Ly6C and Sca-1 (Ly6A/E) are Ly6 family GPI-anchored surface molecules that are differentially expressed by multiple immune populations. Ly6C expression has been used to distinguish short-lived effector CD4+ T cells from memory precursor effector cells, whereas Sca-1 has been used in the identification of CD8+ memory stem cells. This study examines the expression patterns of these molecules and establishes that, in vitro, IL-27, type I IFN, and IFN-γ are potent inducers of Ly6C and Sca-1 in naive mouse CD4+ and CD8+ T cells, whereas TGF-β limits their expression. The induction of Ly6C and Sca-1 by IL-27 and IFN-γ is dependent on STAT1, but not STAT3 or T-bet. In mouse splenocytes, at homeostasis, Ly6C and Sca-1 expression was not restricted to effector cells, but was also found at various levels on naive and memory populations. However, in response to infection with Toxoplasma gondii, pathogen-specific T cells expressed high levels of these molecules and in this context, endogenous IL-27 and IFN-γ were required for the expression of Ly6C but not Sca-1. Together, these findings highlight the TCR-dependent and cytokine-mediated signals that modulate T cell expression of Ly6C and Sca-1 in vitro and in vivo during infection.
The T cell response to infection or immunization involves the generation of minimally differentiated memory cells as well as highly differentiated effector cells (1). Effector T cells produce the cytokines, granzymes, and other molecules necessary for immediate pathogen control, whereas central memory T cells are long-lived and can differentiate into effector cells upon rechallenge (1–3). The identification of surface molecules that distinguish effector and memory T cell populations has allowed researchers to track the expansion, evolution, and contraction of the T cell response during infection and has provided insights into how these cells operate. For example, central memory cells upregulate CD44 and express CD62L, which keeps them localized to lymphoid organs. Effector cells are also CD44hi but lose expression of CD62L, allowing them to home to sites of inflammation. Although these definitions have proven useful to define naive, effector, and memory T cells, additional markers, including KLRG1, CXCR3, and Ly6C have been used to further subdivide these populations (4–8). In current models, a subset of highly differentiated, short-lived effector cells, which for CD8+ T cells are often identified by expression of KLRG1 (8), are specialized to control acute infection. These cells produce high levels of cytokines and granzymes and survive poorly upon adoptive transfer to naive hosts. Less-differentiated CD8+ T cells, identified during acute toxoplasmosis as CXCR3+KLRG1−, exhibit the longevity, proliferative capacity, and differentiation potential typical of memory cells (4).
Ly6C and Sca-1 (Ly6A/E) are members of a family of 21 Ly6-like proteins in mice, with 20 Ly6 family homologs in humans (9). Expression of Ly6C has been used to identify highly differentiated effector CD4+ T cells (6, 10, 11). Among virus-specific effector CD4+ T cells, Ly6C+ cells produced more cytokines and effector molecules than Ly6C− cells (6, 10). Conversely, Sca-1, in combination with the IL-2R β-chain (CD122) and Bcl-2, has been used to identify mature CD8+ T cells with stem-like properties, termed memory stem cells (12–14).
The finding that Ly6C is preferentially expressed by CD4+ short-lived effector cells during acute infection suggests that identification of the factors that modulate Ly6C expression could yield insights into the signals that control the development of memory and effector populations. For example, sustained TCR signals have been found to skew T cells toward terminal differentiation and away from memory development (1, 15), whereas multiple cytokines influence the development of effector and memory populations (16). Relevant to this report, type I IFNs, IFN-γ, and IL-27 have previously been implicated in promoting Ly6C+ and Sca-1+ populations in CD4+, CD8+, and regulatory T cells (17–21). However, previous studies have not been able to distinguish whether these stimuli induce Ly6C and Sca-1 expression or simply promote the outgrowth of Ly6C+ or Sca-1+ populations. Furthermore, extant reports have not examined how these cytokines intersect with TCR signaling to impact Ly6C and Sca-1 expression and what roles these signals play in modulating the expression of these molecules in vivo. In utilizing sorted Ly6C−Sca-1− populations, the current study was able to show that TCR stimulation alone induces Sca-1 on CD4+ and CD8+ T cells, but is not sufficient to induce Ly6C on CD4+ T cells. The cytokines IL-27, IFN-γ, and type I IFN were found to broadly promote the expression of Ly6C and Sca-1, whereas TGF-β inhibited this expression. This cytokine-mediated induction of Ly6C and Sca-1 was largely STAT1-dependent and is not dependent on STAT3 or T-bet. A survey of naive and Ag-experienced T cells in a range of differentiation states found that Ly6C expression is not restricted to effector cells and that Sca-1 expression is not limited to naive/memory-like cells. Furthermore, in vivo studies during infection with Toxoplasma gondii identified a role for endogenous IL-27 and IFN-γ in promoting effector cell expression of Ly6C. Together, these studies provide new insights into the signals that modulate the development of Ly6C+ and Sca-1+ T cell populations but indicate caution in the use of these molecules as markers of highly differentiated effector cells (Ly6C) and minimally differentiated memory stem cells (Sca-1).
Materials and Methods
Maintenance and care of experimental animals
Six-week-old female C57BL/6 controls were purchased from Taconic. Mice deficient in Il27ra/WSX-1 (C57BL/6 background) were generated as described (22) and were originally provided by Amgen (Thousand Oaks, CA). STAT1−/− mice (129S6/SvEv-Stat1tm1Rds) and 129S6 control mice were purchased from Taconic. Spleens from CD4-Cre × STAT3−/− mice were provided by A. Villarino and J. J. O’Shea (National Institutes of Health). Mice were housed and bred in specific pathogen-free (SPF) facilities in the Department of Pathobiology at the University of Pennsylvania in accordance with institutional guidelines. The Me49 strain of T. gondii was prepared from chronically infected CBA/ca mice and experimental animals were infected i.p. with 20 cysts.
Cell sorting and in vitro cell culture
Splenocytes from C57BL/6 mice were obtained by mechanically dissociating the spleen, filtering it through a 40-μm nylon strainer, and lysing RBCs with ammonium-chloride-potassium lysis buffer. T cells were enriched using a Mouse CD3+ T Cell Enrichment Column (MTCC-25; R&D Systems). Cells were then stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (L34957; Thermo Fisher), anti-CD4 (GK1.5, 100447; BioLegend), anti-CD8 (53-6.7, 562283; BD Biosciences), anti-CD44 (IM7, 0441-82; eBioscience), anti-CD62L (MEL-14, 47-0621-82; eBioscience), anti-Ly6C (HK1.4, 45-5932-82; eBioscience), and anti–Sca-1 (D7, 56-5981-82; eBioscience) Abs and were sorted on a FACSAria II flow cytometer (BD Biosciences). Cells were plated in tissue culture–treated round-bottom 96-well plates, 1–2 × 105 per well in 200 μl RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 1 mM sodium pyruvate, 1× MEM nonessential amino acids (Life Technologies), 55 μM 2-ME. The tissue culture plates were precoated with 1 μg/ml anti-CD3 (145-2C11; Bio X Cell) for 3 h at 37°C and excess anti-CD3 was rinsed off with PBS. Cells were stimulated in the presence of anti-CD28 (37.N.51.1, 1 μg/ml), IL-2 (100 U/ml; Proleukin), anti–IFN-γ (XMG1.2, 1 μg/ml; Bio X Cell) (except when exogenous IFN-γ was tested), and anti–IL-4 (11B11, 1 μg/ml; Bio X Cell). Recombinant IL-27 (Amgen) was used at a concentration of 50 ng/ml, TGF-β (eBioscience) was used at 5 ng/ml, and Universal type I IFN (PBL Assay Science) was used at a concentration of 2000 U/ml. IFN-γ (R&D Systems), IL-6 (eBioscience), IL-12 (eBioscience), TNF-α (eBioscience), IL-10 (eBioscience), and IL-7 (PeproTech) were used at 10 ng/ml. IL-15 (PeproTech) and IL-15Ra-Fc (R&D Systems) were incubated at 37°C for 30 min at a ratio of 2:9. The resulting IL-15 complexes were used at 55 ng/ml (10 ng/ml IL-15, plus 45 ng/ml IL-15Ra).
Flow cytometric analysis
Cells were stained with the reagents used for cell sorting, described above, as well as Abs specific for CD122 (5H4, 554452; BD Biosciences), CD127 (SB/199, 121105; BioLegend), CD69 (H1-2F3, 12-0691-83; eBioscience), CD25 (PC61, 553866; BD Biosciences), KLRG1 (2F1, 25-5893-82; eBioscience), and CD49d (R1-2, 103617; BioLegend). For analyses postinfection, splenocytes were harvested as detailed above and peritoneal exudate cells were harvested by i.p. lavage with 7 ml PBS. MHC-I monomers loaded with peptide (SVLAFRRL) from the T. gondii protein Tgd-057 were kindly provided by E. J. Wherry (University of Pennsylvania) and tetramerized by incubation with streptavidin-conjugated PE or allophycocyanin. Some experiments used PE- and allophycocyanin-conjugated MHC-I tetramers loaded with the Tgd-057 peptide that were provided by the National Institutes of Health Tetramer Facility. PE- or allophycocyanin-conjugated MHC-II tetramers loaded with the AS15 peptide AVEIHRPVPGTAPPS were also provided by the National Institutes of Health Tetramer Facility.
Cells were collected on an LSRFortessa (BD Biosciences) and analysis was performed with FlowJo (Tree Star). Cells were gated on lymphocytes (by forward scatter [FSC] and side scatter [SSC]), singlets (by FSC width versus FSC height and SSC width versus SSC height), and live cells (by exclusion of Aqua Dead Cell Stain). CD4+ T cells were gated CD4+CD8−Foxp3− and CD8+ T cells were gated CD8+CD4−.
Statistical significance was determined using GraphPad Prism software, using Student t test. The p values <0.05 were considered significant.
IL-27 promotes expression of Ly6C and Sca-1 on CD4+ and CD8+ T cells
Taking into consideration previous studies that implicated IL-27 in the regulation of Ly6C on regulatory T cells (21) and Sca-1 on CD8+ T cells in vitro and CD4+ T cells in vivo (19, 20), initial experiments were performed to determine the relationship between IL-27 signaling and TCR stimulation in modulating expression of these molecules. Multiple experiments using bulk CD4+ or CD8+ splenocyte cultures showed that IL-27 in combination with TCR potently promotes T cell expression of Ly6C and Sca-1 (Supplemental Fig. 1). Because subpopulations of splenic CD4+ and CD8+ T cells express Ly6C and/or Sca-1, these experiments were repeated with sort-purified Ly6C−Sca-1− naive (CD44loCD62L+) T cells (>90% purity). Cells were then labeled with CFSE and cultured in the presence or absence of plate-bound anti-CD3 and soluble anti-CD28 stimulation (henceforth referred to as TCR stimulation) with IL-2, IL-27, and neutralizing anti–IFN-γ and anti–IL-4 Abs (Fig. 1A). After 3 d of culture in the absence of TCR stimulation or IL-27, the cells did not express Ly6C. Culture of CD4+ or CD8+ T cells with IL-27 alone induced modest Ly6C expression. TCR stimulation on its own induced robust proliferation (as seen by CFSE dilution), but did not induce expression of Ly6C on naive CD4+ T cells, and induced Ly6C on a small percentage of CD8+ T cells. However, when naive CD4+ and CD8+ T cells were provided TCR stimulation combined with IL-27, there was a synergistic effect on Ly6C expression, which was apparent even during early divisions. It is notable that among CD4+ T cells, TCR stimulation alone did not induce Ly6C expression, even in those cells that had proliferated. These results demonstrate that Ly6C is not a general activation marker on CD4+ T cells, but in these experiments requires TCR activation in the presence of IL-27.
When the role of IL-27 and TCR stimulation in the regulation of Sca-1 was examined, each stimulus alone was sufficient to promote high expression of Sca-1 by CD4+ and CD8+ T cells (Fig. 1B). However, TCR stimulation in the presence of IL-27 resulted in further induction of Sca-1 expression, which was upregulated in early divisions and maintained as cells divided. We previously reported that IL-2 downregulates the IL-27 receptor (23), raising the possibility that the exogenous IL-2 used in these cultures might limit the effect of IL-27. However, exogenous IL-2 did not limit expression of Ly6C and Sca-1 by CD4+ T cells and enhanced their expression by CD8+ T cells (Supplemental Fig. 2). These results demonstrate that IL-27 has a major impact on Sca-1 and Ly6C expression, and that Sca-1 is more readily induced by these stimuli than Ly6C.
Expression of Ly6C and Sca-1 is modulated by multiple cytokines
Given the impact of IL-27 on Ly6C and Sca-1, studies were performed to assess whether other cytokines (IFN-γ, type I IFN, IL-6, IL-12, IL-4, IL-10, IL-7, IL-15, and TNF-α) modulate expression of these molecules. Ly6C−Sca-1− naive (CD62L+CD44lo) T cells were sort purified as described above and were given TCR stimulation in the presence of the different cytokines for 72 h before being assayed for expression of Ly6C or Sca-1. Across multiple experiments, IFN-γ, type I IFN, and IL-12 induced expression of Ly6C on naive Ly6C− CD4+ T cells, although not as robustly as IL-27 (Fig. 2A). For naive CD8+ T cells, both IFN-γ and type I IFN induced expression of Ly6C but type I IFN was consistently the strongest inducer of Ly6C in these experiments (Fig. 2B). Stimulation of naive CD4+ or CD8+ T cells with TCR alone induced high expression of Sca-1, but this was not further upregulated by cytokines (data not shown). IL-4, IL-10, IL-7, IL-15, and TNF-α did not modulate expression of Sca-1 or Ly6C on CD4+ or CD8+ T cells (Fig. 2, bar charts). Although there is variation in the impact of individual cytokines on different T cell populations, when taken together, these data identify two groups of related cytokines (the IFNs and the IL-6 family members) that use similar signaling pathways that promote T cell expression of Ly6C and Sca-1.
Requirements for IL-27–mediated upregulation of Ly6C and Sca-1
There are several common elements to the signaling pathways used by the cytokines that most robustly induced expression of Ly6C and Sca-1, specifically the ability to activate STAT1 and to upregulate expression of T-bet (24–26). We previously reported that Ly6C expression is T-bet–dependent in vivo during toxoplasmosis (27), but culture of T-bet−/− T cells demonstrated that T-bet is not required for IL-27–mediated induction of Ly6C and Sca-1 in vitro (Supplemental Fig. 3A, 3B). These findings agree with an earlier study that found that stimulation with anti-CD3/28 Abs overcomes the need for T-bet in the induction of Ly6C (28). A single experiment using T cells from STAT3fl/fl × CD4-Cre mice suggested that STAT3 is not required for IL-27–mediated expression of Ly6C or Sca-1 on CD4+ or CD8+ T cells (Supplemental Fig. 3C, 3D). In contrast, when naive CD8+ Ly6C−Sca-1− T cells from STAT1−/− mice were given TCR stimulation in the presence of IL-27 or IFN-γ, the induction of Ly6C was found to be almost entirely STAT1-dependent (Fig. 3A), as was the induction of Sca-1 (Fig. 3B). However, the type I IFN–mediated induction of Ly6C and Sca-1 was not STAT1-dependent in this system (Fig. 3A, 3B). Similar results were seen for CD4+ T cells (Fig. 3C, 3D). These results demonstrate a key role for STAT1 in IL-27– and IFN-γ–mediated induction of Ly6C and Sca-1.
TGF-β antagonizes Ly6C and Sca-1 expression
Although multiple cytokines promote expression of Ly6C and Sca-1, this screening process revealed that TGF-β was a potent inhibitor of the expression of these molecules. The addition of TGF-β reduced IL-27–mediated expression of Ly6C on CD4+ T cells (Fig. 4A, 4C) and CD8+ T cells (Fig. 4B, 4C), which is similar to results from a previous study that used P14 cells in the context of lymphocytic choriomeningitis virus infection (29). Addition of TGF-β also limited proliferation of CD4+ and CD8+ T cells, as illustrated by the reduced dilution of CFSE (Fig. 4A, 4B, 4D, 4E). This was true in the presence and absence of IL-27 and is consistent with the ability of TGF-β to limit the proliferation and differentiation of naive T cells into terminally differentiated effector cells (30). Addition of TGF-β also reduced TCR-mediated induction of Sca-1 on CD4+ T cells (Fig. 4D, 4F) and on CD8+ T cells (Fig. 4E, 4F), but did not affect the expression of Sca-1 in the presence of IL-27. The relatively modest effect that TGF-β has on IL-27–mediated expression of Sca-1 is consistent with the data in earlier figures that Sca-1 is more robustly expressed than Ly6C, and also indicates that the inhibitory effects of TGF-β are most closely associated with reduced TCR signaling.
Analysis of Ly6C and Sca-1 expression on diverse T cell populations
Ly6C has been used to identify terminally differentiated effector CD4+ T cells during infection (6, 10, 11), but its expression during homeostasis is not well described. To determine which T cell populations express Ly6C under homeostatic conditions, a survey of uninfected SPF mice was conducted. The expression of high levels of CD44 was used to identify Ag-experienced cells (31) and CD62L was used to identify cells that home to lymph nodes, which are primarily naive and central memory cells (32). In this setting, 20% of naive (CD62L+CD44lo) and 10% of memory (CD62L+CD44hi) CD4+ T cells expressed Ly6C and only 10% of CD4+ T cells with an effector phenotype (CD62L−CD44hi) were Ly6C+ (Fig. 5A). For CD8+ T cells, 20% of naive (CD44loCD62L+) cells expressed Ly6C, whereas ∼30% of CD44hiCD62L− and 90% of CD44hiCD62L+ CD8+ T cells expressed Ly6C. Thus, Ly6C expression was concentrated on the Ag-experienced cells, but further subsetting based on T cell expression of CD25, KLRG1, CD127, CD69, or CD49d indicated that Ly6C expression is not restricted to a particular effector/memory T cell population (data not shown). However, it is notable that in this survey, the majority of CD122+ CD8+ T cells expressed Ly6C, regardless of their expression of CD62L and CD44 (Supplemental Fig. 4). CD122 is the β subunit of the IL-2 and IL-15 receptors and, along with CD44 and Ly6C, is upregulated on T cells undergoing homeostatic proliferation (2, 33, 34).
When a similar survey was performed to assess the expression of Sca-1 on naive, effector, and memory T cells in SPF mice, 20% of naive (CD44loCD62L+) CD4+ T cells expressed Sca-1, whereas 80% of CD44hiCD62L− effector CD4+ T cells were Sca-1+ (Fig. 5B). Similarly, minimal Sca-1 expression was seen in CD44loCD62L+ CD8+ T cells, whereas the highest Sca-1 expression by CD8+ T cells (15%) was seen in the CD44hiCD62L− population (Fig. 5B). These findings indicate that Sca-1 was enriched on CD44hi CD4+ and CD8+ T cells, but in this survey was not exclusively expressed by any particular effector/memory population examined.
Ly6C and Sca-1 are upregulated on pathogen-specific T cells during toxoplasmosis
The above survey used established surface markers to distinguish Ag-experienced and naive cells, but in this setting, it is difficult to determine how expression of Ly6C or Sca-1 correlates with previous Ag exposure. To examine an effector population with a well-defined history, mice were infected with T. gondii and the expression of Ly6C and Sca-1 on parasite-specific T cells during acute toxoplasmosis was examined. Mice were infected i.p. with T. gondii and spleens and peritoneal exudate cells were harvested 10 d postinfection. Toxoplasma-specific T cells were identified by staining with parasite-specific MHC-I or MHC-II tetramers in combination with high expression of LFA-1 (35). At day 10 postinfection, the majority of parasite-specific CD4+ and CD8+ T cells in the peritoneum expressed Ly6C, demonstrating that toxoplasmosis promotes Ly6C expression by parasite-specific T cells (Fig. 6A).
A recent study (4) proposed that during toxoplasmosis, minimally differentiated memory CD8+ T cells that are CXCR3+KLRG1− give rise to an intermediate CXCR3+KLRG1+ population that in turn downregulates CXCR3 when it differentiates into terminally differentiated effector cells. Consistent with this previous report, at day 10 of infection, 20% of splenic parasite-specific CD8+ T cells were CXCR3+KLRG1−, 60% were CXCR3+KLRG1+ and <10% were CXCR3−KLRG1+. This analysis was extended to CD4+ T cells, in which 20% were CXCR3+KLRG1−, 30% were CXCR3+KLRG1+ and 30% were CXCR3−KLRG1+ (Fig. 6B). Seventy percent of parasite-specific CD4+ T cells expressed Ly6C (Fig. 6A). When the cells were subsetted by expression of CXCR3 and KLRG1, Ly6C was expressed by 60% of CXCR3+KLRG1−, 75% of CXCR3+KLRG1+, and 70% of CXCR3−KLRG1+ parasite-specific CD4+ T cells. Similar results were seen for CD8+ T cells, as Ly6C was expressed by 90% of CXCR3+KLRG1−, 95% of CXCR3+KLRG1+, and 85% of CXCR3−KLRG1+ parasite-specific CD8+ T cells. Therefore, Ly6C expression on these individual subsets was not exclusive to the CXCR3−KLRG1+ population for CD4+ or CD8+ T cells, demonstrating that Ly6C and KLRG1 are not interchangeable markers of differentiation (Fig. 6C).
When a similar analysis was performed for Sca-1, 85% of parasite-specific CD4+ and CD8+ T cells expressed Sca-1 (Fig. 6D). When these cells were subsetted by their expression of KLRG1 and CXCR3, ∼90% of the CXCR3+KLRG1− and CXCR3+KLRG1+ populations expressed Sca-1 in both CD4+ and CD8+ T cells (Fig. 6E). Seventy percent of CXCR3−KLRG1+ CD4+ T cells and 60% of CXCR3−KLRG1+ CD8+ T cells expressed Ly6C, indicating that Sca-1 is present at a lower frequency on more highly differentiated cells. Nonetheless, Sca-1 is more widely expressed during toxoplasmosis than KLRG1 or CXCR3, and does not enable the ready differentiation of distinct Ag-experienced T cell populations in this experimental system.
IL-27 and IFN-γ promote T cell expression of Ly6C during toxoplasmosis
IL-27 and IFN-γ are key cytokines during toxoplasmosis (36, 37) and are two of the strongest inducers of Ly6C and Sca-1 in vitro. To determine the contribution of IL-27 and IFN-γ signaling to the expression of Ly6C and Sca-1 during infection, wild-type (WT) and IL-27 receptor (Il27ra)-deficient mice were infected with T. gondii. Mice were also treated with an isotype Ab or a neutralizing anti–IFN-γ Ab on days 3 and 6 of infection. When peritoneal tetramer-positive T cells were examined at day 9 of infection, the percentage of parasite-specific CD4+ T cells expressing Ly6C was substantially lower in Il27ra–deficient mice than in WT mice. Ly6C levels were significantly reduced in CD8+ T cells as well, providing evidence that IL-27 promotes the Ly6C+ population in this system (Fig. 7A, 7B). Additionally, neutralizing IFN-γ resulted in less Ly6C expression by CD4+ T cells, but in Il27ra–deficient mice did not result in a complete ablation of Ly6C expression. In contrast, the absence of the IL-27 receptor did not limit the expression of Sca-1 by parasite-specific T cells (Fig. 7C, 7D). Surprisingly, the blockade of IFN-γ increased Sca-1 expression in both WT and Il27ra−/− mice, possibly because the absence of IFN-γ leads to a marked increase in parasite replication and Ag load that might lead to increased T cell activation. Together, these studies establish that IL-27 and IFN-γ are involved in the regulation of Ly6C expression during toxoplasmosis, but in this setting they were not required for maximal Sca-1 expression.
Studies to understand the functions of Ly6 molecules have been performed since the 1970s, but questions still remain about their functions and the factors that influence their expression (38). The association of Ly6C expression with short-lived effector T cells has been reported in multiple experimental systems (6, 10) and the combination of Sca-1, CD122, and Bcl-2 can be used to identify CD8+ memory stem cells (12–14). A previous study showed that treatment of mice with IL-27 promotes the development of a memory precursor population of tumor Ag-specific CD8+ T cells, characterized by high expression of Bcl-6, SOCS3, and Sca-1 (19). However, the expression patterns of Ly6C and Sca-1 have not been compared and there is a limited appreciation of how cytokine and TCR-mediated signals are integrated to promote their expression. The in vitro experiments performed here used a defined population of naive Ly6C− CD4+ and CD8+ T cells to address any concerns that the stimuli used here might preferentially expand a Ly6C+ population. This approach showed quite modest effects of IL-27, the IFNs, or TCR alone on Ly6C expression, but the combination of cytokine plus TCR synergistically promoted the expression of Ly6C by a subset of activated CD4+ and CD8+ T cells. That this activity was STAT1-dependent correlated well with the range of cytokines that could modulate Ly6C, whereas cytokines that predominantly use STAT3, STAT4, or STAT6 had minimal effects. TGF-β was the only signal identified that suppressed the induction of Ly6C and Sca-1, which correlates with its ability to suppress T cell activation and proliferation (39, 40).
Because Ly6C and Sca-1 have been used as markers to identify T cells at different stages of differentiation, it was notable that the expression of these two molecules was upregulated by the same cytokine signals, in the same cells. Sca-1 was potently induced by either TCR stimulation or cytokine signaling and consequently appeared to be more widely expressed than Ly6C. Indeed, a survey to determine if either of these molecules could be associated with different effector or memory populations found that it was difficult to link them to memory-like or terminally differentiated effector cells based on differential expression of KLRG1 and CXCR3. The ability of these cytokines to induce Ly6C and most notably Sca-1 in the absence of TCR stimulation suggests the need for caution in using these molecules alone to identify Ag-experienced populations.
Infection with T. gondii is dominated by the generation of parasite-specific CD4+ and CD8+ T cells that produce IFN-γ, but this is also a system in which endogenous IL-27 is required to limit the inflammatory response (37). Although the loss of either cytokine signal during infection reduces Ly6C expression, it was relevant to note that in the setting of IL-27R deficiency, there are markedly elevated levels of IFN-γ (37), but IFN-γ blockade did not result in a further reduction in the numbers of parasite-specific effectors that expressed Ly6C. The observation that IL-27 was more important in driving Ly6C in CD4+ T cells than in CD8+ T cells during toxoplasmosis is consistent with the larger effect of IFNs in promoting Ly6C in CD8+ T cells seen in vitro. Nevertheless, the in vivo studies presented here indicate that IL-27 and IFN-γ are not redundant in promoting Ly6C expression during toxoplasmosis.
Although the section above focuses on the regulation of Ly6C, the in vitro and in vivo studies identified common pathways that influence expression of Ly6C and Sca-1, but also highlighted some notable differences. Again, the ability to use a defined, naive Sca-1− starting population helped establish the profound impact of TCR stimulation alone or in combination with cytokines on Sca-1 expression. As seen for Ly6C, not every cytokine was a potent inducer of Sca-1 and those that activated STAT1 seemed dominant, although the use of STAT1-deficient cells does indicate the presence of additional pathways that are involved in this process. Potential STAT1-independent pathways relevant to IL-27 and type I IFNs include p38 MAPK and ERK1/2 (24, 25). However, unlike Ly6C, Sca-1 expression was not attenuated by the loss of IL-27 or IFN-γ in in vivo studies. This contrasts with an earlier report using a model of colitis, in which in vivo administration of an adeno-associated virus vector encoding IL-27 was associated with reduced inflammation and increased expression of Sca-1 by CD4+ T cells (20). This contradiction likely reflects the complexity in trying to distinguish a role for endogenous IL-27 or IFN-γ in a systemic infection, in which many signals including TCR and other cytokines readily promote Sca-1 expression, versus a dominant signal provided by overexpression of IL-27.
There have been few studies that have directly addressed the function of Ly6C and Sca-1 in the immune response. Ly6C has been proposed to be involved in T cell homing to secondary lymphoid organs, possibly through an association with LFA-1 (41–43), and ImageStream analysis of T. gondii–specific effectors shows colocalization of Ly6C and CD11a (a subunit of LFA-1) on the surface of these cells (27). IL-27 has been proposed to modulate T cell homing through upregulation of ICAM-1 and selectin ligands on conventional CD4+ T cells (21, 44, 45), as well as CXCR3 on regulatory T cells (21). Thus, upregulation of Ly6C may be an additional mechanism by which IL-27 and/or IFNs modulate T cell trafficking. There is also a literature that suggests a regulatory role for Ly6C and Sca-1 in limiting the T cell response. A mutation in the promoter of Ly6C reduces its expression in NOD, NZB/W, and ST mice, which are strains that spontaneously develop autoimmune diseases (46). Moreover, in the context of TCR stimulation, Abs that crosslink Ly6C or Sca-1 on the surface of T cells limit their ability to produce IL-2 and proliferate (47–49). Furthermore, in mice genetically engineered to lack Sca-1 expression, T cells exhibit enhanced proliferation in response to TCR stimulation (50). Additional evidence for a regulatory function of Sca-1 is the finding that transgenic overexpression of Sca-1 limits T cell proliferation (48, 49, 51) and suppresses lymphoproliferation and autoimmunity in lpr/lpr mice (51). Together, these findings suggest that Ly6C and/or Sca-1 may have a role in limiting T cell responses. A suppressive function for Ly6C and Sca-1 would complement reports that IL-27 promotes inhibitory pathways including IL-10 and LAG-3 (52–58), and that IL-27 and IFN-γ promote expression of PD-L1 (58–60). Additional studies are needed to determine whether Ly6C and Sca-1 primarily function to promote T cell activation and migration, or if any of the shared immune regulatory effects of IL-27 and the IFNs are mediated through the induction of Ly6C and/or Sca-1.
We thank the members of the Hunter laboratory for intellectual and experimental contributions to this study. We thank the University of Pennsylvania Flow Cytometry and Cell Sorting Facility for support.
This work was supported by National Institutes of Health Grant T32 5T32AR007442-28 (to J.H.D.), American Society of Nephrology Ben J. Lipps Research Fellowship (to G.M.C.), University of Pennsylvania School of Medicine Measey Senior Research Fellowship (to G.M.C.), National Institutes of Health Grant R01 5R01AI110201-03 (to C.A.H.), and the Commonwealth of Pennsylvania.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.