Supraphysiological Levels of IL-2 in Jak3-Deficient Mice Promote Strong Proliferative Responses of Adoptively Transferred Naive CD8+ T Cells

The antigen-independent, strong proliferative responses of naive CD8+ T cells have been well demonstrated in a particular strain of mice lacking IL-2 receptors. This type of proliferation is mainly driven by common gamma-chain (γc) cytokines, such as IL-2, IL-7, and IL-15, present at abnormally high levels in these mice. Similarly, in the present study, we showed that mice lacking Janus kinase 3 (Jak3), a tyrosine kinase crucial for γc cytokine signaling, could induce strong proliferation of adoptively transferred naive CD8+ T cells. This proliferation was also independent of antigenic stimulation, but heavily dependent on IL-2, as evidenced by the failure of proliferation of adoptively transferred IL-2 receptor alpha- and beta-chain-deficient naive CD8+ T cells. Consistent with this, Jak3 –/– mice showed elevated serum levels of IL-2 compared to wild-type mice, and interestingly, IL-2 production was due to high levels of accumulation of activated CD4+ T cells in Jak3 –/– mice along with defective CD4+ T regulatory cells. Collectively, these findings reveal previously unidentified unique immune contexts of Jak3 –/– mice that cause robust IL-2-driven T cell expansion and have a clinical implication for designing a treatment strategy for human patients with loss-of-function genetic mutations of Jak3.


INTRODUCTION
Janus kinase 3 (Jak3) is a member of the Janus family of protein tyrosine kinases and is specifically associated with a cytoplasmic domain of the common receptor gamma-chain (g c ) shared by cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (1,2). When these cytokines bind to their specific receptors containing g c , Jak3 is activated and initiates a cascade of signal transduction through the phosphorylation of signal transducer and activator of transcription (STAT) proteins to induce the expression of various genes and subsequent biological functions (3)(4)(5). Based on these roles of g c cytokines, Jak3-deficient mice have several abnormalities similar to those observed in mice lacking g c cytokines and their corresponding receptors (6)(7)(8). In particular, defects of T and B cells and NK cells typically observed in mice lacking Jak3 can be largely attributed to failures in inducing IL-7 and IL-15 signaling, respectively (9)(10)(11). Likewise, defects in the development of CD4 + T regulatory cells (Tregs) are also apparent in Jak3-deficient mice owing to the inability to induce IL-2 signaling that is involved in the development, maintenance, and function of Tregs (12).
Although all these aforementioned alterations are adequately documented in both mice and humans lacking functional Jak3, the human cases are in general more clearly associated with severe combined immunodeficiency (13)(14)(15). Thus, Jak3deficient mice, unlike patients with loss-of-function mutations of Jak3 (16), are not completely lymphopenic for T cells in particular, but their numbers (for CD4 + but not CD8 + T cells) are almost fully restored over time with age (9,10,17). Moreover, the T cells restored in Jak3-deficient mice have been shown to display an activated T cell phenotype, such as high and low levels of CD44 and CD62L, respectively (9). Although these large numbers of activated T cells are likely to be associated with a defect in Treg-mediated immunosuppression in Jak3-deficient mice (12,18), whether these cells indeed contribute to shaping altered immune contexts in these mice remains to be addressed.
In this study, we focused on this issue by investigating proliferative responses of naive CD4 + and CD8 + T cells adoptively transferred into Jak3-deficient mice. We demonstrated that these mice have a unique IL-2-rich immune environment and thus stimulate a fast and robust form of antigen-independent, but IL-2-dependent, T cell proliferative responses. Our findings highlight the important role of Jak3 in restraining the spontaneous activation of CD4 + T cells and thus lowering the production of in vivo IL-2 below a certain physiological level at which abnormal T cell proliferation is inhibited while Tregs homeostasis is preserved.

Adoptive Transfer
After purification, T cells were labeled with 5 µM of CFSE (Invitrogen) as previously described (20) and injected intravenously (i.v.) into the indicated hosts. For inducing lymphopenia, the indicated mice were treated with 700 rad of whole-body irradiation (1 day before adoptive transfer).

Administration of Antibodies In Vivo
For the in vivo CD4 + T cell depletion experiment, 100 µg of anti-CD4 mAb (GK1.5) was injected intraperitoneally (i.p.) four times every 2 days for 7 days before adoptive cell transfer into Jak3 −/− hosts.

Cytokine ELISA
For detection of in vivo IL-2, sera from the indicated mice were collected and analyzed by a standard protocol using a cytokine sandwich ELISA kit for IL-2 (BD Biosciences) according to the manufacturer's instructions.

Real-Time (RT) PCR
1-2 × 10 6 spleen cells or FACS-purified CD4 + T cells from the indicated mice were used for RNA extraction with NucleoZOL (Macherey-Nagel) and stored at -80°C before the further steps. Isolation of mRNA was done according to the manufacturer's instructions. cDNA was synthesized with the M-MLV reverse transcriptase and oligo dT (TAKARA). Real-time RT-PCR was performed with the TaqMan Gene Expression Master Mix using StepOnePlus Real-Time PCR System with TaqMan probe for Il2 mRNA (Mm00434256_m1; Applied Biosystems).

Statistical Analysis
An unpaired two-tailed Student's t-test was performed to test statistical significance using Prism (GraphPad Software). Differences in mean values were considered statistically significant at a P value of less than 0.05.

Robust Proliferative Responses of Naive T Cells in Jak3 -/-Hosts
Given the functional association of Jak3 for mediating the signal transduction of g c cytokines, it has been previously demonstrated that mice lacking Jak3 (or its binding receptor CD132) have a defect in T cell development, especially the CD8 + T cell population, due to IL-7 signaling failure (9,10,17). To further examine the altered immune contexts of these mice, we adoptively transferred FACS-purified, CFSE-labeled C57BL/6 (B6) naive CD4 + or CD8 + T cells into Jak3 -/mice ( Figure  1A). Notably, the donor CD4 + and CD8 + T cells showed marked proliferation from day 3 and a more potent response on day 7, as evidenced by CFSE dye dilution ( Figure 1B, bottom, and Supplementary Figure 1A). As a control, when compared to > 6 divisions in Jak3 -/mice, the donor cells adoptively transferred into irradiated B6 mice (700 rad) showed only 2-3 rounds of cell divisions ( Figure 1B, top, and Supplementary Figure 1A). These data suggest that the tempo and type of proliferation of T cells observed in Jak3 -/mice are distinctly different from the typically slow rate of lymphopenia-induced homeostatic proliferation (LIP) (22).
To examine whether the aforementioned fast and robust proliferative responses are dependent on abnormal stimulation by undefined antigens that may be present in Jak3 -/hosts, we performed the adoptive transfer experiment using monoclonal T cell receptor (TCR) transgenic CD4 + or CD8 + T cells. Jak3 -/mice were co-transferred with a mixture of either OT-II and SMARTA CD4 + T cells (specific for I-A b -restricted ovalbumin 323-339 and lymphocyte choriomeningitis virus glycoprotein 61-80 peptide, respectively) or 2C and OT-I CD8 + T cells (specific for H-2K b -restricted SIYRYYGL peptide and ovalbumin 257-264 peptide, respectively) ( Figure 1C). All donor cells showed proliferation, although to varying extents depending on their intrinsic self-reactivity-known to be correlated with expression levels of CD5, a negative regulator of TCR signaling, i.e., SMARTA > OT-II, and OT-I > 2C (23-25)-with SMARTA and OT-I donors demonstrating a greater proliferation than OT-II and 2C donors, respectively ( Figure 1D and Supplementary Figure 1B). These findings suggest that the strong T cell proliferative response observed in Jak3 -/hosts is antigen-independent but dependent on tonic TCR contacts with self-ligands.

Role of gc Cytokines in Naive T Cell Expansion in Jak3 -/-Hosts
Given the antigen-independent strong T cell proliferation in Jak3 -/hosts, we next examined the involvement of certain g c cytokines, especially IL-2 and IL-15, both of which have been shown to induce antigen-independent robust T cell proliferation (19). To this end, a mixture of FACS-purified, CFSE-labeled Cd122 -/-(IL-2 receptor beta-chain shared for IL-2 and IL-15) and wild-type (WT) naive 2C CD8 + T cells was cotransferred into Jak3 -/or control Jak3 +/hosts (Figure 2A). At day 7 after transfer, in control Jak3 +/hosts, both WT and Cd122 -/-2C donor cells remained undivided ( Figure 2B, top left two). However, in Jak3 -/hosts, while WT 2C donor cells showed strong proliferative responses, Cd122 -/-2C donor cells failed to proliferate ( Figure 2B, top right two, and Supplementary Figure 2). When this adoptive transfer experiment was repeated in the same but irradiated (700 rad) hosts, both WT and Cd122 -/-2C donors showed similar degrees of slow LIP in control Jak3 +/hosts ( Figure 2B, bottom left two), which is known to be IL-7 (and self)dependent (26)(27)(28)(29). However, in irradiated Jak3 -/hosts, WT 2C donor cells showed markedly faster and greater proliferative responses than Cd122 -/-2C donor cells showing only slow rate of LIP ( Figure 2B, bottom right two, and Supplementary  Figure 2), suggesting a role of CD122-dependent cytokines other than IL-7.
The implication from the above data with Cd122 -/cells as a donor is that IL-2 and/or IL-15 are crucial requirements for driving strong T cell proliferation in Jak3 -/hosts, as the CD122 is a receptor subunit shared for both IL-2 and IL-15. To further examine whether both cytokines are equally required, we performed the same aforementioned adoptive transfer experiments with CD25 (IL-2 receptor alpha-chain)-deficient 2C cells as the donor. For this purpose, a mixture of FACS-purified, CFSE-labeled Cd25 -/and WT naive 2C CD8 + T cells was cotransferred into Jak3 -/or control Jak3 +/hosts ( Figure 3A). WT 2C donor cells showed robust proliferation at day 7 in Jak3 -/hosts; however, Cd25 -/-2C donor cells remained undivided ( Figure 3B, top right two, and Supplementary Figure 3).
Similarly, for experiments with the same but irradiated Jak3 -/hosts, WT 2C donor cells showed markedly faster and more robust proliferation than Cd25 -/-2C counterparts ( Figure 3B, bottom right two, and Supplementary Figure 3). The latter Cd25 -/-2C cells showed only slow rate of LIP comparable to that of both WT and Cd25 -/-2C donor cells in irradiated Jak3 +/hosts, indicating similar responses of these cells to IL-7 under this lymphopenic condition ( Figure 3B, bottom left two). Together, these findings strongly suggest that the antigen-independent robust proliferation of naive CD8 + T cells in Jak3 -/hosts is heavily dependent on the direct engagement of both receptors CD25 and CD122 that are specific for only IL-2. Role of CD4 + T Cells in High IL-2 Production in Jak3 -/-Hosts Based on the aforementioned strict dependency on IL-2, we tested the in vivo levels of IL-2 in Jak3 -/mice. For this purpose, serum samples were collected from WT and Jak3 -/mice, which were then analyzed for IL-2 detection using ELISA. As shown in Figure 4A, Jak3 -/mice showed high serum levels of IL-2 compared to their WT counterparts. Consistent with this finding, Il2 mRNA expression levels were also markedly more enhanced in Jak3 -/spleen cells than in their WT counterparts ( Figure 4B). Il15 mRNA levels were also tested and were no increase (but rather decreased) in Jak3 -/cells compared to WT counterparts (Supplementary Figure 4A), suggesting a negligible role of IL-15.
The direct requirement of IL-2 was also confirmed by an in vivo blocking experiment, in which Jak3 -/hosts were administered with a monoclonal antibody to either IL-2 or IL-2 receptor beta-chain (clones JES6.1 and TM-b1, respectively; Supplementary Figure 4B); the results clearly showed that the JES6.1 and TM-b1 injection (relative to PBS control) are both suppressive to a similar extent for the expansion of OT-I donor cells adoptively transferred into Jak3 -/hosts. These data, together with the aforementioned in vivo data with 2C donor cells lacking IL-2 receptors (i.e., CD25 and CD122) (Figures 2,  3), further support our conclusion that strong T cell proliferative responses are driven exclusively by higher concentrations of in vivo IL-2 present in Jak3 -/mice.
Given the rich supply of in vivo IL-2 in Jak3 -/mice, our prior data that, despite similar degree of cell divisions, WT 2C cells exhibited~3-5 times lower expansion in irradiated Jak3 -/hosts than in non-irradiated counterparts (Figures 2, 3 and Supplementary Figures 2, 3) suggest a radiosensitive nature of IL-2-producing cells. With respect to the radiosensitive source of producing such high levels of IL-2 in vivo, the role of Jak3 -/-CD4 + T cells was considered of particular importance, as these cells exhibited highly activated phenotypes (i.e., CD44 hi CD62L lo ) and were shown to gradually accumulate in sufficient numberscomparable or even higher than those of WT controls-in Jak3 -/mice over time with age (9). In support of this, Il2 mRNA expression levels and in vivo IL-2 productions were higher in Jak3 -/-CD4 + T cells than in their WT counterparts (Figures 4C,  D). To further confirm the possible role of Jak3 -/-CD4 + T cells as a major source of high levels of in vivo IL-2, Jak3 -/mice were intraperitoneally administered a monoclonal antibody against CD4 for depleting host CD4 + T cell populations (clone GK1.5) for 7 days prior to starting adoptive transfer experiments ( Figure  4E, top). The antibody-treated Jak3 -/mice were adoptively transferred with FACS-purified, CFSE-labeled OT-I naive CD8 + T cells, and 5 days later, the proliferation of these cells was analyzed. Proliferation of OT-I donor cells was substantially reduced in GK1.5 mAb-treated Jak3 -/hosts compared to control Ab-treated Jak3 -/hosts ( Figure 4E, bottom, and Supplementary Figure 4C), suggesting a role for Jak3 -/-CD4 + T cells as an in vivo IL-2 resource. Based on this role of in vivo IL-2, which is produced by activated CD4 + T cells in Jak3 -/mice, we tested the effect of CD4 + Tregs, as this inhibitory CD4 + population is absent in Jak3 -/mice (12,18). To this end, FACS-purified, CFSE-labeled OT-I naive CD8 + T cells were adoptively transferred with or without FACS-purified CD4 + Tregs (from Foxp3-GFP mice) into Jak3 -/hosts ( Figure 4F, top). Proliferative responses of OT-I donor cells were markedly inhibited by the co-injection of Tregs compared to control non-injected Jak3 -/hosts ( Figure 4F, bottom, and Supplementary Figure 4D), and this finding was consistent with the prior notion that high level of CD25 expression on Tregs could deprive IL-2 from effector T cells and inhibit their proliferation by predominantly consuming IL-2 in vivo (30)(31)(32).
Collectively, these data indicate that Jak3 -/mice have a unique immune environment, where large numbers of activated CD4 + T cells (and conversely a scarcity of CD4 + Tregs) result in abnormally high levels of IL-2 production, which can induce the strong proliferation of adoptively transferred naive T cells in an antigen-independent manner.

DISCUSSION
Jak3 -/mice have been demonstrated to experience profound immunodeficiency, with developmental defects in various immune cell types, including T cells (both ab and gd T cells), B cells, and NK cells (9)(10)(11). These defects have been largely attributed to defective downstream signaling of Jak3-associated g c cytokines, particularly IL-7 and IL-15 (6)(7)(8). In addition, defective IL-2 signaling of Jak3-deficient mice leads to Treg deficiency, which is consistent with mice lacking IL-2 or its receptor components (i.e., CD25, CD122, and CD132) (12,33,34). Although the latter is associated with the gradual accumulation of activated CD4 + T cell populations in Jak3 -/mice (9, 10, 17), its physiological impacts remain unclear. Here, we demonstrated a previously unidentified phenotype of Jak3deficient mice by showing that Jak3 -/-CD4 + T cells produced extremely high amounts of IL-2 in vivo, and consequently, adoptively transferred naive T cells could undergo a rapid and intense form of antigen-independent proliferation. Notably, this type of proliferation-even though antigen-independent-was still influenced by the intrinsic TCR affinity/avidity for selfligands. Such self-dependency was in close agreement with previous findings demonstrating strong IL-2-driven T cell proliferation both in vitro and in vivo (19,20). Therefore, our findings reveal a unique immune context, i.e., supraphysiological levels of in vivo IL-2, of Jak3-deficient mice and further strengthen the prevailing notion that even a single g c cytokine (IL-2) plays a crucial role in promoting massive bystander expansions of naive T cell populations.
The strong proliferation of adoptively transferred naive CD8 + T cells observed in Jak3 -/mice closely resembles those observed in mice lacking CD122 (IL-2 receptor beta, IL-2Rb) and CD132 (common g c receptor) in terms of their tempo and robustness as well as strict dependency on two stimuli, namely g c cytokines and TCR contacts with self-ligands (19,35). However, the difference was the type of cytokines involved; while IL-2 is a major driver in Jak3 -/mice, mixtures of 2-3 g c cytokines-either IL-2 and IL-15 or IL-7, IL-2, and IL-15-act as stimulators in Cd122 -/and Cd132 -/mice, respectively. Moreover, the reason for such enhanced levels of these cytokines in Cd122 -/and Cd132 -/mice was largely accounted for by the lack of the receptors involved in specific binding to either IL-2 and IL-15 (for Cd122 -/mice) or IL-7, IL-2, and IL-15 (for Cd132 -/mice), thereby leading to in vivo accumulation of cytokines that would otherwise be consumed (19,35). In this regard, high levels of in vivo IL-2 in Jak3 -/mice was odd at first glance, since these mice have a defect not in the IL-2 receptors but in the downstream signaling. However, based on our data showing the reduced T cell expansion caused by CD4 + T cell depletion (using GK1.5 mAb) and co-transfer with Tregs, we propose that CD4 + T cells in Jak3 -/mice are overtly activated-presumably by their uncontrolled reactivity to self-antigens-due to the lack of Tregs, which in turn produces IL-2 markedly beyond a physiological level that is sufficient for stimulating intense proliferative responses of adoptively transferred naive CD8 + T cells. Whether prior Tregs reconstitution can restore abnormal immune contexts (i.e., high levels of in vivo IL-2) of Jak3-deficient mice will thus be interesting to address in future studies.
Despite a few cases of Jak3 mutations in humans, whether the phenomenon observed in Jak3 -/mice is similarly reproduced in clinical patients lacking functional Jak3 remains elusive. In this regard, clinical cases, albeit a few, showing that activated/ memory T cells have been associated with Jak3 mutations support this possibility (36,37). Nevertheless, the majority of patients with defective Jak3 are associated with severe combined immunodeficiency and thus compounding effects due to increased susceptibility to infection cannot be easily excluded (13)(14)(15)(16)38). In summary, our results further extend our understanding of the physiological impact of Jak3 deficiency, which can cause the uncontrolled activation and accumulation of self-reactive CD4 + T cells and accordingly abnormally high levels of in vivo IL-2 production. These findings also have an implication for rationale treatment strategies for patients with a loss-offunction Jak3 mutation-especially those concerning graftversus-host reaction from transplacentally acquired maternal or donor allogeneic T cells after bone marrow transfusions.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Experimental and Ethic Committee of the Chonnam National University.

AUTHOR CONTRIBUTIONS
G-WL performed all major experiments. G-WL and J-HC designed experiments and analyzed and interpreted the data. S-WL, JK, and Y-JJ helped some experiments, and C-HY and H-OK contributed to this study with valuable discussion and comments. G-WL and J-HC wrote the manuscript. All authors contributed to the article and approved the submitted version.