IL-21 Is Important for Induction of KLRG1⁺ Effector CD8 T Cells during Acute Intracellular Infection Free

Interleukin 21 is a member of the common γ-chain family, which is composed of IL-2, IL-4, IL-7, IL-9, and IL-15. This pleiotropic cytokine is produced by activated CD4 T cells, in particular follicular Th (Tfh), Th17, and activated NKT cells (1). In a murine model of lymphocytic choriomeningitis virus (LCMV) infection, IL-21 produced by virus-specific CD4 T cells was essential for sustaining the CD8 T cell response after they lost their effector abilities (2–4). In patients with HIV-1 infection, reduced IL-21 production could be a contributing factor to the compromised cellular and humoral response (5). Collectively, these studies underline the importance of IL-21 in long-term CD8 T cell immunity needed for restricting the chronic infection, but the absence of IL-21 does not seem to affect the development of a potent effector immunity against viral infections.

Microsporidia causes a self-limiting disease in immunocompetent individuals but results in progressive infection in HIV-infected and other immunocompromised individuals (6). Symptoms in these high-risk groups can be severe, ranging from chronic diarrhea to encephalitis and hepatitis (7). Evidence has recently emerged suggesting that microsporidiosis is a latent infection. In an animal model, corticosteroid-induced immunosuppression led to the reactivation and dissemination of the parasite (8), confirming older data, which document the frequent relapse of infection in patients with HIV who discontinued therapy after pathogen clearance (9, 10). In a mouse model of microsporidial infection using Encephalitozoon cuniculi, induction of a robust CD8 T cell immunity has been observed, and the CD8 T cells exhibit polyfunctional characteristics manifested by IFN-γ production and cytolytic activity against infected targets (11). Studies from our laboratory have demonstrated that protective immunity against i.p. infection is exclusively dependent on CD8 T cell immunity. However, during per-oral or natural route of infection, depletion of CD8 T cells alone does not result in the mortality of infected animals, and both CD4 and CD8 T cells are needed for protection (12). Consequently, the generation of effector CD8 T cell immunity against microsporidia is an important component of protective immunity, and factors needed for eliciting this response need to be studied. Interestingly, the data presented in this manuscript show that among the γ-chain cytokines, only IL-21 was elevated during the acute infection and played an important intrinsic role in the development of polyfunctional CD8 effector response against the pathogen. In the absence of functional IL-21, CD8 T cell effectors were unable to acquire polyfunctional ability and unable to expand optimally. The data demonstrate a critical role for IL-21 in the development of a robust effector CD8 T cells during microsporidial infection.

C57BL/6J (wild-type [WT]) and B6.SJL-Ptprc^a/BoyAiTac (CD45.1) 6–8-wk-old mice were purchased from Taconic Farms. IL-21R knockout (KO) and CD4 KO mice, purchased from The Jackson Laboratory (B6N.129-Il21r^tm1Kopf/J), were bred in-house. Mice were housed under specific pathogen-free conditions at the Animal Research Facility at The George Washington University (Washington, D.C.). All animal experiments were approved by the George Washington School of Medicine and Health Sciences Institutional Animal Care and Use Committee.

For some experiments, WT/IL-21R KO mixed bone marrow chimera animals were generated. Bone marrow was extracted from IL-21R KO (CD45.2) and B6.SJL (CD45.1) mice as previously described (11), and 5 × 10⁶ total cells (at a 1: 1 ratio for WT/IL-21R KO) were injected i.v. into lethally irradiated recipients (8 Gy/20 g body weight). Recipient animals received sulfamethoxazole- and trimethoprim-supplemented (Hi-Tech Pharmacal) water for 5 wk posttransfer. Experiments were performed 8 wk postreconstitution.

For BrdU experiments, mice were injected with BrdU (1 mg/mouse) i.p. starting at day 7 postinfection (p.i.) and every other day thereafter until the end of the experiment.

A rabbit isolate of E. cuniculi (genotype III) was maintained as previously described (13). Animals were infected with 2 × 10⁷ spores/mouse orally.

Antigenic extract was prepared by mechanical disruption of freshly harvested E. cuniculi spores in presence of 0.5-mm zirconia/silica beads (BioSpec Products) using six pulses of 1 min each in a mini bead beater. Insoluble Ag and residual spores were removed by centrifugation, and solution was sterile filtered before use.

Splenocytes were prepared as previously described (11). Cell suspension was labeled for surface markers before fixation (IC fixation buffer; Invitrogen).

For in vitro function assay, overnight restimulation of splenocytes was performed in presence of 20 μg/ml E. cuniculi–specific antigenic extract, followed by 4-h incubation in the presence of protein transport inhibitor mixture as well as fluorochrome-conjugated anti-CD107a. Surface staining was followed by fixation with IC fixation buffer/IC permeabilization buffer (Invitrogen) according to the manufacturer’s instruction and intracellular staining for IFN-γ and Ki67.

Intracellular staining for IL-21 was performed using rIL-21R/Fc fusion protein (R&D Systems) followed by PE-conjugated F(ab’)₂ goat anti-human Fcγ (Jackson ImmunoResearch Laboratories) according to a previously published report (4).

For T-bet detection, splenocytes were stimulated overnight with antigenic extract as described above and labeled for surface Ags. T-bet staining was performed following fixation and permeabilization with Foxp3/transcription factor staining buffer set (Affymetrix and eBioscience).

Annexin V staining was performed according to the manufacturer’s instruction after 4-h incubation at 37°C (BioLegend).

Abs used for flow cytometry analysis were: CD8β (eBioH35-17.2), CD4 (GK1.5), KLRG1 (2F1), CD44 (IM7), CD62L (MEL-14), CD11a (M17/4), CD127 (A7R34), IL-21R (eBio4A9), ICOS (7E.17G9), IFN-γ (XMG1.2), Ki67 (SolA15), CD107a (eBio1D4B), and T-bet (eBio4B10) (purchased from Affymetrix and eBioscience). Abs for CXCR5 (L38D7) and PD-1 (RMP1-14) were obtained from BioLegend. Live/Dead Aqua staining (Invitrogen) was systematically performed prior to any flow cytometry analysis. Cell acquisition was performed with an FACSCalibur cytometer (BD Biosciences) with a Cytek upgrade (Cytek Development). Data analysis was accomplished using FlowJo software (Tree Star). After data analysis, computation of different functions (IFN-γ, Ki67, and CD107a) was performed using the SPICE program (provided by M. Roederer, National Institutes of Health, Bethesda, MD). Fluorescence-minus-one controls were performed for all experiments.

BrdU staining was performed according to the manufacturer’s instruction (BD Pharmingen).

For real-time PCR detection of IL-21, IL-7, IL-15, and Il-2, total RNA was extracted from the spleen using TRIzol (Invitrogen). After DNAse I treatment and RNA cleanup with the RNeasy kit (Qiagen), cDNA was prepared using SuperScript II reverse Transcriptase (Invitrogen). The primers used were: IL-21, 5′-TCA TCA TTG ACC TCG TGG CCC-3′ and 5′-ATC GTA CTT CTT CAC TTG CAA TCC C-3′; IL-2, 5′-AGC AGC TGT TGA TGG ACC TA-3′ and 5′-CGC AGA GGT CCA AGT TCA T-3′; IL-15, 5′-ATC AAC ACG TCC TGA CT-3′ and 5′-GTC TGA GAC GAG CTC TT-3′; IL-7, 5′-TCC TCC ACT GAT CCT TGT TC-3′ and 5′-CTT CAA CTT GCG AGC AGC AC-3′; and β-actin 5′-CTC TGG CTC CTA GCA CCA TGA AGA-3′ and 5′-GTA AAA CGC AGC TCA GTA ACA GTC CG-3′. Real-time PCR analysis was performed with a myIQ PCR (Bio-Rad) using published primers (14). Amplification was performed for 40 cycles as follows: 95°C for 45 s, 61.4°C for 50 s, and 72°C for 45 s. Gene expression was normalized using β-actin as endogenous control.

Results are presented as mean ± SD. Comparison between two groups was performed by Student t test throughout the study.

The central role of IL-21 in memory CD8 T cells maintenance in a number of chronic viral infections is well accepted (2–4, 15, 16). To assess if IL-21 was upregulated during microsporidia infection, the message levels of the cytokine was analyzed in the spleens of infected animals. As shown in Fig. 1A, IL-21 mRNA transcript was transiently but significantly increased in the spleen of infected mice at day 14 p.i. (acute phase) and returned to background level as early as day 21 p.i. There was no significant increase in the message levels of other common γ-chain cytokines like IL-2, IL-7, or IL-15 (Supplemental Fig. 1), which have been reported to be important for the development and maintenance of CD8 T cell immunity (17). As Tfh are known to be major producers of IL-21 (18), their induction in response to infection was measured. Increase in both frequency and number of Tfh cells (based on CXCR5, ICOS, and programmed cell death-1 expression) was observed at day 14 p.i. (Fig. 1B–D), which correlates with the peak of IL-21 message. Using intracellular staining, we observed that at day 14 p.i., CXCR5⁺ICOS⁺ CD4 T cells expressed significantly increased levels of IL-21 compared with the same subset from naive animals (Fig. 1E, 1F). In contrast, IL-21 staining was not observed in CD8, γδ, NK, or NKT cells after E. cuniculi infection (data not shown). These data suggest that per-oral E. cuniculi infection leads to upregulated Tfh response, and these cells are an important source of IL-21 during the acute phase of the infection.

Next, we determined the role of IL-21 in the generation of Ag-specific CD8 T cells, which represent a critical component of the protective immunity during microsporidial infection (19). Although the role of IL-21 in the survival of a primary CD8 T cell response has been suggested (20, 21), its contribution to the induction of a CD8 T cell response against a parasite has not been studied. For this purpose, IL-21R KO and WT mice were infected and the CD8 T cell response evaluated at day 14 p.i. As tetramers for E. cuniculi CD8 T cells are not yet available, the Ag-specific response was measured using a previously described surrogate markers strategy (22). First, to validate this approach, analysis of CD44 and CD11a expression by CD8 T cells from WT animals was assessed (Supplemental Fig. 2). As expected, we observed an increase in CD44^hiCD11a^hi CD8 T cells at day 14 p.i., and this subset exhibited strong functionality characteristic (Supplemental Fig. 2A, 2B). In addition, IFN-γ⁺CD107a⁺ CD8 T cells expressed elevated CD11a and CD44 markers (Supplemental Fig. 2C, 2D), confirming that CD44^hiCD11a^hi CD8 T cells represent the Ag-specific CD8 T cell population. As shown in Fig. 2A, a significant increase in the frequency of CD44^hiCD11a^hi CD8 T cells in response to the infection was detected in both WT and IL-21R KO animals at day 14 p.i. However, this response was significantly lower in the absence of IL-21 signaling (Fig. 2A, 2B). Furthermore, the majority of the Ag-specific CD8 T cells expressed KLRG1 but not CD127, a hallmark of short-lived effector cell (SLEC) population (11), as expected during the acute phase of the infection (Fig. 2B). However, yet again, lack of IL-21 signaling led to lower KLRG1 expression among Ag-specific CD8 population (Fig. 2F). However, KLRG1⁺ CD8 T cells from both IL-21R KO and WT exhibit the same phenotypic attribute for Ag specificity (Fig. 2C). Subsequently, to further dissect the defect in induction of CD8 T cell effectors in the absence of IL-21 signaling, we investigated early effectors (EE): CD44^hi, CD62L^lo, KLRG1^lo, CD127^lo, and CD11a^hi) at day 5 p.i. This cell type has been categorized as the first effector population and governs the differentiation of all other effector subsets (23). There was no significant difference in frequency or total numbers of EE between IL-21R KO and WT mice at day 5 p.i. (Fig. 2G). Also, there was no change in frequency of memory precursor effector cells (MPEC) between WT and mutant animals. However, IL-21R KO SLEC were significantly reduced compare with WT as early as day 5 p.i. (Fig. 2F). Therefore, our data suggest that the lack of IL-21 signaling exclusively compromises the development of Ag-specific KLRG1⁺ CD8 response against E. cuniculi, and this defect cannot be attributed to reduced generation of early precursor population.

Because previous studies from our laboratory reported that the majority of polyfunctional CD8 T cells belongs to the KLRG1⁺ CD8 T cell subset (11), this population was subsequently analyzed in both IL-21R KO mice and WT mice. At day 14 p.i., IFN-γ⁺CD107a⁺ IL-21R KO effector KLRG1⁺ CD8 T cells were significantly reduced compared with WT controls (Fig. 3A, 3B). These data strongly suggest that IL-21 play a role in the initiation of the effector CD8 immunity in response to microsporidia infection. A recent study revealed that IL-21 was involved in the induction of T-bet in CD8 T cells, and this transcription factor has been reported to be critical for the development of the Th1 response against different pathogens (24). Interestingly, lack of IL-21 signaling did not hamper the Th1 differentiation of CTL response as KLRG1⁺ CD8 T cells from both WT and KO animals exhibited similar expression of T-bet (Fig. 3C, 3D). To establish the role of CD4 in the development of KLRG1⁺ CD8 T cell response, experiments were performed using CD4 KO mice. Similar to IL-21R KO, CD4-deficient mice exhibited reduced Ag-specific CD8 response at day 14 p.i. (Supplemental Fig. 3A, 3B). Also, KLRG1 expression on the Ag-specific CD8 T cells from these mutant animals was significantly decreased (Supplemental Fig. 3C–E), and they displayed lower bifunctionality in response to the infection (Supplemental Fig. 3F, 3G). These results demonstrate an important role for CD4 T cells in the induction of the effector CD8 T cell response, which is most likely mediated by IL-21.

To establish that polyfunctional attributes of CD8 effectors are dependent on IL-21 signaling, we analyzed their IL-21R expression postmicrosporidial infection. At day 14 p.i., a fraction of KLRG1⁺ CD8 T cells from WT animals exhibited significantly higher staining for IL-21R compared with KLRG1⁻ or total CD8 T cell population (Fig. 4A). Also, IL-21R^hiKLRG1⁺ subset exhibited significantly higher frequency of IFN-γ⁺ (Fig. 4B), CD107a⁺ (Fig. 4C), and Ki67⁺ (Fig. 4D) population than IL-21R^loKLRG1⁺ CD8 T cells. The increased functionality of IL-21R^hiKLRG1⁺ CD8 population was also reflected by the significantly higher mean fluorescence intensity (MFI) for IFN-γ, CD107a, and Ki67. The upregulation of IL-21R by KLRG1⁺ CD8 subset was correlated with their increased polyfunctionality capability (Fig. 4E). Similar to the above findings (with KLRG1⁺ CD8 T cells from IL-21R KO mice), IL-21R^hi and IL-21R^loKLRG1⁺ CD8 T cells displayed comparable frequencies of T-bet⁺–expressing cells, and expression for T-bet as shown by MFI was also comparable (Fig. 4F). Nevertheless, the data obtained from these studies demonstrate that IL-21 signaling is required for the optimal generation of polyfunctional KLRG1⁺ CD8 effector response, but the induction of Th1 effector response as demonstrated by T-bet expression is not affected.

To determine if IL-21 plays a cell intrinsic role in the induction of optimal KLRG1⁺ CD8 response, a well-established mixed bone marrow chimeric approach was used (11). Bone marrow from WT (CD45.1) and IL-21R KO (CD45.2) donors were injected at 1:1 ratio into lethally irradiated WT animals (CD45.1). As shown in Fig. 5A, in mixed bone marrow chimera, frequency of IL21R KO KLRG1⁺ CD8 T cells was significantly lower than WT population. Comparable to the data obtained from IL-21R KO mice, frequency of bifunctional KLRG1⁺ CD8 T cells in IL-21R KO compartment was significantly reduced in the chimeric animals (Fig. 5B). Similar to previously described reports demonstrating the importance IL-21 in long-term CD8 T cell response (2–4), the reduced bifunctionality of CD8 T cells in the IL-21R KO compartment from the chimeric mice was observed at day 30 p.i. (Supplemental Fig. 4). Interestingly, as compared with WT CD8 KLRG1⁺ cells, both frequency and MFI for T-bet was significantly decreased in the cells from the IL-21R KO compartment (Fig. 5C). These findings are different from those observed in IL-21R KO mice (Fig. 3C, 3D) and could be attributed to the existence of a compensatory mechanism in the KO animals. IL-2 and IL-21 are closely related cytokines, and the role of IL-2 in the generation of effector CD8 T cell response is well documented (25). To determine if normal T-bet levels in the CD8 T cells from the IL-21R KO mice can be attributed to increased IL-2 levels in these animals, the cytokine response was evaluated by real-time PCR and intracellular staining. However, no upregulation of the cytokine message in the total splenocyte population or protein expression by CD4 or CD8 T cells was detected in IL-21R KO mice (data not shown). Overall, our data demonstrate a cell-intrinsic role for IL-21 signaling in the generation of bifunctional KLRG1⁺ CD8 effector response during E. cuniculi infection.

Previous studies have reported the role of IL-21 in promoting T cell survival in the absence of Ag (26). Furthermore, in vivo studies with an acute infection model demonstrated that IL-21 signaling was essential for the survival of activated CD8 T cells (21). Therefore, assays were performed to better assess the role of IL-21 signaling in the elicitation of an optimal KLRG1⁺ CD8 response during E. cuniculi infection using the mixed bone marrow approach mentioned above. As expected, terminally differentiated WT KLRG1⁺ CD8 T cells exhibited high levels of Annexin V (Fig. 5A). However, surprisingly, a smaller frequency of early apoptotic (live cells expressing Annexin V) KLRG1⁺ CD8 T cells was detected in IL-21R KO compartment (Fig. 6A). Because IL-21–deficient signaling did not affect survival of KLRG1⁺ CD8 T cells, the proliferative capability of these cells was analyzed via expression of Ki67, a nuclear protein expressed in all active phases of cell division. Previous reports have shown that IL-21 can synergize with either IL-7 or IL-15 to induce proliferation of CD8 T cells in vitro (27, 28). As shown in Fig. 6B, using mixed bone marrow chimera model, frequencies of Ki67⁺KLRG1⁺ CD8 T cell were similarly increased for both WT and IL21R KO at day 14 p.i. These results were confirmed with in vivo BrdU assay (Fig. 6C), and no difference in frequency of BrdU⁺ Ag-specific CD8 T cells between WT and IL-21R KO mice was observed. However, the total number of BrdU⁺ CD44^hiCD11a^hi CD8 T cells from mutant mice was significantly lower than WT (Fig. 6D). Thus, our findings establish that in a microsporidial model, lack of IL-21 signaling led to increased survival of KLRG1⁺ CD8 T cells, and the expansion of the population from the mutant animals was similar to WT animals as demonstrated by Ki67 expression and BrdU incorporation. The fact that lower numbers of KLRG1⁺ CD8 T cells in response to infection was noted as early as day 5 p.i. strongly suggests a role for IL-21 in the generation of KLRG1⁺ CD8 effector response during microsporidial infection.

The role of IL-21 in the maintenance of memory CD8 T cells during chronic infectious disease models (especially in viral infections) is well established. In this regard, several studies conducted with LCMV infection have demonstrated that although mice with defective IL-21 signaling are able to develop an initial CD8 T cell immunity, the memory CD8 T cell response was subsequently impaired, leading to their inability to control the chronic infection (2–4). Using a microsporidial infection model, the evidence presented in this manuscript demonstrates an important role for IL-21 in the development of a robust Ag-specific CD8 T cell effector immunity against E. cuniculi. The induction of IL-21 was observed during the acute phase of infection and led to the generation of optimal levels of CD8 T cell effectors. To the best of our knowledge, this novel report establishes the critical function of IL-21 in the development of CD8 effectors. Using a mixed bone marrow chimeric approach, we established a cell-intrinsic role for IL-21 in the development of KLRG1⁺ CD8 effector response during microsporidial infection. Although a robust functional effector CD8 T cell immunity in the cells from the WT compartment of the mixed bone marrow chimera was noted, the response was severely impaired in the KO effector CD8 T cells. Interestingly, in contrast to CD8 effectors from KO mice, the IL-21R KO cells from the chimeric mice exhibited lower expression of T-bet (an important transcription factor for effector CD8 T cells) as compared with their WT counterparts. This could be explained by compensatory mechanisms in the KO mice, which could, to a certain extent, rescue the CD8 effector response in the absence of functional IL-21. This is also emphasized by the fact that although, CD8 T cell effector response in IL-21R KO mice was subdued compared with WT animals, the differences in the chimeric animals (between WT and IL-21R KO compartment) were more pronounced. The interpretation that can be drawn from these observations is as follows: although the compensatory mechanisms taking place in the KO mice are able to support the CD8 effector immunity, and the cells exhibit normal levels of T-bet, IL-21 signaling is still critical for the development of an optimal effector CD8 response.

Infections, like LCMV or Listeria monocytogenes, induce strong inflammatory cytokines, and the primary CD8 T cells response is relatively independent of CD4 T cells (29). However, CD8 effector response against noninflammatory Ags is reliant on CD4 T cell help, and IL-2, a cytokine primarily produced by CD4 T cells (30), is often important for the generation of this help-dependent primary CD8 T cell response (31). Early CD25 upregulation, which was critically dependent on third cytokine signaling (32), combined with increased IL-2 expression is known to promote effector CD8 T cell differentiation (33, 34). Furthermore, in an infectious model using mixed bone marrow chimeric mice, it was demonstrated that IL-2Rα deficiency impaired the differentiation of effector CD8 T cells, and these cells exhibited reduced T-bet expression and CTL response (34). Also, in a low inflammatory situation, induction of the transcription factor B lymphocyte–induced maturation protein 1 [required for proper acquisition of effector functions by SLEC (35)] was contingent on IL-2 expression (36). In the case of microsporidia infection, which does not induce a strong inflammation, CD4 T cells appear to play a very important role in the control of the pathogen. Depletion of both CD4 and CD8 T cells led to host mortality, whereas the mice treated with anti-CD4 or anti-CD8 alone survived the challenge (12). Interestingly, the role of IL-2 in priming the effector CD8 T cell response appears to be minimal, as this cytokine was not detected either by real-time PCR or intracellular staining of T cells from infected animals (data not shown). In contrast, in the case of microsporidial infection, we observed an increase in CXCR5⁺ICOS⁺ CD4 T in the WT mice at day 14 p.i., and this population produced IL-21 after infection. Based on these findings, we can interpret that one of the important roles of CD4 T cells during microsporidial infection is their ability to secrete IL-21, which is needed for the generation of a robust effector CD8 T cell response. However, the ability of IL-21 to generate this response cannot be generalized given that this cytokine limits the terminal differentiation of effector CD8 T cells in a cancer model, favoring the development of immature effector cells that are more effective in suppressing tumor growth (37). Conversely, several studies have demonstrated the role of IL-21 in the survival, expansion, and/or function of CD8 T cells in general, alone or in combination with IL-2. Recent observations have shown that IL-21 was able to rescue CD8 T cells cultured under IL-2 deprivation conditions. In these studies, blockade of both IL-2 and IL-21 signaling pathway led to reduced CD8 T cell survival (38). Similarly, in another study, IL-21 alone promoted expansion and cytolytic development of OT1 T cells (39). Comparable to these in vitro studies, our data revealed that mice with defective IL-21 signaling displayed lower frequency of Ag-specific and KLRG1⁺ CD8 T cells. Studies conducted in vitro have demonstrated that IL-21 can cause an upregulation of the transcription factor T-bet in CD8 T cells, which promotes their functionality (24). In another study, addition of IL-21 in vitro rapidly upregulated IFN-γ, T-bet, IL-2Rα, and IL-12Rβ2 mRNA synthesis, all genes associated with a Th1 response (40). The data presented in this manuscript demonstrate that although effector CD8 T cells from IL-21R KO mice exhibited normal levels of T-bet, significantly lower expression was observed in the IL-21R KO compartment from the chimeric animals. As mentioned above, the discrepancy in these observations can be attributed to the existence of compensatory mechanism in the KO mice. As it has been recently reported that IL-21 can inhibit IL-2 production by conventional T cells (41), we suspected that in the absence of functional IL-21, upregulation of IL-2 may rescue the CD8 T cell effector response during microsporidial infection. However, this was not the case, as an increase in message for IL-2 was not observed, and CD4 or CD8 T cells from infected animals failed to secrete the protein as determined by flow cytometry, and therefore, IL-2 was not involved in the rescue of KLRG1⁺ CD8 effectors generated in the absence of IL-21 signaling.

Although IL-21R KO KLRG1⁺ CD8 T cells proliferate as well as their WT counterpart, they exhibited higher survival rate. This is somewhat unexpected as KLRG1⁺ CD8 T cells are terminally differentiated and subsequently die after the peak of the response (42). As far as the role of IL-21 on T cell apoptosis is concerned, the situation is unclear and most likely dependent on the nature of stimulus. In one of the studies using a noninfectious model, it was reported that the cytokine can promote T lymphocyte survival by activating the PI3K cascade and inducing Bcl-2 expression (26). In contrast, in an SIV model of infection, IL-21 was reported to drive CD8 T cell to apoptosis by downregulating Bcl-2 (43). Similar to the observations made with SIV, in our model, a lower frequency of Annexin V⁺KLRG1⁺ CD8 T cells was observed in the absence of IL-21 signaling, which could be attributed to proapoptotic properties of the cytokine in this model. Also, as fewer effector CD8 T cells are generated in the KO compartment, the decreased apoptosis may be due to a delay in the contraction phase. The potential role of IL-21 in the contraction of CD8 T cell effector response is an interesting area that needs to be investigated.

Overall, our findings demonstrate an important and previously unidentified role for IL-21 in the development of a polyfunctional effector CD8 T cell response in an infectious disease model. These studies extend the role of IL-21 to the generation of CD8 effectors T cells in addition to its already established importance in the development and maintenance of the memory response (2–4). The data obtained with chimeric animals clearly underline the critical importance of IL-21 in the generation of CD8 T cell response and strongly suggest that, like type I IFN and IL-12, it could be considered a third signal important for the development of effector CD8 T cells against intracellular infection. The findings presented in this manuscript raise important questions related to the alternative mechanisms responsible for CD8 T cell effector development in the IL-21R KO mice. As our studies rule out IL-2 in the elicitation of CD8 effectors in these animals, it is possible that other CD4 subsets like Th1 or Th17 may be able to compensate in the absence of IL-21. Furthermore, how IL-21 plays a role in the generation of KLRG1⁺ population is an interesting question, which will be addressed in future studies conducted in our laboratory. Ongoing studies in our laboratory will provide answers to these important questions. Our observations have significant implications for the HIV-infected population in whom IL-21–producing Tfh populations are major targets (44), and, as a result, depleted cytokine levels could severely compromise the CD8 T cell effectors against this important opportunistic infection.

The authors have no financial conflicts of interest.