T Cells from Leishmania major-Susceptible BALB/c Mice Have a Defect in Efficiently Up-Regulating CXCR3 upon Activation¹

The Leishmania are obligate intracellular parasites that cause a wide range of diseases such as cutaneous, mucocutaneous, and visceral leishmaniasis. In humans, cutaneous leishmaniasis caused by Leishmania major usually manifests as a localized, self-resolving lesion associated with development of long-term immunity (1, 2). The murine model of cutaneous L. major infection has been well characterized and frequently has been used as a functional model of Th1 and Th2 cell responses (3). Most inbred mice such as C57BL/6, C3H, and CBA/J are genetically resistant to L. major and spontaneously resolve infection (4) because they mount a protective IL-12-induced Th1-type response. In contrast, susceptible BALB/c mice develop large nonhealing lesions and mount a Th2 response that is associated with the production of the cytokines IL-4 and IL-10. Nevertheless, previous studies have shown that BALB/c mice are capable of mounting a Th1 response and contain a significant number of IFN-γ-producing T cells in their lymph nodes (LN)³ comparable to C57BL/6 mice during the early phase of L. major infection (5, 6). C57BL/6 mice also produce high levels of IL-4 similar to BALB/c mice early after infection. Furthermore, some studies have found that deletion of the IL-4 or IL-4R gene in BALB/c mice has no effect on the outcome of L. major infection (7, 8). Taken together, these findings suggest that genetically regulated mechanisms other than Th1/Th2 cytokine production may also control the outcome of L. major infection in BALB/c mice.

We had previously found that the CXCR3, which binds CXCL9, CXCL10, and CXCL11, plays a critical role in immunity against L. major in C57BL/6 mice by mediating recruitment of IFN-γ-producing T cells from the LN to lesion (9). This was perhaps not surprising since CD4⁺ Th1 cells have been shown to preferentially express CXCR3 (10). Because CXCR3 is critical for Th1 cell migration and L. major-infected BALB/c mice fail to restrict early parasite growth in the skin despite containing a high number of IFN-γ-producing T cells in their LN, we hypothesized that BALB/c mice may have a defect in up-regulating CXCR3 on T cells. Such a defect may prevent recruitment of Th1 cells to the lesion and, therefore, contribute to L. major susceptibility. To test this hypothesis, we analyzed CXCR3-expressing T cells the lesions and LN from BALB/c and C57BL/6 mice during L. major infection. In addition, we compared the ability of naive CD4⁺ and CD8⁺ T cells from BALB/c and C57BL/6 mice to up-regulate CXCR3 upon anti-CD3/anti-CD28 activation in vitro. Our results indicate that both CD4⁺ and CD8⁺ T cells from susceptible BALB/c, but not resistant C57BL/6, mice have an intrinsic defect in efficiently up-regulating CXCR3 upon activation, which may contribute to susceptibility to L. major.

Sex- and age-matched C57BL/6 and BALB/c mice were purchased from Harlan Sprague Dawley and maintained at The Ohio State University according to institutional regulations. IL-4Rα−/− mice were provided by Alison Finnegan, Chicago, IL. For intradermal infections, 1 × 10⁴ L. major promastigotes (LV39) were injected into the ears of C57BL/6 and BALB/c mice.

At time points post infection, three to four C57BL/6 and BALB/c mice were sacrificed. The draining LN (dLN) were excised and single-cell suspensions of LN cells were obtained by teasing through a 70-μm mesh. Lesion leukocytes were isolated as described previously (9). Cells were stained with PE-conjugated anti-mouse CXCR3 (R&D Systems) and either FITC-labeled anti-CD4 or anti-CD8 Abs (Biolegend). Flow cytometric analysis of LN and lesion cells was performed with a BD FACSCalibur.

Cell suspensions were obtained from the excised LNs and spleens of uninfected mice. Then, 90–95% pure CD3⁺ T cell or CD4⁺ and CD8⁺ T cell populations were obtained via nylon wool columns and immunomagnetic isolation (Mitenyi Biotec) from either wild-type (WT) C57BL/6, BALB/c, IL-4R−/−, or IL-10−/− BALB/c mice (11). T cells were incubated 48 h at 0.5–2.5 × 10⁶ cells/well in a 24-well plate precoated with 3 and 4 μg/ml anti-CD3 (clone 145–2C11) and anti-CD28 (clone 37.51) Abs (Biolegend), respectively. Following in vitro activation, cells were transferred to uncoated wells and rested in their conditioned media for 24 h before flow cytometry analysis. In brief, in vitro activated cells were recovered and washed in cold PBS before staining with PE-labeled anti-mouse CXCR3 Abs or a rat IgG2a-PE control (R&D Systems). Stained cells were either analyzed immediately or fixed in 1% paraformaldehyde for short-term storage.

Culture supernatants were sampled after 48 h of incubation at 37°C, 5% CO₂ and then analyzed for the presence of IFN-γ, IL-4, and IL-10 by sandwich ELISA as described previously (9).

Total RNA was extracted from T cells using TRIzol Reagent (Invitrogen), and cDNA was synthesized and amplified as described previously (12). Primer sequences were found using the Harvard’s PRIMER BANK website, and oligonucleotides were purchased.

Statistically significant differences were determined by an unpaired Student’s t test. A p-value <0.05 was considered significant.

CXCR3 is preferentially expressed on activated Th1-promoting cells (10). Previously, our group found that during L. major infection, CXCR3 deficiency results in more severe nonresolving disease compared with WT mice. This heightened susceptibility coincided with deficient T cell mobilization and local IFN-γ production (9). Others have reported that resistant C57BL/6 mice produce high levels of CXCR3 ligands in response to L. major infection, whereas susceptible BALB/c mice do not (13). These findings indicate that CXCR3 plays a critical role in immunity against L. major in a resistant mouse and that L. major-susceptible and -resistant mice differ in their ability to produce CXCR3 ligands. However, whether CXCR3 is differentially expressed by susceptible and resistant mice remains to be investigated.

Therefore, we infected L. major-susceptible BALB/c and -resistant C57BL/6 mice intradermally by inoculating 1 × 10⁴ L. major promastigotes into ear dermis and frequency of CXCR3 expressing T cells in the dLN and lesions temporally using flow cytometry. Throughout the course of infection, L. major-infected C57BL/6 mice displayed higher frequencies of CXCR3 expressing CD4⁺ and CD8⁺ lymphocytes in their dLNs compared with BALB/c mice (Fig. 1,A). Similar trends in CXCR3⁺ T cell frequencies were also noted between the lesions of BALB/c and C57BL/6 mice (Fig. 1,B). Furthermore, analysis of the dLN and lesion revealed that CXCR3⁺ T cells comprised greater percentages of the CD4⁺ and CD8⁺ compartments of infected C57BL/6 mice than BALB/c mice (Fig. 1,C, and data not shown). Also, C57BL/6 mice contained more CXCR3⁺ CD4⁺ and CD8⁺ T cells in their dLN (Fig. 2, bottom panels) and lesions than BALB/c mice (Fig. 2, top panels), although L. major-infected C57BL/6 mice showed a decline in CXCR3-expressing T cells in the dLNs and lesions as they resolved the infection (data not shown). These results demonstrate that resolution of L. major infection in C57BL/6 mice is associated with an increase in CXCR3⁺ T cells in their dLNs and lesions, whereas disease progression in BALB/c mice is associated with no or only modest increase in these cell populations.

To determine whether T cells from C57BL/6 and BALB/c mice inherently differ in their ability to up-regulate CXCR3, naive T cells were isolated from the spleens of naive mice and stimulated with plate-bound anti-CD3/anti-CD28 Abs as described previously (11). Expression of CXCR3 on these activated T cells was compared by flow cytometry. Following in vitro stimulation, T cells from C57BL/6 mice efficiently up-regulated CXCR3. In contrast, the increase in CXCR3 was minimal in similarly stimulated T cells from BALB/c mice (Fig. 3,A). Moreover, low CXCR3 expression on activated BALB/c T cells also correlated with low CXCR3 mRNA levels suggesting that suppression of CXCR3 expression was at the transcript level (Fig. 3 B). This defect in CXCR3 expression was observed in CD4⁺ and CD8⁺ T cells of BALB/c mice purified and activated separately (data not shown). Baseline levels of CXCR3 were low on prestimulated T cells from both BALB/c and C57BL/6 mice (data not shown) as previously reported (12, 14).

It has been shown that IFN-γ and the transcription factor T-bet are required for efficient induction of CXCR3 on certain T cell subsets (14, 15, 16). To determine whether failure of BALB/c T cells to up-regulate CXCR3 was associated with reduced production of IFN-γ, we analyzed levels of Th1-associated IFN-γ as well as Th2-associated IL-4 and IL-10 in the above culture supernatants by ELISA. Also we quantified mRNA levels of T-bet in T cells by real-time PCR.

Both C57BL/6- and BALB/c-derived T cells produced high and comparable levels of IFN-γ upon activation (Fig. 4,A). However, activated T cells from BALB/c mice produced significantly more IL-4 and IL-10 as compared with similarly activated T cells from C57BL/6 mice (Fig. 4, B and C). Also, mRNA levels of T-bet and IFN-γR (Fig. 4, D and E) as well as surface levels of IFN-γR (data not shown) were comparable in T cells from both groups. These results show that even though BALB/c T cells produce higher levels of Th2-associated cytokine, they are not deficient in IFN-γ production, T-bet induction, or IFN-γR expression compared with C57BL/6-derived cells. Furthermore, they suggest that neither lack of IFN-γ production or deficient expression of IFN-γR is responsible for the inability of BALB/c-derived T cells to up-regulate CXCR3.

Between 10 and 48 h post stimulation, C57BL/6-derived T cells rapidly induced CXCR3 surface protein (Fig. 5,A) while their BALB/c-derived counterparts expressed only marginal levels of CXCR3 after 48 h (Fig. 5,B). Interestingly, the slight increase in CXCR3 staining of BALB/c-derived T cells coincided with reduction in IL-4 concentrations at later time points poststimulation (Fig. 4, B and C).

To determine whether IL-4 was involved in preventing up-regulation of CXCR3 on BALB/c T cells, we stimulated BALB/c and C57BL/6 T cells in the presence of 10 μg/ml anti-IL-4 or an isotype-matched control Ab and examined the expression of CXCR3 by flow cytometry. In addition, we analyzed CXCR3 expression on T cells from WT BALB/c and IL-4Rα−/− BALB/c mice following in vitro activation with anti-CD3/CD28. Blockade of IL-4 (Fig. 6,A) or lack of IL-4Rα (Fig. 6,B) failed to increase expression of CXCR3 on BALB/c-derived T cells, indicating that IL-4 and IL-13 were not responsible for suppression of CXCR3. As expected, expression of CXCR3 on the T cells of C57BL/6 mice were unaffected by IL-4 neutralization (Fig. 6 C).

Because T cells from BALB/c mice also produced significantly more IL-10 than C57BL/6 T cells after stimulation, we investigated whether the cytokine was involved in preventing up-regulation of CXCR3 on BALB/c T cells. To this end, we blocked IL-10 signaling by including anti-IL-10R Abs in the culture media of BALB/c- and C57BL/6-derived T cells and examined its effect on CXCR3 expression. Additionally, we activated T cells from WT BALB/c and IL-10^−/− BALB/c mice in vitro with anti-CD3/CD8 and compared levels of CXCR3. Flow cytometric analysis showed that CXCR3 expression by anti-IL-10-treated BALB/c T cells was greater than that of isotype control-treated cells yet still lower than C57BL/6 T cells (Fig. 7,A). Not surprisingly, C57BL/6-derived T cells were unaffected by IL-10R blockade (data not shown). Furthermore, activated T cells from IL-10−/− BALB/c mice expressed more CXCR3 than WT controls (Fig. 7,B). Additionally, increased CXCR3 expression in the absence of IL-10 was also noted on T cells in the dLN of IL-10−/− mice infected with L. major (Fig. 7 C). Collectively, these results indicate that IL-10 is partially responsible for inhibiting CXCR3 expression on T cells in BALB/c mice.

Since we found that IL-10 was partially involved in preventing up-regulation of CXCR3 on BALB/c T cells, we determined whether IL-10 can block induction of CXCR3 on T cells in resistant mice. Naive T cells from C57BL/6 mice were stimulated in vitro with anti-CD3/CD28 as described above in the presence of rIL-10 and expression of CXCR3 was analyzed by flow cytometry. Interestingly, rIL-10 failed to suppress expression of CXCR3 on C57BL/6 T cells (Fig. 4,C) suggesting that these T cells were refractory to IL-10-induced suppression of CXCR3 expression. We therefore compared levels of IL-10R on activated T cells from BALB/c and C57BL/6 mice using real-time RT-PCR as well as flow cytometry. Anti-CD3/CD28-activated CD4⁺ and CD8⁺ T cells from C57BL/6 mice displayed less induction of IL-10R mRNA and expressed less IL-10R as compared with BALB/c-derived T cells (Fig. 8, A and B). These data suggest that low levels of IL-10R on C57BL/6 mice may be responsible for their refractoriness to IL-10-mediated down-regulation of CXCR3.

We had previously shown that CXCR3 plays a critical role in immunity against L. major by controlling T cell recruitment and IFN-γ levels at the site of infection. The novel finding in the present study is that CD4⁺ and CD8⁺ T cells from L. major-susceptible BALB/c, but not -resistant C57BL/6, mice have a defect in efficient up-regulation of CXCR3 upon activation. It is well documented that Th1-associated cytokines IL-12 and IFN-γ are crucial for resolution of L. major infection in C57BL/6 mice, whereas Th2-associated cytokines IL-4, IL-10, and IL-13 have been implicated as susceptibility factors in BALB/c mice. However, a previous study has documented that both BALB/c and C57BL/6 mice contain comparable frequencies of IFN-γ-producing T cells in their dLNs during early phase of L. major infection, yet BALB/c mice fail to control infection. One explanation for this observation could be that IFN-γ-producing T cells in the LN of BALB/c mice migrate less efficiently to the infected skin because BALB/c mice produce less CXCR3 ligand CXCL10 than C57BL/6 mice during L. major infection (13). However, intralesional administration of rIP-10 (CXCL10) to BALB/c mice shortly after L. major infection increases NK cell cytotoxicity in the LN but fails to limit disease progression (13). These data suggest that IFN-γ-producing T cells in BALB/c mice may not be responsive to CXCL10 and fail to migrate to lesion. In the present study, we found that resolution of L. major infection in C57BL/6 mice was associated with an influx CXCR3⁺ T cells in the LN as well as lesions, whereas disease progression in BALB/c mice was associated with reduced frequency of CXCR3⁺ T cells at both these sites. We also found that naive T cells from BALB/c mice up-regulated CXCR3 less efficiently than C57BL/6 T cells following in vitro activation with anti-CD3/CD28. Furthermore, low CXCR3 expression on BALB/c T cells also correlated with low CXCR3 mRNA levels suggesting that suppression of CXCR3 was at the level of transcription. In vitro stimulation studies performed using purified CD4⁺ and CD8⁺ T cells revealed that both these cell types in BALB/c mice had a defect in up-regulating CXCR3 (data not shown). Taken together, these findings suggest that a defect in CXCR3 up-regulation on T cells could contribute to disease progression in BALB/c mice. In addition, they indicate that the inability of activated T cells from BALB/c mice to up-regulate CXCR3 is not due to lack of IFN-γ.

Previous studies have shown that IFN-γ, STAT1, and the transcription factor T-bet are required for efficient induction of CXCR3 on certain T cell subsets (14, 15, 16). IFN-γ, which signals via IFN-γR/STAT1 pathway, induces expression of T-bet (17), which in turn enhances expression of CXCR3 on T cells (15, 16). To determine whether reduced expression of CXCR3 on BALB/c T cells is due to lack of IFN-γ production, we measured levels of IFN-γ as well as Th2-associated IL-4 and IL-10 in culture supernatant of activated T cells from BALB/c and C57BL/6 mice. In addition, we quantified mRNA levels of T-bet as well as IFN-γR by real-time RT-PCR. Upon activation with anti-CD3/CD28, T cells from C57BL/6 and BALB/c mice produced significant and comparable amounts of IFN-γ, but later produced significantly more IL-4 and IL-10. Both groups showed significant induction of T-bet and comparable levels of T-bet mRNA. Levels of IFN-γR mRNA were also comparable in T cells from BALB/c and C57BL/6 mice and correlated with surface expression of IFN-γR as assessed by flow cytometry (data not shown). Collectively, these findings show that lack of IFN-γ production or impaired expression of T-bet or IFN-γR are not responsible for failure of CXCR3 up-regulation in T cells from BALB/c mice. In addition, they suggest that Th2-associated cytokines may be involved in antagonizing IFN-γ and T-bet-induced expression of CXCR3 in BALB/c T cells.

Th2-associated cytokines IL-4 and IL-10 that mediate susceptibility to L. major, also regulate expression of several chemokine receptors and their ligands. However, previous studies have reported that IL-4 and IL-10 play distinct roles in regulation of expression of CXCR3 and its ligands CXCL9 and CXCL10. For example, Albanesi et al. (18) found that IL-4 enhances CXCL9 and CXCL10 production in keratinocytes and induces migration of CXCR3⁺ T cells in murine model of allergic contact dermatitis. IL-4 treatment also exacerbated disease in a Th1 cells transfer model of colitis, which was associated with up-regulation of CXCR3 and its ligands in the colon (19). In contrast, IL-10 was found to inhibit expression of CXCR3 in microglial cells as well as human eosinophils (20). In the present study, a decrease in the levels of IL-4 in the culture supernatants from BALB/c mice was associated with a modest increase in levels of CXCR3 suggesting that IL-4 may be involved in suppressing expression of CXCR3. However, blockade of IL-4 using neutralizing Abs failed to restore expression of CXCR3 to levels observed on C57BL/6 T cells. Additionally, T cells from IL-4Rα−/− BALB/c mice were deficient in up-regulating CXCR3 upon activation. In contrast, blockade of IL-10R using anti-IL-10R Ab enhanced expression of CXCR3 on BALB/c-derived T cells. Furthermore, in vitro-activated T cells from IL-10−/− BALB/c mice expressed CXCR3 more readily than WT controls. Expression of CXCR3 on the T cells of C57BL/6 mice were unaffected by IL-4 neutralization or IL-10R blockade or rIL-4 treatment. Together, these results demonstrate that IL-10 is partly responsible for preventing up-regulation of CXCR3 on T cells in BALB/c mice. Furthermore, they show that IL-4 is not involved in inducing CXCR3 expression on T cells. It is interesting to note that levels of IL-4 in culture supernatants dropped rapidly when activated T cells of BALB/c mice were rested. This may be due to rapid consumption of IL-4 by these cells or simply due to degradation of this cytokine. Nonetheless, despite the presence of abundant “pro-Th1 factors” (i.e., T-bet and IFN-γ) and decrease in IL-4 levels, BALB/c-derived T cells failed to up-regulate CXCR3 levels. Furthermore, blockade of IL-10R or lack of IL-10 only partially restored expression of CXCR3 on BALB/c T cells indicating that other molecular mechanisms may be involved in negatively regulating induction of CXCR3 on these cells. One such potential mechanism is that a Th2-associated transcription factor GATA3, which directly binds to T-bet, may be involved in preventing T-bet-induced induction of CXCR3 in BALB/c T cells. We are currently investigating this possibility in ongoing studies in our laboratory.

Because IL-10/IL-10R pathway was found to be involved in suppressing CXCR3 expression on BALB/c T cells, we investigated whether IL-10 can block induction of CXCR3 on T cells in L. major-resistant mice. In addition, we analyzed expression of IL-10R and measured IL-10R mRNA levels in activated T cells from both strains. We found that rIL-10 failed to prevent up-regulation of CXCR3 on C57BL/6 T cells, suggesting that these cells were resistant to IL-10-mediated CXCR3 suppression. Furthermore, BALB/c T cells expressed higher levels of IL-10R and contained more IL-10R mRNA than C57BL/6 T cells. Taken together, these results suggest that lower levels of IL-10R on C57BL/6 T cells may render them resistant to IL-10-mediated CXCR3 suppression and aid in up-regulation of CXCR3. Our finding are supportive of a previous study which showed that C57BL/6 leukocytes are more resistant to the IL-10-mediated immune-suppressive effect of regulatory T cells than those of BALB/c mice (21).

Although IL-10 has been observed to inhibit CXCR3 expression by mouse microglia and human eosinophils (22), no such role is known for T cells. Interestingly, in the murine colitis model, forced expression of IL-10 results in amelioration of this Th1- (and CXCL10-) mediated disease that is associated with reduced expression of CXCR3 and its ligands (23). Our observations are particularly interesting in light of recent studies revealing the IL-10-producing capacity of Th1 cells as a means of limiting their own inflammatory potential (24). One such consequence of T cell-derived IL-10 may be to limit CXCR3-mediated cell homing – a recruitment axis critical for Th1 trafficking in numerous disease models.

In conclusion, resolution of L. major infections by resistant C57BL/6 mice is associated with an increase in CXCR3 expressing T cells in their LN and lesions whereas disease progression in susceptible BALB/c mice correlates with fewer CXCR3⁺ T cells in these tissues. Certainly, extrapolating the results of this and other mouse model intensive studies to human leishmaniasis should be done with caution and further study is needed to confirm the protective role of CXCR3 expression and signaling in the human response to Leishmania infection. Nevertheless, our present study has elucidated some previously unrecognized aspects of CXCR3 regulation and its dependence on elements of the Th1 and Th2 response. Naive T cells from BALB/c mice and C57BL/6 mice markedly differ in their abilities to up-regulate CXCR3 upon activation. Anti-CD3/CD28-activated T cells from BALB/c mice produce as much IFN-γ as T cells from C57BL/6 mice but they secrete more IL-4 and IL-10 and fail to up-regulate CXCR3 efficiently. Blockade of IL-4 or lack of IL-4Rα fails to increase CXCR3 levels on BALB/c T cells but blockade of IL-10R or IL-10 deficiency partially restores CXCR3 expression. These results indicate that L. major-susceptible BALB/c mice, but not -resistant C57BL/6 mice, have a defect in efficiently up-regulating CXCR3 on T cells, which is partly mediated by IL-10. Yoon et al. (25) has described differences in CXCR3 transcript level in the stressed ocular tissues of C57BL/6 and BALB/c mice. Furthermore, a recent study has found that C57BL/6 mice, which are susceptible to cerebral malaria, contain more CXCR3⁺ T cells in spleens compared with cerebral malaria-resistant BALB/c mice during infection (26). Given our findings, differential expression of CXCR3 on T cells of BALB/c and C57BL/6 mice may explain the observations reported in above studies. Further studies are needed to determine whether this defect in CXCR3 up-regulation in BALB/c mice contributes to susceptibility to L. major as well as their phenotype in above models.

We thank Sarah E. Walker for critical review of this manuscript and Dr. Alison Finnegan for providing IL-4Rα−/− BALB/c mice.

The authors have no financial conflict of interest.