Absence Of T-bet Research Articles

MicroRNA-142 regulates gut associated lymphoid tissues and group 3 innate lymphoid cells

The transcriptomic signatures that shape responses of innate lymphoid cells (ILCs) have been well characterised, however post-transcriptional mechanisms which regulate their development and activity remain poorly understood. We demonstrate that ILC groups of the intestinal lamina propria express mature forms of microRNA-142 (miR-142), an evolutionarily conserved microRNA family with several non-redundant regulatory roles within the immune system. Germline Mir142 deletion alters intestinal ILC compositions, resulting in the absence of T-bet+ populations and significant defects in the cellularity and phenotypes of ILC3 subsets including CCR6+ LTi-like ILC3s. These effects were associated with decreased pathology in an innate-immune cell driven model of colitis. Furthermore, Mir142−/− mice demonstrate defective development of gut-associated lymphoid tissues, including a complete absence of mature Peyer’s patches. Conditional deletion of Mir142 in ILC3s (RorcΔMir142) supported cell-intrinsic roles for these microRNAs in establishing or maintaining cellularity and functions of LTi-like ILC3s in intestinal associated tissues. RNAseq analysis revealed several target genes and biological pathways potentially regulated by miR-142 microRNAs in these cells. Finally, lack of Mir142 in ILC3 led to elevated IL-17A production. These data broaden our understanding of immune system roles of miR-142 microRNAs, identifying these molecules as critical post-transcriptional regulators of ILC3s and intestinal mucosal immunity.

Human CD4 T Cells From Thymus and Cord Blood Are Convertible Into CD8 T Cells by IL-4.

Commitment to the CD4+ or CD8+ T cell lineages is linked to the acquisition of a functional program broadly defined by helper and cytotoxic properties, respectively. The mechanisms underlying these processes in the human thymus remain largely unclear. Moreover, recent thymic emigrants are thought to have some degree of plasticity, which may be important for the shaping of the immune system and adjustment to specific peripheral needs. We show here that IL-4 induces proliferation-independent de novo synthesis of CD8αβ in human CD4 single-positive (SP) thymocytes, generating a stable CD8SP population that features a diverse TCRαβ repertoire, CD4 expression shut-down and ThPOK downregulation. IL-4 also promotes an innate-like program in both CD4SP and CD8SP thymocytes, characterized by Eomes upregulation in the absence of T-bet, in line with its recognized role in the generation of thymic innate-like CD8+ T cells. The clinical relevance of these findings is further supported by the profile of IL-4 production and IL-4 receptor expression that we identified in the human thymus. Importantly, human cord blood CD4+ T cells preserve the ability to generate Eomes+ CD8+ T cells in the presence of IL-4, with implications in neonatal immunity. Our results support a role for IL-4 in the dynamic regulation of human thymocyte plasticity and identify novel strategies to modulate immune responses.

Effective clearance of Chlamydia from the female reproductive tract in the absence of T-bet+ Th1 cells

Abstract Protective immune responses to Chlamydia infection within the female reproductive tract (FRT) are incompletely understood. MHC class II-restricted CD4 Th1 responses are thought to be vital for bacterial clearance, due to their capacity to secrete IFN-γ, but Th1 responses as defined by the master transcription factor T-bet had not yet been fully interrogated in primary infection. Here, we investigated T-bet and IFN-γ involvement in primary infection clearance. We found that IFN-γ producing CD4+ T cells from the FRT exhibited surprisingly low levels of T-bet throughout primary infection, indicating low involvement of classical T-bet+ Th1 cells. Mice deficient for T-bet either globally or specifically in CD4+ cells both competently cleared infection along wild-type kinetics. T-bet-deficient mice also showed significant skewing towards Th17 responses, indicating potential compensation pathways through alternate Th fates. On the other hand, IFN-γ-, and IFN-γR-deficient mice resolved much of FRT infection, but experienced systemic dissemination and 100% mortality with an average median survival of approximately 4 weeks. Together, this indicates that while IFN-γ is important to protect mice from death, classical T-bet+ Th1 cells are ultimately not required for primary clearance. Studies are underway to examine which alternative CD4 T cell mechanisms contribute to bacterial clearance within the FRT. Supported by grants from NIH (RO1 A1103422, T32 AI060555)

T-bet+ B cells promote adipose tissue inflammation and metabolic disorder during obesity.

Abstract Obesity is a public health crisis affecting a large and ever-growing portion of the global population. Obesity is associated with an increased risk of death and comorbidities, resulting in lower quality of life for obese patients and increased economic costs on healthcare systems. Obesity is linked with a low-grade and chronic inflammation in the adipose tissue, which further exacerbates metabolic dysfunctions. Understanding the mechanisms and immune cell types promoting this inflammation would allow for the development of new therapies to restore immune regulation in obese patients. Our lab has identified a subset of B lymphocytes which is expanded in the adipose tissue of humans and mice during obesity. This subset is characterized by the co-expression of T-bet and CD11c, production of IgG2c, and its expansion is dependent on the presence of iNKT cells. In vivo studies with mice fed a high fat diet reveal that these Tbet+ B cells promote inflammation. Serum transfer experiments and flow cytometry analysis revealed that this effect is mediated through the production of CXCL10 and antibodies. The absence of T-bet+ B cells does not appear to affect adipose CD4+ T cells or NK cells, but it reduces macrophage polarization towards a more proinflammatory phenotype. Transgenic mice engineered to lack T-bet+ B cells not only have reduced adipose tissue inflammation, but present improved metabolic function as well, gaining less weight on a high-fat diet than littermate controls and presenting improved glucose tolerance. Based on these results, T-bet+ B cells represent an interesting potential target to reduce inflammation in obese patients and improve their metabolic functions.

CD11c+ T-bet+ B Cells Require IL-21 and IFN-γ from Type 1 T Follicular Helper Cells and Intrinsic Bcl-6 Expression but Develop Normally in the Absence of T-bet.

CD11c+ T-bet+ B cells generated during ehrlichial infection require CD4+ T cell help and IL-21 signaling for their development, but the exact T cell subset required had not been known. In this study, we show in a mouse model of Ehrlichia muris that type 1 T follicular helper (TFH1) cells provide help to CD11c+ T-bet+ B cells via the dual secretion of IL-21 and IFN-γ in a CD40/CD40L-dependent manner. TFH1 cell help was delivered in two phases: IFN-γ signals were provided early in infection, whereas CD40/CD40L help was provided late in infection. In contrast to T-bet+ T cells, T-bet+ B cells did not develop in the absence of B cell-intrinsic Bcl-6 but were generated in the absence of T-bet. T-bet-deficient memory B cells were largely indistinguishable from their wild-type counterparts, although they no longer underwent switching to IgG2c. These data suggest that a primary function of T-bet in B cells during ehrlichial infection is to promote appropriate class switching, not lineage specification. Thus, CD11c+ memory B cells develop normally without T-bet but require Bcl-6 and specialized help from dual cytokine-producing TFH1 cells.

Effective clearance of Chlamydia from the female reproductive tract in the absence of T-bet+ Th1 cells

Abstract Protective immune responses to Chlamydia infection within the female reproductive tract (FRT) are incompletely understood. MHC class II-restricted CD4 Th1 responses are thought to be vital for bacterial clearance, due to their capacity to secrete IFN-γ. However, other studies suggest protective or pathogenic roles for additional CD4 T helper subsets. We used a variety of gene-deficient mice to re-examine bacterial clearance following primary genital infection of C57BL/6 mice with Chlamydia muridarum. As seen previously, IFN-γ-, and IFN-γR-deficient mice resolved much of FRT infection, but experienced systemic dissemination and 100% mortality with an average median survival of approximately 4 weeks. Mice lacking the Th1 subset defining transcription factor T-bet or the Th2 transcription factor STAT6 both effectively cleared infection from the reproductive tract, suggesting that neither Th1 nor Th2 cells are essential for local bacterial control. In contrast, RORγt mutant mice selectively deficient in Th17 responses exhibited a week delay in clearance compared to wild-type mice. Th17 subset markers were not observed to be upregulated in T-bet−/− mice and markers of Th1 subsets were minimally upregulated in RORγt mutant mice, indicating that compensation between these subsets does not explain bacterial clearance. In conclusion, timely local clearance of Chlamydia muridarum from the reproductive tract requires Th17 CD4 T cells, but will ultimately clear independent of classical T-bet+ Th1 and RORγt+ Th17 cells individually. Studies are underway to examine which alternative CD4 T cell mechanisms contribute to bacterial clearance within the FRT.

Transcription Factor T-bet in B Cells Modulates Germinal Center Polarization and Antibody Affinity Maturation in Response to Malaria

Despite the key role that antibodies play in protection, the cellular processes mediating the acquisition of humoral immunity against malaria are not fully understood. Using an infection model of severe malaria, we find that germinal center (GC) B cells upregulate the transcription factor T-bet during infection. Molecular and cellular analyses reveal that T-bet in B cells is required not only for IgG2c switching but also favors commitment of B cells to the dark zone of theGC. T-bet was found to regulate the expression of Rgs13 and CXCR3, both of which contribute tothe impaired GC polarization observed in the absence of T-bet, resulting in reduced IghV gene mutations and lower antibody avidity. These results demonstrate that T-bet modulates GC dynamics, thereby promoting the differentiation of B cells with increased affinity for antigen.

Tumoral BRD4 expression in lymph node-negative breast cancer: association with T-bet+ tumor-infiltrating lymphocytes and disease-free survival

BackgroundWe previously observed that T-bet+ tumor-infiltrating T lymphocytes (T-bet+ TILs) in primary breast tumors were associated with adverse clinicopathological features, yet favorable clinical outcome. We identified BRD4 (Bromodomain-Containing Protein 4), a member of the Bromodomain and Extra Terminal domain (BET) family, as a gene that distinguished T-bet+/high and T-bet−/low tumors. In clinical studies, BET inhibitors have been shown to suppress inflammation in various cancers, suggesting a potential link between BRD4 and immune infiltration in cancer. Hence, we examined the BRD4 expression and clinicopathological features of breast cancer.MethodsThe cohort consisted of a prospectively ascertained consecutive series of women with axillary node-negative breast cancer with long follow-up. Gene expression microarray data were used to detect mRNAs differentially expressed between T-bet+/high (n = 6) and T-bet−/low (n = 41) tumors. Tissue microarrays (TMAs) constructed from tumors of 612 women were used to quantify expression of BRD4 by immunohistochemistry, which was analyzed for its association with T-bet+ TILs, Jagged1, clinicopathological features, and disease-free survival.ResultsMicroarray analysis indicated that BRD4 mRNA expression was up to 44-fold higher in T-bet+/high tumors compared to T-bet−/low tumors (p = 5.38E-05). Immunohistochemical expression of BRD4 in cancer cells was also shown to be associated with T-bet+ TILs (p = 0.0415) as well as with Jagged1 mRNA and protein expression (p = 0.0171, 0.0010 respectively). BRD4 expression correlated with larger tumor size (p = 0.0049), pre-menopausal status (p = 0.0018), and high Ki-67 proliferative index (p = 0.0009). Women with high tumoral BRD4 expression in the absence of T-bet+ TILs exhibited a significantly poorer outcome (log rank test p = 0.0165) relative to other subgroups.ConclusionsThe association of BRD4 expression with T-bet+ TILs, and T-bet+ TIL-dependent disease-free survival suggests a potential link between BRD4-mediated tumor development and tumor immune surveillance, possibly through BRD4’s regulation of Jagged1 signaling pathways. Further understanding BRD4’s role in different immune contexts may help to identify an appropriate subset of breast cancer patients who may benefit from BET inhibitors without the risk of diminishing the anti-tumoral immune activity.

The Transcription Factor T-bet Limits Amplification of Type I IFN Transcriptome and Circuitry in T Helper 1 Cells

Host defense requires the specification of CD4+ helper T (Th) cells into distinct fates, including Th1cells that preferentially produce interferon-γ (IFN-γ). IFN-γ, a member of a large family of anti-pathogenic and anti-tumor IFNs, induces T-bet, a lineage-defining transcription factor for Th1 cells, which in turn supports IFN-γ production in a feed-forward manner. Herein, we show that a cell-intrinsic role of T-bet influences how Tcells perceive their secreted product in the environment. In the absence of T-bet, IFN-γ aberrantly induced atype I IFN transcriptomic program. T-bet preferentially repressed genes and pathways ordinarily activated by type I IFNs to ensure that its transcriptional response did not evoke an aberrant amplification of type I IFN signaling circuitry, otherwise triggered by its own product. Thus, in addition to promoting Th1 effector commitment, T-bet acts as a repressor in differentiated Th1 cells to prevent abberant autocrine type I IFN and downstream signaling.

Novel benzoxazole derivatives DCPAB and HPAB attenuate Th1 cell-mediated inflammation through T-bet suppression

Interferon-γ (IFN-γ), a critical inflammatory cytokine, is primarily produced by T helper 1 (Th1) cells and accelerates the pathogenesis of inflammatory colitis. Pharmacological suppression of IFN-γ production attenuates dysregulated inflammatory responses and may be beneficial for treating inflammatory disease. In this study, we aimed to discover potent anti-inflammatory compounds that suppress IFN-γ production and found that the novel benzoxazole derivatives, 2-((3,4-dichlorophenyl) amino) benzo[d]xazol-5-ol (DCPAB) and 2-((3,4-hydroxyphenyl) amino) benzo[d]xazol-5-ol (HPAB), suppressed IFN-γ production by T cells. Treatment of CD4+ T cells with DCPAB and HPAB selectively inhibited Th1 cell development, and DCPAB more potently suppressed IFN-γ than HPAB did. Interestingly, DCPAB and HPAB significantly suppressed the expression of T-box containing protein expressed in T cells (T-bet) that activates IFN-γ gene transcription. DCPAB additionally suppressed transcriptional activity of T-bet on IFN-γ gene promoter, whereas HPAB had no effect on T-bet activity. IFN-γ suppressive activity of DCPAB and HPAB was impaired in the absence of T-bet but was retrieved by the restoration of T-bet in T-bet-deficient T cells. Furthermore, DCPAB and HPAB attenuated inflammatory colitis development that was induced by CD4+ T cells in vivo. We suggest that the novel benzoxazole derivatives, DCPAB and HPAB, may have therapeutic effects on inflammatory colitis.

Natural Killer Cells Induce the Formation of Neutrophil Extracellular Traps (NETs) in Venous Thrombosis

Thrombosis is considered to be a pathological deviation of physiologic hemostasis involving similar mechanisms. Interestingly, recent work demonstrates that innate immune cells promote venous thrombosis. Innate immune cells were shown to collaborate to induce the activation of the coagulation cascade and platelets. In particular, neutrophils contribute to venous thrombosis through the release of neutrophil extracellular traps (NETs). However, the mechanism triggering the formation of NETs during venous thrombosis remain unknown. Of interest, a study showed that IFNγ induced the formation of NETs. Thus, we investigated the role of IFNγ-producing cells in the development of thrombosis.We used mice lacking IFNγ, Tbet (the transcription factor regulating the expression of IFNγ) or wild type mice. Venous thrombosis was induced using the flow restriction model in the inferior vena cava , as has been previously published. In Tbet-/-, IFNγ-/- and WT mice, we show that the absence of Tbet or IFNγ decreases the formation of thrombi after venous thrombosis induction, suggesting that the Tbet+/IFNγ producing cells are required for the early development of venous thrombosis. Comparing the composition of the thrombi from Tbet-/-, IFNγ-/- and WT mice, we show that, in all mice, neutrophils are the main cellular component of thrombi followed by monocytes; however, the number of neutrophil extracellular traps (NETs) formed during thrombosis is significantly lower in Tbet-/- and IFNγ-/- mice. Furthermore, NET formation is also decreased in WT mice specifically depleted of IFNγ and increases in Tbet-/- and IFNγ-/- mice injected with recombinant IFNγ. In vitro, we show that stimulation of WT murine neutrophils with recombinant IFNγ triggers the formation of NETs demonstrating that Tbet and IFNγ are crucial for NET formation by neutrophils.Natural killer (NK) cells are the main producers of IFNγ . Thus, we investigated the role of NK cells in venous thrombosis induced by flow restriction. NK cells were specifically depleted with an antibody during the development of venous thrombosis. The absence of NK cells results in smaller thrombi suggesting that NK cells are required for early thrombus development. Additionally, depletion in NK cells results in decreased in-situ IFNγ production and decreased NET formation. To directly link NK cells to the formation of NETs, WT neutrophils were co-cultured with Tbet-/- and IFNγ-/- NK cells. We show that WT neutrophils release less NETs when cultured with Tbet-/- and IFNγ-/- NK cells as compared to WT NK cells. These data suggest that NK cells trigger the formation of NETs by neutrophils through the production of IFNγ.Hence, we demonstrate that, in a partial flow restriction model of venous thrombosis, Tbet and IFNγ are crucial for thrombus development by promoting the formation of NETs by neutrophils and that NK cells are key effector cells in this process. DisclosuresBlostein:boehringer-ingelheim: Research Funding.

T-bet inhibits innate lymphoid cell–mediated eosinophilic airway inflammation by suppressing IL-9 production

Innate lymphoid cells (ILCs) are emerging subsets of immune cells that produce large amounts of cytokines upon cytokine and/or alarmin stimulation. Recent studies have shown that T-bet plays pivotal roles in the development of ILC3s and type 1 ILCs; however, the roles of T-bet in lung type 2 innate lymphoid cells (ILC2s) remain unknown. We sought to determine the role of T-bet in ILC2-mediated airway inflammation. The expression of T-bet in lung ILCs (defined as Thy1.2+ Lin- cells) was examined. The roles of T-bet in the development of lung ILC2s and airway inflammation induced by IL-33 administration were examined by using T-bet-deficient (T-bet-/-) mice. Gene expression profiles of T-bet-/- lung ILCs were analyzed by RNA sequencing. T-bet was expressed in lung ILC2s (defined as Thy1.2+ Lin- cells expressing ST2 or CD25) and IFN-γ enhanced its expression. Although the development of lung ILC2s at steady-state conditions was normal in T-bet-/- mice, IL-33-induced accumulation of lung ILC2s and eosinophilic airway inflammation were exacerbated in T-bet-/- mice. The exacerbated accumulation of ILC2s and eosinophilic airway inflammation by the absence of T-bet were evident even in a RAG2-/- background, suggesting that T-bet expressed in non-T/non-B population is involved in the suppression of IL-33-induced eosinophilic airway inflammation. Transcriptome analysis revealed that IL-9 expression in IL-33-stimulated lung ILCs was upregulated in T-bet-/- mice compared with that in wild-type mice. Importantly, neutralization of IL-9 markedly attenuated IL-33-induced accumulation of lung ILC2s and eosinophilic inflammation in T-bet-/- mice. T-bet suppresses IL-9 production from lung ILC2s and thereby inhibits IL-33-induced eosinophilic airway inflammation.

Cyclosporine A Inhibits the T-bet-Dependent Antitumor Response of CD8(+) T Cells.

Transplant recipients face an increased risk of cancer compared with the healthy population. Although several studies have examined the direct effects of immunosuppressive drugs on cancer cells, little is known about the interactions between pharmacological immunosuppression and cancer immunosurveillance. We investigated the different effects of rapamycin (Rapa) versus cyclosporine A (CsA) on tumor-reactive CD8(+) T cells. After adoptive transfer of CD8(+) T cell receptor-transgenic OTI T cells, recipient mice received either skin grafts expressing ovalbumin (OVA) or OVA-expressing B16F10 melanoma cells. Animals were treated daily with Rapa or CsA. Skin graft rejection and tumor growth as well as molecular and cellular analyses of skin- and tumor-infiltrating lymphocytes were performed. Both Rapa and CsA were equally efficient in prolonging skin graft survival when applied at clinically relevant doses. In contrast to Rapa-treated animals, CsA led to accelerated tumor growth in the presence of adoptively transferred tumor-reactive CD8(+) OTI T cells. Further analyses showed that T-bet was downregulated by CsA (but not Rapa) in CD8(+) T cells and that cancer cytotoxicity was profoundly inhibited in the absence of T-bet. CsA reduces T-bet-dependent cancer immunosurveillance by CD8(+) T cells. This may contribute to the increased cancer risk in transplant recipients receiving calcineurin inhibitors.

T-bet modulates the antibody response and immune protection during murine malaria.

CD4(+) T-cell subtypes govern the synthesis of different Ab isotypes and other immune functions. The influence of CD4(+) T-cell differentiation programs on isotype switching and other aspects of host immunological networks during malaria infection are currently poorly understood. Here, we used Tbx21(-/-) mice deficient for T-bet, a regulator of Th1 CD4(+) T-cell differentiation, to examine the effect of Th1 CD4(+) T cells on the immune protection to nonlethal murine malaria Plasmodium yoelii 17XNL. We found that Tbx21(-/-) mice exhibited significantly lower parasite burden that correlated with elevated levels of IgG1, indicating that T-bet-dependent Ab isotype switching may be responsible for lower parasite burden. Absence of T-bet was also associated with a transient but significant loss of T cells during the infection, suggesting that T-bet may suppress malaria-induced apoptosis or induce proliferation of T cells. However, Tbx21(-/-) mice produced greater numbers of Foxp3(+) CD25(+) regulatory CD4(+) T cells, which may contribute to the early contraction of T cells. Lastly, Tbx21(-/-) mice exhibited unimpaired production of IFN-γ by a diverse repertoire of immune cell subsets and a selective expansion of IFN-γ-producing T cells. These observations may have implications in malaria vaccine design.

T-bet controls intestinal chronic inflammation via regulation of IL-10 production by CD4 T cells (MUC8P.806)

Abstract The dynamics of pro- and anti-inflammatory cytokine secretion by CD4 T cells is critical for the maintenance of intestinal homeostasis. Disruption of this equilibrium can result in chronic inflammation in the intestine, causing inflammatory bowel disease (IBD). Among the various subsets of CD4 T cells, the Th1 and Th17 subsets are increased in IBD patients and are proposed to be pathogenic. Therefore, understanding the mechanisms by which these cells are regulated is important to expand current treatments of IBD. Using the CD4 T cell dependent adoptive transfer model of colitis, we have investigated the Th1-associated transcription factor, T-bet, in the context of intestinal inflammation. Loss of T-bet results in Th17 skewed immune responses; however, despite wild-type Th17 CD4 T cells being colitogenic, transfer of T-bet deficient naïve CD4 T cells fails to induce colitis in a lymphopenic recipient. Surprisingly, we show that IL-10 production is significantly increased in CD4 T cells in the absence of T-bet. Furthermore, deficiency of both T-bet and IL-10 negates the protective effects of T-bet deficiency alone, as recipients of T-bet-/-IL-10-/- CD4 T cells develop colitis with similar weight loss and histological damage to recipients of wild-type CD4 T cells. Therefore, our data suggests that T-bet plays crucial role in the maintenance of intestinal homeostasis by regulating the intricate balance of effector and regulatory cytokines, in particular IL-10 levels.

Genome-Wide Regulatory Analysis Reveals That T-bet Controls Th17 Lineage Differentiation through Direct Suppression of IRF4

The complex relationship between Th1 and Th17 cells is incompletely understood. The transcription factor T-bet is best known as the master regulator of Th1 lineage commitment. However, attention is now focused on the repression of alternate T cell subsets mediated by T-bet, particularly the Th17 lineage. It has recently been suggested that pathogenic Th17 cells express T-bet and are dependent on IL-23. However, T-bet has previously been shown to be a negative regulator of Th17 cells. We have taken an unbiased approach to determine the functional impact of T-bet on Th17 lineage commitment. Genome-wide analysis of functional T-bet binding sites provides an improved understanding of the transcriptional regulation mediated by T-bet, and suggests novel mechanisms by which T-bet regulates Th cell differentiation. Specifically, we show that T-bet negatively regulates Th17 lineage commitment via direct repression of the transcription factor IFN regulatory factor-4 (IRF4). An in vivo analysis of the pathogenicity of T-bet-deficient T cells demonstrated that mucosal Th17 responses were augmented in the absence of T-bet, and we have demonstrated that the roles of T-bet in enforcing Th1 responses and suppressing Th17 responses are separable. The interplay of the two key transcription factors T-bet and IRF4 during the determination of T cell fate choice significantly advances our understanding of the mechanisms underlying the development of pathogenic T cells.

Virus-specific CD4 and CD8 T cell responses in the absence of Th1-associated transcription factors

Effector and memory CD4 and CD8 T cell responses are critical for the control of many intracellular pathogens. The development of these populations is governed by transcription factors that molecularly control their differentiation, function, and maintenance. Two transcription factors known to be involved in these processes are Tbet and STAT4. Although Tbet has been shown to regulate CD8 T cell fate decisions and effector CD4 T cell choices, the contribution of STAT4 is less well understood. To address this, we examined the impact of STAT4 on T cell responses in the presence or absence of Tbet, following LCMV infection by using mice lacking Tbet, STAT4, or both transcription factors. STAT4 was not required for Tbet or Eomes expression; however, virus-specific effector CD8 T cells are skewed toward a memory-precursor phenotype in the absence of STAT4. This altered proportion of memory precursors did not result in an increase in memory CD8 T cells after the resolution of the infection. We also demonstrate that virus-specific effector and memory CD4 T cells formed independently of Tbet and STAT4, although a slight reduction in the number of antigen-specific CD4 T cells was apparent in mice lacking both transcription factors. Collectively, we have discovered distinct roles for Tbet and STAT4 in shaping the phenotype and function of virus-specific T cell responses.

The Transcription Factor T-bet Regulates Parasitemia and Promotes Pathogenesis duringPlasmodium bergheiANKA Murine Malaria

The pathogenesis of experimental cerebral malaria (ECM) is an immunologic process, mediated in part by Th1 CD4(+) T cells. However, the role of the Th1 CD4(+) T cell differentiation program on the ability to control parasitemia and susceptibility to ECM disease during blood stage malaria has never been assessed directly. Using the Plasmodium berghei ANKA murine model of ECM and mice deficient for the transcription factor T-bet (the master regulator of Th1 cells) on the susceptible C57BL/6 background, we demonstrate that although T-bet plays a role in the regulation of parasite burden, it also promotes the pathogenesis of ECM. T-bet-deficient (Tbx21(-/-)) mice had higher parasitemia than wild type controls did during the ECM phase of disease (17.7 ± 3.1% versus 10.9 ± 1.5%). In addition, although 100% (10/10) of wild type mice developed ECM by day 9 after infection, only 30% (3/10) of Tbx21(-/-) mice succumbed to disease during the cerebral phase of infection. Resistance to ECM in Tbx21(-/-) mice was associated with diminished numbers of IFN-γ-producing CD4(+) T cells in the spleen and a lower accumulation of CD4(+) and CD8(+) T cells in the brain. An augmented Th2 immune response characterized by enhanced production of activated GATA-3(+) CD4(+) T cells and elevated levels of the eotaxin, MCP-1, and G-CSF cytokines was observed in the absence of T-bet. Our results suggest that in virulent malarias, immune modulation or therapy resulting in an early shift toward a Th2 response may help to ameliorate the most severe consequences of malaria immunopathogenesis and the prospect of host survival.

Double Deficiency for RORγt and T-bet Drives Th2-Mediated Allograft Rejection in Mice

Although Th1, Th2, and Th17 cells are thought to be major effector cells in adaptive alloimmune responses, their respective contribution to allograft rejection remains unclear. To precisely address this, we used mice genetically modified for the Th1 and Th17 hallmark transcription factors T-bet and RORγt, respectively, which allowed us to study the alloreactive role of each subset in an experimental transplant setting. We found that in a fully mismatched heterotopic mouse heart transplantation model, T cells deficient for T-bet (prone to Th17 differentiation) versus RORγt (prone to Th1 differentiation) rejected allografts at a more accelerated rate, indicating a predominance of Th17- over Th1-driven alloimmunity. Importantly, T cells doubly deficient for both T-bet and RORγt differentiated into alloreactive GATA-3-expressing Th2 cells, which promptly induced allograft rejection characterized by a Th2-type intragraft expression profile and eosinophilic infiltration. Mechanistically, Th2-mediated allograft rejection was contingent on IL-4, as its neutralization significantly prolonged allograft survival by reducing intragraft expression of Th2 effector molecules and eosinophilic allograft infiltration. Moreover, under IL-4 neutralizing conditions, alloreactive double-deficient T cells upregulated Eomesodermin (Eomes) and IFN-γ, but not GATA-3. Thus, in the absence of T-bet and RORγt, Eomes may salvage Th1-mediated alloimmunity that underlies IL-4 neutralization-resistant allograft rejection. We summarize that, whereas Th17 cells predictably promote allograft rejection, IL-4-producing GATA-3(+) Th2 cells, which are generally thought to protect allogeneic transplants, may actually be potent facilitators of organ transplant rejection in the absence of T-bet and RORγt. Moreover, Eomes may rescue Th1-mediated allograft rejection in the absence of IL-4, T-bet, and RORγt.

Active STAT5 Regulates T-bet and Eomesodermin Expression in CD8 T Cells and Imprints a T-bet–Dependent Tc1 Program with Repressed IL-6/TGF-β1 Signaling

In adoptive therapy, CD8 T cells expressing active STAT5 (STAT5CA) transcription factors were found to be superior to unmanipulated counterparts in long-term persistence, capacity to infiltrate autochthonous mouse melanomas, thrive in their microenvironment, and induce their regression. However, the molecular mechanisms sustaining these properties were undefined. In this study, we report that STAT5CA induced sustained expression of genes controlling tissue homing, cytolytic granule composition, type 1 CD8 cytotoxic T cell-associated effector molecules granzyme B(+), IFN-γ(+), TNF-α(+), and CCL3(+), but not IL-2, and transcription factors T-bet and eomesodermin (Eomes). Chromatin immunoprecipitation sequencing analyses identified the genes possessing regulatory regions to which STAT5 bound in long-term in vivo maintained STAT5CA-expressing CD8 T cells. This analysis identified 34% of the genes differentially expressed between STAT5CA-expressing and nonexpressing effector T cells as direct STAT5CA target genes, including those encoding T-bet, Eomes, and granzyme B. Additionally, genes encoding the IL-6R and TGFbRII subunits were stably repressed, resulting in dampened IL-17-producing CD8 T cell polarization in response to IL-6 and TGF-β1. The absence of T-bet did not affect STAT5CA-driven accumulation of the T cells in tissue or their granzyme B expression but restored IL-2 secretion and IL-6R and TGFbRII expression and signaling, as illustrated by IL-17 induction. Therefore, concerted STAT5/T-bet/Eomes regulation controls homing, long-term maintenance, recall responses, and resistance to polarization towards IL-17-producing CD8 T cells while maintaining expression of an efficient type 1 CD8 cytotoxic T cell program (granzyme B(+), IFN-γ(+)).