CD8+ virtual memory T (TVM) cells rapidly respond to infection via antigen-independent bystander effector functions. While it is recognized that TVM cells arise independently of foreign antigen encounter, the mechanisms governing their development are not fully understood. Here, we identify the Ikaros transcription factor Aiolos as a negative regulator of TVM cell programming. We observe enhanced frequencies and numbers of TVM in the spleen, liver, and blood of unchallenged Aiolos-deficient (Ikzf3-/-) mice and in the lungs 1-day post-infection with influenza A virus (IAV). Furthermore, Ikzf3-/- TVM cells produce elevated IFN-γ and granzyme B in response to cytokine stimulation. Importantly, Aiolos-deficient mice control IAV more rapidly and exhibit reduced morbidity, indicating enhanced TVM cell functionality. Mechanistically, Aiolos represses the expression of the transcription factor Eomes and the IL-15R subunit CD122, known positive regulators of TVM gene program. Collectively, these findings establish Aiolos as a molecular repressor of TVM programming and responses. The mechanism(s) controlling CD8+ virtual memory T cell (TVM) development are still under investigation. Here, using uninfected or IAV-challenged Ikzf3-deficient mice, the authors identify the transcription factor Aiolos as a negative regulator of TVM cell development by repressing Eomes and IL-15/STAT5 signaling.

In contrast to the "helper" activities of most CD4+ T effector subsets, CD4+ cytotoxic T lymphocytes (CD4-CTLs) perform functions normally associated with CD8+ T and NK cells. Specifically, CD4-CTLs secrete cytotoxic molecules and directly target and kill compromised cells in an MHC class II-restricted fashion. The functions of these cells have been described in diverse immunological contexts, including their ability to provide protection during antiviral and antitumor responses, as well as being implicated in autoimmunity. Despite their significance to human health, the complete mechanisms that govern their programming remain unclear. In this article, we identify the Ikaros zinc finger transcription factor Eos (Ikzf4) as a positive regulator of CD4-CTL differentiation during murine immune responses against influenza virus infection. We find that the frequency of Eos+ cells is elevated in lung CD4-CTL populations and that the cytotoxic gene program is compromised in Eos-deficient CD4+ T cells. Consequently, we observe a reduced frequency and number of lung-residing, influenza virus-responsive CD4-CTLs in the absence of Eos. Mechanistically, we determine that this is due, at least in part, to reduced expression of IL-2 and IL-15 cytokine receptor subunits on the surface of Eos-deficient CD4+ T cells, both of which support the CD4-CTL program. Finally, we find that Aiolos, a related Ikaros family member and known CD4-CTL antagonist, represses Eos expression by antagonizing STAT5-dependent activation of the Ikzf4 promoter. Collectively, our findings reveal a mechanism wherein Eos and Aiolos act in opposition to regulate cytotoxic programming of CD4+ T cells.

CD4+ T helper 1 (Th1) cells coordinate adaptive immune responses to intracellular pathogens, including viruses. Key to this function is the ability of Th1 cells to migrate within secondary lymphoid tissues, as well as to sites of inflammation, which relies on signals received through the chemokine receptor CXCR3. CXCR3 expression is driven by the Th1 lineage-defining transcription factor T-bet and the cytokine-responsive STAT family members STAT1 and STAT4. Here, we identify the Ikaros zinc finger (IkZF) transcription factor Aiolos (Ikzf3) as an additional positive regulator of CXCR3 both in vitro and in vivo using a murine model of influenza virus infection. Mechanistically, we found that Aiolos-deficient CD4+ T cells exhibited decreased expression of key components of the IFN-γ/STAT1 signaling pathway, including JAK2 and STAT1. Consequently, Aiolos deficiency resulted in decreased levels of STAT1 tyrosine phosphorylation and reduced STAT1 enrichment at the Cxcr3 promoter. We further found that Aiolos and STAT1 formed a positive feedback loop via reciprocal regulation of each other downstream of IFN-γ signaling. Collectively, our study demonstrates that Aiolos promotes CXCR3 expression on Th1 cells by propagating the IFN-γ/STAT1 cytokine signaling pathway.

CD4 +cytotoxic T lymphocytes, or CD4-CTLs, comprise a CD4 +subset capable of performing functions normally observed in CD8 +T and Natural Killer cells. CD4-CTLs play critical roles in many immune contexts, including protective anti-viral responses to viruses such as SARS-CoV-2 and influenza. Despite their well-documented importance, the complete mechanisms that underlie their formation remain unclear. Here, we define the transcription factor Aiolos as a novel repressor of CD4-CTL differentiation. We find that Aiolos deficiency results in increased CD4-CTL responses in lungs of influenza-infected mice, as assessed by elevated expression of Granzyme B and Perforin, as well as the CTL marker NKG2A/C/E. We further find that Aiolos-deficient CD4-CTLs exhibit increased expression of transcription factors associated with cytotoxic programming, including Eomes and Blimp-1. Mechanistically, we find that Aiolos-deficient cells have a heightened sensitivity to IL-2/STAT5 signaling due to enhanced expression of IL-2 receptor subunits. This translates into increased chromatin accessibility and STAT5 association at regulatory regions of hallmark CD4-CTL genes in the absence of Aiolos. Consistent with these data, in silico analyses demonstrate that STAT5 DNA-binding motifs are significantly enriched at these same regions. Intriguingly, the STAT5 motif partially overlaps with that of the core Aiolos DNA binding motif, suggesting that Aiolos may function to broadly antagonize STAT5 activity throughout the genome. Collectively, this work establishes Aiolos as a novel repressor of cytotoxic programming in CD4 +T cells and highlights its potential as a therapeutic target for enhancing anti-viral immunity. This work was supported by grants from The National Institutes of Health AI134972 and AI127800 to K.J.O, AI156411 to P.L.C. K.A.R. is supported by funding through The Ohio State University College of Medicine Advancing Research in Infection and Immunity Fellowship Program. J.A.T. and S.P. are supported through funding from the Infectious Disease Institute T32 in Host-Microbe Interactions fellowship.

The Ikaros zinc-finger transcription factor Eos has largely been associated with sustaining the immunosuppressive functions of regulatory T cells. Paradoxically, Eos has more recently been implicated in promoting proinflammatory responses in the dysregulated setting of autoimmunity. However, the precise role of Eos in regulating the differentiation and function of effector CD4(+) T cell subsets remains unclear. In this study, we find that Eos is a positive regulator of the differentiation of murine CD4(+) T(H)2 cells, an effector population that has been implicated in both immunity against helminthic parasites and the induction of allergic asthma. Using murine in vitro T(H)2 polarization and an in vivo house dust mite asthma model, we find that Eos(KO) T cells exhibit reduced expression of key T(H)2 transcription factors, effector cytokines, and cytokine receptors. Mechanistically, we find that the IL-2/STAT5 axis and its downstream T(H)2 gene targets are one of the most significantly downregulated pathways in Eos-deficient cells. Consistent with these observations, we find that Eos forms, to our knowledge, a novel complex with and supports the tyrosine phosphorylation of STAT5. Collectively, these data define a regulatory mechanism whereby Eos propagates STAT5 activity to facilitate T(H)2 cell differentiation.

The Ikaros zinc finger transcription factor Eos has largely been associated with chromatin regulation promoting immunosuppressive regulatory T cells. However, Eos’ role in the differentiation and function of pro-inflammatory T cells has remained unclear. Surprisingly, our work reveals that Eos is a positive regulator of CD4 +T helper 2 (T H2) cells—effector T cells that participate in anti-helminthic immunity but are also implicated in inducing allergic asthma. Using in vitro-generated T H2 cells and an in vivo allergic asthma mouse model, we found that Eos-deficient T cells had reduced gene and protein expression of critical T H2 transcription factors (including the lineage-defining transcription factor Gata3), effector cytokines, and differentiation receptors. Among the various T H2-polarizing pathways, the IL-2/STAT5 axis and its downstream T H2 gene targets emerged as one of the most significantly downregulated networks in Eos deficiency. Using in vitro-generated T H2 cells and overexpression of Eos zinc-finger-domain mutants, we discovered that Eos forms a novel complex with and promotes the tyrosine-phosphorylated activation of STAT5. Additionally, we showed that components of the IL-2/STAT5 pathway participate in a feed-forward loop to promote Eos expression in T H2 cells, further supporting Eos’ regulatory connection with the IL-2/STAT5 axis. Together, these data define a novel mechanism whereby Eos mediates IL-2/STAT5 activity to facilitate T H2 differentiation. This work is significant, as its findings reframe our understanding of Eos’ role in T cell differentiation. K.J.O. is supported by a grant from The National Institutes of Health (NIH) AI134972, as well as from The Ohio State University College of Medicine and The Ohio State University Comprehensive Cancer Center. J.A.T. is supported by funding through the Susan Huntington Dean’s Distinguished University Fellowship and the NIH T32 “Interdisciplinary Program in Microbe-Host Biology” pre-doctoral fellowship administered through the OSU Infectious Diseases Institute and OSU Department of Microbial Infection and Immunity. K.M.G. is supported by NIH grant 1R01ES028829-01A1. L.M.C. and K.J.O. were supported in part by the Jeffress Trust Awards Program in Interdisciplinary Research. K.A.R. is supported by funding through The Ohio State University College of Medicine Advancing Research in Infection and Immunity Fellowship Program.

During intracellular infection, T follicular helper (T FH ) and T helper 1 (T H 1) cells promote humoral and cell-mediated responses, respectively. Another subset, CD4-cytotoxic T lymphocytes (CD4-CTLs), eliminate infected cells via functions typically associated with CD8 + T cells. The mechanisms underlying differentiation of these populations are incompletely understood. Here, we identify the transcription factor Aiolos as a reciprocal regulator of T FH and CD4-CTL programming. We find that Aiolos deficiency results in downregulation of key T FH transcription factors, and consequently reduced T FH differentiation and antibody production, during influenza virus infection. Conversely, CD4-CTL programming is elevated, including enhanced Eomes and cytolytic molecule expression. We further demonstrate that Aiolos deficiency allows for enhanced IL-2 sensitivity and increased STAT5 association with CD4-CTL gene targets, including Eomes, effector molecules, and IL2Ra. Thus, our collective findings identify Aiolos as a pivotal regulator of CD4-CTL and T FH programming and highlight its potential as a target for manipulating CD4 + T cell responses.

Chemokine signaling is an integral component of lymphocyte migration, activation, and survival. The chemokine receptor CXCR3 is highly expressed on CD4 +T helper 1 (T H1) cells, which play a critical role in the adaptive immune response against intracellular pathogens. While CXCR3 helps direct T H1 cells to sites of inflammation or infection, it has also been implicated in a multitude of autoimmune diseases and cancers. Thus, understanding the molecular mechanisms that regulate CD4 +T cell migration is imperative for preventing immunopathology. Here, we identify the Ikaros zinc finger (IkZF) transcription factor Aiolos (IkZF3) as a positive regulator of CXCR3 expression in CD4 +T cells in a murine influenza model. Initially, we show decreased numbers of Aiolos-deficient antigen-specific CD4 +T cells in the lung, which correlates with decreased CXCR3 expression. RNA-seq and ATAC-seq analyses of in vitro-generated WT and Ikzf3 −/−T H1 cells revealed decreased Cxcr3 transcript and reduced chromatin accessibility at regulatory regions of Cxcr3 in the absence of Aiolos, respectively. Collectively, these data imply a novel role for Aiolos in positively regulating CXCR3 expression in T H1 cells. Given these findings, we seek to define the functional effects of Aiolos deficiency on T cell migration and the molecular mechanism(s) by which Aiolos directly and/or indirectly regulates migration. Future work is aimed at determining whether Aiolos affects CXCR3 expression through direct association or indirectly via impacts on other factors. K. J. O. is supported by a grant from The National Institutes of Health (NIH) AI134972, as well as from The Ohio State University College of Medicine and The Ohio State University Comprehensive Cancer Center.

Classically, CD4+ T cells have been defined as “helper” type cells, providing aid to other immune cell populations via the secretion of cytokines and direct cell-cell interactions. More recently, a subset of CD4+ T cells with cytotoxic capabilities, termed CD4+ cytotoxic T lymphocytes (CD4-CTLs), have been observed in both mice and humans, performing protective functions in the settings of infection and cancer while conversely contributing to the pathogenesis of autoimmunity. Despite their well-documented importance in several disease contexts, the complete mechanisms that underlie their differentiation and function remain unknown. Here, we identify the Ikaros family member, Aiolos, as a novel regulator of CD4+ CTL differentiation and function. We find that Aiolos deficiency results in increased expression of key CD4+ CTL transcription factors and effector molecules both in vitro and in an in vivo murine model of influenza infection. Mechanistically, we find that Aiolos deficiency results in increased IL-2/STAT5 signaling, supporting a repressive role for Aiolos in CD4+ CTL differentiation via negative regulation of the IL-2/STAT5 pathway. Collectively, this work identifies Aiolos as a novel negative regulator of CD4+ cytotoxic gene programming, and thus may represent a therapeutic target for the treatment of autoimmune diseases and enhanced anti-tumor immunity. Sponsored by a grant from NIAID (NIH-RO1 AI134972) and funds through The Ohio State University College of Medicine

CD4+ central memory T cells (TCM) are key players in recall immune responses and thus are critical to long-term immunity. Despite their importance, much remains to be defined regarding the mechanisms that promote their differentiation and survival. Ikaros zinc finger (IkZF) transcription factors are known regulators of T and B cell differentiation programs. To date, the IkZF factor Eos has been mainly linked to regulatory T cell suppressive functions. Now, we have found that Eos is expressed at elevated levels in CD4+ TCM populations. Comparison between WT and Eos-deficient T cells in a murine model of influenza infection revealed a decrease in the percentage of CD4+ TCM cells in the absence of Eos, suggesting that Eos promotes TCM differentiation and/or survival. Using an established in vitro model of CD4+ TCM-like cell differentiation, we found that loss of Eos correlated with reduced expression of genes encoding key TCM transcription factors and cell surface markers. Further, Eos deficiency resulted in decreased STAT5 activation downstream of IL-7 signaling, a known positive regulator of TCM populations. In WT cells, IL-7 treatment increased Eos expression, suggesting a positive feed-forward relationship exists between Eos expression and IL-7/STAT5 signaling. Overall, our findings demonstrate that Eos is an important regulator of TCM populations. Understanding the mechanisms by which Eos regulates TCM function and survival may therefore be of interest in exploiting TCM protection against re-infection for therapeutic benefit. Supported by a grant from NIAID (NIH- R01 AI134972) and funds through The Ohio State University College of Medicine

The Ikaros zinc finger transcription factor Eos has been commonly implicated in regulatory T cells to promote their immunosuppressive functions. Paradoxically, a new role is emerging for Eos in promoting pro-inflammatory responses of conventional CD4 + T cells in the dysregulated setting of autoimmunity. Even so, the precise role of Eos in regulating the differentiation and function of healthy effector CD4 + T cell subsets remains unclear. Here, we find that Eos is a positive regulator of CD4 + T helper 2 (T H 2) cells—effector T cells implicated in the induction of allergic asthma. Using murine in vitro T H 2 cells and an in vivo house dust mite asthma model, we found that Eos-deficient T cells had reduced expression of key T H 2 transcription factors, effector cytokines, and differentiation receptors. Mechanistically, among various T H 2-polarizing pathways, the IL-2/STAT5 axis and its downstream T H 2 gene targets emerged as one of the most significantly downregulated networks in Eos deficiency. Using in vitro T H 2 cells and overexpression of Eos zinc-finger-domain mutants, we discovered that Eos forms a novel complex with and supports the tyrosine-phosphorylated signaling activity of STAT5. Overall, these data define a novel regulatory mechanism whereby Eos promotes IL-2/STAT5 activity to facilitate T H 2 differentiation.

Effective immunity to influenza virus and other respiratory viruses requires the generation of CD4 + T cell subsets that coordinate multiple aspects of the immune response. These subsets include T follicular helper (T FH ) and T helper 1 (T H 1) cells, which promote humoral and cell-mediated responses, respectively. A third population, CD4 + cytotoxic T lymphocytes (CD4-CTLs) facilitates clearance of infection via mechanisms normally associated with CD8 + T cells. Here, we identify the transcription factor Aiolos as a regulator of T FH and CD4-CTL responses. We demonstrate that Aiolos deficiency compromises T FH differentiation and antibody production during influenza virus infection. Conversely, we find that CD4 + T cells acquire a cytotoxic-like program in the absence of Aiolos, including increased expression of the CTL-associated transcription factors Eomes and Blimp-1. We further show that while Aiolos positively regulates the T FH transcriptional regulators Zfp831, TCF-1 and Bcl-6, it also directly represses expression of IL-2Rα and IL-2/STAT5-driven expression of the cytotoxic gene program. Thus, our findings identify Aiolos as a pivotal regulator of T FH and CD4-CTL differentiation and highlight its potential as a target for manipulating CD4 + T cell humoral and cytotoxic responses.

Abstract CD4+ T follicular helper (TFH) cells are critical for the generation of robust humoral immune responses, as they provide help to B cells to support the generation of both pathogen-neutralizing antibodies and long-lived plasma cell populations. To date, the mechanisms underlying TFH differentiation are incompletely understood. Here, we identify the transcription factor Aiolos as a key regulator of TFH responses. We find that Aiolos expression is increased in TFH cell populations generated during influenza infection and that Aiolos deficiency results in compromised TFH cell differentiation and B cell helper activity. Mechanistically, loss of Aiolos results in diminished expression of TFH genes, including those encoding the key transcription factors Bcl-6, TCF-1, and Tox. Conversely, expression of genes associated with IL-2/STAT5 signaling, a known antagonist of the TFH gene program, were significantly elevated in Aiolos-deficient settings. Consistent with these data, antigen-specific Aiolos-deficient effector CD4+ T cell populations generated in response to influenza infection exhibited significantly elevated IL-2Rα surface expression. Together, our findings suggest that repression of IL-2/STAT5 signaling represents a novel mechanism by which Aiolos regulates the TFH gene program. These findings are important, as they provide critical insight that may ultimately be leveraged for the development of novel TFH-focused immunotherapies and strategies to improve vaccination approaches. Supported by a grant from the NIAID (NIH; R01 AI134972), funds through The Ohio State University College of Medicine, and funds through The Ohio State University College of Medicine Advancing Research in Infection and Immunity Fellowship Program

CCR5 KO kidney transplant (KTx) recipients are extraordinarily high alloantibody producers and develop pathology that mimics human antibody-mediated rejection (AMR). C57BL/6 and CCR5 KO mice (H-2(b)) were transplanted with A/J kidneys (H-2(a)); select cohorts received adoptive cell therapy (ACT) with alloprimed CXCR5(+)CD8(+) T cells (or control cells) on day 5 after KTx. ACT efficacy was evaluated by measuring posttransplant alloantibody, pathology, and allograft survival. Recipients were assessed for the quantity of CXCR5(+)CD8(+) T cells and CD8-mediated cytotoxicity to alloprimed IgG(+) B cells. Alloantibody titer in CCR5 KO recipients was four-fold higher than in C57BL/6 recipients. The proportion of alloprimed CXCR5(+)CD8(+) T cells 7 days after KTx in peripheral blood, lymph node, and spleen was substantially lower in CCR5 KO compared to C57BL/6 recipients. In vivo cytotoxicity towards alloprimed IgG(+) B cells was also reduced six-fold in CCR5 KO recipients. ACT with alloprimed CXCR5(+)CD8(+) T cells (but not alloprimed CXCR5(-)CD8(+) or third-party primed CXCR5(+)CD8(+) T cells) substantially reduced alloantibody titer, ameliorated AMR pathology, and prolonged allograft survival. These results indicate that a deficiency in quantity and function of alloprimed CXCR5(+)CD8(+) T cells contributes to high alloantibody and AMR in CCR5 KO recipient mice, which can be rescued with ACT.

Hepatocyte transplant represents a treatment for metabolic disorders but is limited by immunogenicity. Our prior work identified the critical role of CD8+ T cells, with or without CD4+ T cell help, in mediating hepatocyte rejection. In this study, we evaluated the influence of invariant NKT (iNKT) cells, uniquely abundant in the liver, upon CD8-mediated immune responses in the presence and absence of CD4+ T cells. To investigate this, C57BL/6 (wild-type) and iNKT-deficient Jα18 knockout mice (cohorts CD4 depleted) were transplanted with allogeneic hepatocytes. Recipients were evaluated for alloprimed CD8+ T cell subset composition, allocytotoxicity, and hepatocyte rejection. We found that CD8-mediated allocytotoxicity was significantly decreased in iNKT-deficient recipients and was restored by adoptive transfer of iNKT cells. In the absence of both iNKT cells and CD4+ T cells, CD8-mediated allocytotoxicity and hepatocyte rejection was abrogated. iNKT cells enhance the proportion of a novel subset of multipotent, alloprimed CXCR3+CCR4+CD8+ cytolytic T cells that develop after hepatocyte transplant and are abundant in the liver. Alloprimed CXCR3+CCR4+CD8+ T cells express cytotoxic effector molecules (perforin/granzyme and Fas ligand) and are distinguished from alloprimed CXCR3+CCR4-CD8+ T cells by a higher proportion of cells expressing TNF-α and IFN-γ. Furthermore, alloprimed CXCR3+CCR4+CD8+ T cells mediate higher allocytotoxicity and more rapid allograft rejection. Our data demonstrate the important role of iNKT cells in promoting the development of highly cytotoxic, multipotent CXCR3+CCR4+CD8+ T cells that mediate rapid rejection of allogeneic hepatocytes engrafted in the liver. Targeting iNKT cells may be an efficacious therapy to prevent rejection of intrahepatic cellular transplants.

Hepatocyte transplant (HcTx) is a treatment for metabolic diseases and a bridge to liver transplant but is limited by immunogenicity. Our prior work identified the critical role of CD4-dependent and CD4-independent CD8+ T cells in HcTx rejection. Herein, we evaluated the influence of invariant natural killer T cells (iNKTs), abundant in the liver, upon CD8+ T cell responses in the presence and absence of CD4+ T cells. To investigate this, C57BL/6 (wild-type; WT) or iNKT deficient Jα18 KO (H-2b) received FVB/N (H-2q) HcTx. Cohorts were depleted of CD4+ T cells and/or reconstituted with iNKTs. Recipients were evaluated for cytotoxicity, CD8+ T cell phenotype, and HcTx rejection. By comparing CD4-sufficient WT and Jα18 KO recipients, we found that the presence of iNKTs significantly enhanced in vivo CD8-mediated cytotoxicity (83±3% vs 23±3%; p<0.001) but did not affect survival (10 days vs 14 days; p=ns). However, in CD4-deficient recipients, iNKTs were critical, since without CD4+ T cells and iNKTs, CD8-mediated in vivo cytotoxicity was completely abrogated (31±3% vs 2±1%; p<0.001) and HcTx survival was significantly prolonged (14 days vs 56 days; p<0.001). iNKTs enhanced the quantity of a novel CD8+ T cell subset, CD44+IFN-γ+CXCR3+CCR4+CD8+ T cells, in the liver (2-fold) in all HcTx recipients (p<0.03 for all comparisons). CXCR3+CCR4+CD8+ T cells, when compared to CXCR3+CCR4−CD8+ T cells, express higher IFN-γ (32±3% vs 9±1%; p<0.001) and TNF-α (37±3% vs 6±1%; p<0.001). They also exhibit greater in vivo cytotoxicity (13±1% vs 5±1%; p<0.001) and mediate rapid HcTx rejection in RAG1 KO recipients (5 days vs 16 days; p=0.02). We show that iNKTs enhance the expansion of highly cytotoxic CXCR3+CCR4+CD8+ T cells that mediate rapid rejection of HcTx.

CD4+ T helper cells are capable of differentiating into subsets that regulate distinct aspects of the immune response. The differentiation of these populations is driven by subset-specific cytokine signals and downstream transcription factor networks that are responsible for promoting the unique gene expression programs of each cell type. It is well-established that cytokine signals are propagated by Signal Transducer and Activator of Transcription (STAT) factors. Recently, we demonstrated that STAT3 and Aiolos, a member of the Ikaros Zinc Finger (IkZF) family, form a novel transcriptional complex that directly induces expression of the T follicular helper (TFH) cell lineage defining factor, Bcl-6. Interestingly, we have also found that a second IkZF factor Eos interacts with STAT5 and forms an opposing complex in T helper 1 (TH1) cells that promotes expression of the Bcl-6 antagonist, Blimp-1. However, whether additional IkZF/STAT complexes function downstream of other cytokine signals in these cell populations is unclear. Here, we report that in vitro-generated TH1 and TFH1-like cells similarly express Ikaros concurrent with increased STAT4 activation downstream of IL-12 signaling. Using co-immunoprecipitation analyses, we were also able to detect interactions between Ikaros and STAT4. Furthermore, as with the other IkZF/STAT complexes, we find that interactions between Ikaros and STAT4 were dependent upon the IkZF C-terminal ZF domain, suggesting that this is a conserved regulatory feature of IkZF/STAT complex formation. Current efforts are now aimed at identifying whether mechanistic interplay between the Ikaros/STAT4 complex and the other IkZF/STAT factors may ultimately dictate TH1 or TFH cell fate and function.

Abstract Purpose CCR5 KO kidney transplant (KT) mice produce high titer alloantibody (alloAb) associated with severe antibody-mediated rejection (AMR) that reproduces AMR histology observed in human KT recipients. We previously reported the novel alloAb suppressor activity of alloprimed CXCR5+IFNγ+CD8+ T (Tab-supp) cells following hepatocellular transplant in mice. Here, we investigated the biologic impact of alloprimed CD8+ Tab-supp cells and their capacity to suppress alloAb following adoptive cell transfer (ACT) into CCR5 KO KT mice. Methods CCR5 KO (H-2b) mice were transplanted with allogeneic A/J (H-2a) kidneys, and on postoperative day (POD) 5 underwent ACT of alloprimed CD8+ Tab-supp cells (retrieved from C57BL/6 mice alloprimed with A/J alloantigen). A second cohort received concomitant bilateral native nephrectomy, and allograft survival was monitored with serial serum creatinine (>100 μmol/L defines KT graft loss). Untreated CCR5 KO KT mice served as controls. Results CCR5 KO KT mice developed high alloAb titer compared to wild-type (WT) recipients (5,800±700 vs 1,200±100; p=0.0003). ACT significantly inhibited alloAb production by 5-fold (1,200±200; p<0.0001) and POD14 AMR pathology (peritubular capillary margination and C4d deposition, arteritis) in CCR5 KO KT mice (composite histologic score 3.6±1.5 vs 8.4±0.2 in untreated controls; p=0.006). Following ACT, alloAb remained suppressed beyond POD 30, correlating with enhanced allograft survival (MST 52 vs 14 days; p=0.006). Conclusion ACT of CD8+ Tab-supp cells effectively inhibits alloAb production and AMR not only after allogeneic hepatocellular transplant, but also after vascularized solid organ transplant such as in CCR5 KO KT recipients.