bottom) in Arm and cl13 infection. (E) Quantification of induced chemokine production by epitope-specific CD8⁺ and CD4⁺T_E; 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⁺T_M compartments according to epitope specificity (NP₃₉₆ > GP₃₃ > GP₂₇₆) but also a trend toward increased specific CD4⁺T_M numbers in comparison with the corresponding CD4⁺T_M pool in Arm-immune mice (Fig. 7B, 7C). Although the effects of cl13 infection on compromised CD8⁺T_E expansions are well known, a direct enumeration of specific T_M 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⁺T_M 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⁺T_M is reasonably well preserved. In contrast, a minor numerical decrease of D^bGP₂₇₆⁺ CD8⁺T_M is associated with 2.5-fold fewer IFN-γ–producing, GP₂₇₆-specific CD8⁺T_M, demonstrating a considerable functional incapacitation (Fig. 7D). Similar comparisons of GP₆₄-specific CD4⁺T_M further complicate this picture because in both LCMV cl13 and Arm infection, ∼3.5-fold fewer IFN-γ–producing than tetramer⁺ CD4⁺T_M were recovered from the spleen (Fig. 7D). Thus, in the dynamic interplay between specific T_M and persisting virus that may constrain (CD8⁺T_M) or expand (CD4⁺T_M) 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.

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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 T_M frequencies and numbers by tetramer and IFN-γ stains; the arrows/values indicate the factor by which respective T_E frequencies or numbers differ between Arm- and cl13-infected mice (black font, significant differences; gray font, NS). (D) Comparison of epitope-specific T_M 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 T_M enumeration. (E) Cytokine and TNFSF ligand production by GP_33/4-specific CD8⁺T_M. (F) Ex vivo (top) and induced (bottom) chemokine expression by GP₂₇₆-specific CD8⁺T_M 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 T_M populations in cl13 infection. At the level of inducible cytokines and TNFSF ligands, GP_33/4-specific CD8⁺T_M, and to a similar, although lesser, extent also GP₆₄-specific CD4⁺T_M, 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⁺T_M 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⁺T_M 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⁺ T_M subsets demonstrated impaired chemokine responses (CCL1/3/4/5 and XCL1) in cl13 infection that were more pronounced in NP₃₉₆- as compared with GP_33/4- and GP₂₇₆-specific CD8⁺T_M; differences were less evident in the CD4⁺T_M compartment, although similar to the T_E 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⁺T_M subsets as determined solely by IFN-γ production largely disappears (Figs. 7D, 8B, left). Thus, in the context of these complementary analyses, CD8⁺T_M 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⁺T_M 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⁺T_M is underway) (63, 68) revealed a slightly smaller functional GP₂₇₆-specific CD8⁺T_M 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⁺T_M pool, likely as a consequence of its protracted recovery, exhibited particularly prominent fractions of IFN-γ⁻/chemokine⁺ subsets (Fig. 8B, right).

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FIGURE 8.

Total T_M chemokine responses and viral persistence under conditions of systemic chemokine deficiency. (A) Quantification of chemokine production by specific CD8⁺ and CD4⁺T_M on d30 after Arm or cl13 infection (significant differences are indicated by asterisks). As before, these analyses depict chemokine-producing T_M subsets as a fraction of the respective IFN-γ⁺ epitope–specific T_M populations. (B) Quantification of chemokine-producing CD8⁺T_M subsets on d30 (left; Arm and cl13) and d107 (right; cl13 only). In this study, the percentage of D^bGP₂₇₆⁺ and D^bNP₃₉₆⁺ CD8⁺T_M 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⁺T_M 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 T_M 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 T_M-derived CCL1 or CCL3, the absence of a phenotype under conditions of systemic chemokine deficiency strongly suggests that T_M-produced CCL1 or CCL3 are unlikely to contribute to the partial control of a chronic LCMV infection.