DX5+CD4+ T cells modulate CD4+ T‐cell response via inhibition of IL‐12 production by DCs

DX5⁺CD4⁺ T cells have been shown to dampen collagen‐induced arthritis and delayed‐type hypersensitivity reactions in mice. These cells are also potent modulators of T‐helper cell responses through direct effects on CD4⁺ T cells in an IL‐4 dependent manner. To further characterize this T‐cell population, we studied their effect on DCs and the potential consequences on T‐cell activation. Here, we show that mouse DX5⁺CD4⁺ T cells modulate DCs by robustly inhibiting IL‐12 production. This modulation is IL‐10 dependent and does not require cell contact. Furthermore, DX5⁺CD4⁺ T cells modulate the surface phenotype of LPS‐matured DCs. DCs modulated by DX5⁺CD4⁺ T‐cell supernatant express high levels of the co‐inhibitor molecules PDL‐1 and PDL‐2. OVA‐specific CD4⁺ T cells primed with DCs exposed to DX5⁺CD4⁺ T‐cell supernatant produce less IFN‐γ than CD4⁺ T cells primed by DCs exposed to either medium or DX5^?CD4⁺ T‐cell supernatant. The addition of IL‐12 to the co‐culture with DX5⁺ DCs restores IFN‐γ production. When IL‐10 present in the DX5⁺CD4⁺ T‐cell supernatant is blocked, DCs re‐establish their ability to produce IL‐12 and to efficiently prime CD4⁺ T cells. These data show that DX5⁺CD4⁺ T cells can indirectly affect the outcome of the T‐cell response by inducing DCs that have poor Th1 stimulatory function.     

DX5+CD4+ T cells modulate cytokine production by CD4+ T cells towards IL‐10 via the production of IL‐4

CD4⁺ Th cells play a critical role in orchestrating the adaptive immune response. Uncontrolled Th1 responses are implicated in the pathogenesis of autoimmune diseases. T cells with immune‐modulatory properties are beneficial for inhibiting such inflammatory responses. Previously we demonstrated that repetitive injections of immature DC induce expansion of DX5⁺CD4⁺ T cells, which upon adoptive transfer show potent regulatory properties in murine collagen‐induced arthritis as well as in delayed‐hypersensitivity models. However, their regulatory mechanism remains to be defined. Here, we analyzed the effect of DX5⁺CD4⁺ T cells on other CD4⁺ T cells in vitro. Although proliferation of naïve CD4⁺ T cells upon antigenic triggering was not altered in the presence of DX5⁺CD4⁺ T cells, there was a striking difference in cytokine production. In the presence of DX5⁺CD4⁺ T cells, an IL‐10‐producing CD4⁺ T‐cell response was induced instead of a predominant IFN‐γ‐producing Th1 response. This modulation did not require cell–cell contact. Instead, IL‐4 produced by DX5⁺CD4⁺ T cells was primarily involved in the inhibition of IFN‐γ and promotion of IL‐10 production by CD4⁺ T cells. Together, our data indicate that DX5⁺CD4⁺ T cells modulate the outcome of Th‐responses by diverting Th1‐induction into Th responses characterized by the production of IL‐10.     

Differential requirements of CD4+ T‐cell signals for effector cytotoxic T‐lymphocyte (CTL) priming and functional memory CTL development at higher CD8+ T‐cell precursor frequency

Increased CD8⁺ T‐cell precursor frequency (PF) precludes the requirement of CD4⁺ helper T (Th) cells for primary CD8⁺ cytotoxic T‐lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) ‐pulsed dendritic cells (DC_OVA) derived from C57BL/6, CD40 knockout (CD40^?/?) or CD40 ligand knockout (CD40L^?/?) mice were used to immunize C57BL/6, Ia^b?/?, CD40^?/? or CD40L^?/? mice, whose PF was previously increased with transfer of 1 × 10⁶ CD8⁺ T cells derived from OVA‐specific T‐cell receptor (TCR) transgenic OTI, OTI(CD40^?/?) or OTI(CD40L^?/?) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4⁺ T‐cell help and Th‐provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA‐expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4⁺ T‐cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4⁺ T‐cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4⁺ T‐cell functions.     

H2‐M3‐restricted CD8+ T cells augment CD4+ T‐cell responses by promoting DC maturation

Infection with Listeria monocytogenes triggers the activation and expansion of nonconventional CD8⁺ T cells restricted by the MHC class Ib molecule, H2‐M3. H2‐M3‐restricted CD8⁺ T cells exhibit a memory phenotype, rapidly produce cytokines, and reach peak frequencies sooner than conventional MHC class Ia‐restricted CD8⁺ T cells. In this study, we found that simultaneous in vivo priming of H2‐M3‐restricted T cells and adoptively transferred OT‐II CD4⁺ T cells on the same DC enhances the survival of OT‐II cells. Stimulation of H2‐M3‐restricted T cells were found to induce DC maturation resulting in costimulatory molecule upregulation and production of T_H1‐type cytokines, which was dependent on both cell‐to‐cell contact and soluble factors, particularly TNF‐α, produced by activated H2‐M3‐restricted T cells. Interestingly, H2‐M3‐restricted T cells were more efficient than activated NK cells in inducing DC maturation. Furthermore, we found that OVA_323–339‐coated DC matured by coculturing with peptide‐stimulated H2‐M3‐restricted T cells were more efficient in stimulating the proliferation of Ag‐activated OT‐II cells. This study indicates that H2‐M3‐restricted T cells promote immune responses by CD4⁺ T cells by inducing DC maturation and suggests novel mechanisms for vaccine development.     

Antigen presentation by B cells guides programing of memory CD4+ T‐cell responses to a TLR4‐agonist containing vaccine in mice

The contribution of B cells to immunity against many infectious diseases is unquestionably important and well characterized. Here, we sought to determine the role of B cells in the induction of T‐helper 1 (T_H1) CD4⁺ T cells upon vaccination with a tuberculosis (TB) antigen combined with a TLR4 agonist. We used B‐cell deficient mice (μMT^?/?), tetramer‐positive CD4⁺ T cells, markers of memory “precursor” effector cells (MPECs), and T‐cell adoptive transfers and demonstrated that the early antigen‐specific cytokine‐producing T_H1 responses are unaffected in the absence of B cells, however MPEC induction is strongly impaired resulting in a deficiency of the memory T_H1 response in μMT^?/? mice. We further show that antigen‐presentation by B cells is necessary for their role in MPEC generation using B‐cell adoptive transfers from wt or MHC class II knock‐out mice into μMT^?/? mice. Our study challenges the view that B‐cell deficiency exclusively alters the T_H1 response at memory time‐points. Collectively, our results provide new insights on the multifaceted roles of B cells that will have a high impact on vaccine development against several pathogens including those requiring T_H1 cell‐mediated immunity.     

Mouse pancreatic beta cells express MHC class II and stimulate CD4+ T cells to proliferate

Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. Both CD4⁺ and CD8⁺ T cells have been shown to mediate beta‐cell killing. While CD8⁺ T cells can directly recognize MHC class I on beta cells, the interaction between CD4⁺ T cells and beta cells remains unclear. Genetic association studies have strongly implicated HLA‐DQ alleles in human type 1 diabetes. Here we studied MHC class II expression on beta cells in nonobese diabetic mice that were induced to develop diabetes by diabetogenic CD4⁺ T cells with T‐cell receptors that recognize beta‐cell antigens. Acute infiltration of CD4⁺ T cells in islets occurred with rapid onset of diabetes. Beta cells from islets with immune infiltration expressed MHC class II mRNA and protein. Exposure of beta cells to IFN‐γ increased MHC class II gene expression, and blocking IFN‐γ signaling in beta cells inhibited MHC class II upregulation. IFN‐γ also increased HLA‐DR expression in human islets. MHC class II⁺ beta cells stimulated the proliferation of beta‐cell‐specific CD4⁺ T cells. Our study indicates that MHC class II molecules may play an important role in beta‐cell interaction with CD4⁺ T cells in the development of type 1 diabetes.     

Parenchymal cells critically curtail cytotoxic T‐cell responses by inducing Bim‐mediated apoptosis

To develop cytolytic effector functions, CD8⁺ T lymphocytes need to recognize specific Ag/MHC class I complexes in the context of costimuli on Ag‐presenting DC. Thereafter they differentiate into effector and memory CTL able to confer protection against pathogen infection. Using transgenic mice with DC‐selective MHC class I expression and DC‐specific versus ubiquitous vaccination regimen, we found that DC are sufficient to prime CTL responses. However, Ag recognition on parenchymal non‐professional APC negatively affected CD8⁺ T‐cell responses in mice by inducing expression of the pro‐apoptotic bcl2‐family member bim in CTL. This unexpected induction of apoptosis in the early phase of effector CTL accumulation lead to suboptimal clonal burst size and diminished long‐term memory. Thus, our data demonstrate that effector CTL differentiation and apoptosis are regulated independently. Moreover, Ag distribution on cells other than DC critically reduces CTL responses.     

Efficient and versatile manipulation of the peripheral CD4+ T‐cell compartment by antigen targeting to DNGR‐1/CLEC9A

DC NK lectin group receptor‐1 (DNGR‐1, also known as CLEC9A) is a C‐type lectin receptor expressed by mouse CD8α⁺ DC and by their putative equivalents in human. DNGR‐1 senses necrosis and regulates CD8⁺ T‐cell cross‐priming to dead‐cell‐associated antigens. In addition, DNGR‐1 is a target for selective in vivo delivery of antigens to DC and the induction of CD8⁺ T‐cell and Ab responses. In this study, we evaluated whether DNGR‐1 targeting can be additionally used to manipulate antigen‐specific CD4⁺ T lymphocytes. Injection of small amounts of antigen‐coupled anti‐DNGR‐1 mAb into mice promoted MHC class II antigen presentation selectively by CD8α⁺ DC. In the steady state, this was sufficient to induce proliferation of antigen‐specific naïve CD4⁺ T cells and to drive their differentiation into Foxp3⁺ regulatory lymphocytes. Co‐administration of adjuvants prevented this induction of tolerance and promoted immunity. Notably, distinct adjuvants allowed qualitative modulation of CD4⁺ T‐cell behavior: poly I:C induced a strong IL‐12‐independent Th1 response, whereas curdlan led to the priming of Th17 cells. Thus, antigen targeting to DNGR‐1 is a versatile approach for inducing functionally distinct CD4⁺ T‐cell responses. Given the restricted pattern of expression of DNGR‐1 across species, this strategy could prove useful for developing immunotherapy protocols in humans.     

C5a receptor‐deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3⁺ Treg within 12 h after co‐incubation with CD4⁺ T cells from DO11.10/RAG2^?/? mice. Stimulation of C5aR^?/? DC with OVA and TLR2 ligand Pam₃CSK₄ increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4⁺ Th cell into C5aR^?/? mice immunized with OVA and Pam₃CSK₄. The altered cytokine production of C5aR^?/? DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

Stress‐activated dendritic cells interact with CD4+ T cells to elicit homeostatic memory

Evidence is presented that thermal or oxidizing stress‐activated DC interact with CD4⁺ T cells to induce and maintain a TCR‐independent homeostatic memory circuit. Stress‐activated DC expressed endogenous intra‐cellular and cell surface HSP70. The NF‐κB signalling pathway was activated and led to the expression of membrane‐associated IL‐15 molecules. These interacted with the IL‐15 receptor complex on CD4⁺ T cells, thus activating the Jak3 and STAT5 phosphorylation signalling pathway to induce CD40 ligand expression, T‐cell proliferation and IFN‐γ production. CD40 ligand on CD4⁺ T cells in turn re‐activated CD40 molecules on DC, inducing DC maturation and IL‐15 expression thereby maintaining the feedback circuit. The proliferating CD4⁺ T cells were characterized as CD45RA^? CD62L⁺ central memory cells, which underwent homeostatic proliferation. The circuit is independent of antigen and MHC‐class‐II‐TCR interaction as demonstrated by resistance to TCR inhibition by ZAP70 inhibitor or MHC‐class II antibodies. These findings suggest that stress can activate a DC‐CD4⁺ T‐cell interacting circuit, which may be responsible for maintaining a homeostatic antigen‐independent memory.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

Epithelial expression of human papillomavirus type 16 E7 protein results in peripheral CD8 T‐cell suppression mediated by CD4+CD25+ T cells

The role of thymic versus peripheral epithelium in the regulation of the antigen‐specific CD8 T‐cell repertoire is still largely unresolved. We generated TCR‐β chain transgenic mice in which an increased frequency of peripheral CD8 T cells recognizes an epitope from a viral oncoprotein (HPV16E7) in the context of H‐2D^b MHC class I. When T cells from these mice developed through the thymus of mice expressing functional E7 protein from a keratin 14 promoter, no major perturbation to transgenic T‐cell development in the thymus was observed in these double‐transgenic mice. In contrast, peripheral CD8 T‐cell responses in the single‐transgenic, K14E7 mice, including those unrelated to E7 antigen, are reduced whereas CD4 T‐cell responses and antibody production are unchanged in these mice. Peripheral non‐responsiveness among CD8 T cells was mediated largely by CD4⁺CD25⁺ T cells. This suggested that epithelium expressing HPV16E7 protein induces Treg that specifically down‐regulate CD8 T‐cell responses in the periphery. This may have important consequences for the treatment of cervical pre‐cancers and provides a model for understanding differential suppression of T and B lymphocyte subsets by Treg.     

IGRP and insulin vaccination induce CD8+ T cell‐mediated autoimmune diabetes in the RIP‐CD80GP mouse

Autoimmune diabetes is characterized by autoantigen‐specific T cell‐mediated destruction of pancreatic islet beta cells, and CD8⁺ T cells are key players during this process. We assessed whether the bitransgenic RIP‐CD80 x RIP‐LCMV‐GP (RIP‐CD80GP) mice may be a versatile antigen‐specific model of inducible CD8⁺ T cell‐mediated autoimmune diabetes. Antigen‐encoding DNA, peptide‐loaded dendritic cells and antigen plus incomplete Freund's adjuvant were used for vaccination. Of 14 pancreatic proteins tested by DNA vaccination, murine pre‐proinsulin 2 (100% of mice; median time after vaccination, 60 days) and islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP) (77%, 58 days) could induce diabetes. Vaccination with DNA encoding for zinc transporter 8, Ia‐2, Ia‐2β, glutamic acid decarboxylase 67 (Gad67), chromogranin A, insulinoma amyloid polypeptide and homeobox protein Nkx‐2.2 induced diabetes development in 25–33% of mice. Vaccination with DNA encoding for Gad65, secretogranin 5, pancreas/duodenum homeobox protein 1 (Pdx1), carboxyl ester lipase, glucagon and control hepatitis B surface antigen (HBsAg) induced diabetes in <20% of mice. Diabetes induction efficiency could be increased by DNA vaccination with a vector encoding a ubiquitin–antigen fusion construct. Diabetic mice had florid T cell islet infiltration. CD8⁺ T cell targets of IGRP were identified with a peptide library‐based enzyme‐linked immunospot assay, and diabetes could also be induced by vaccination with major histocompatibility complex (MHC) class I‐restricted IGRP peptides loaded on mature dendritic cells. Vaccination with antigen plus incomplete Freund's adjuvant, which can prevent diabetes in other models, led to rapid diabetes development in the RIP‐CD80GP mouse. We conclude that RIP‐CD80GP mice are a versatile model of antigen specific autoimmune diabetes and may complement existing mouse models of autoimmune diabetes for evaluating CD8⁺ T cell‐targeted prevention strategies.     

Patency of Litomosoides sigmodontis infection depends on Toll‐like receptor 4 whereas Toll‐like receptor 2 signalling influences filarial‐specific CD4+ T‐cell responses

BALB/c mice develop a patent state [release of microfilariae (Mf), the transmission life‐stage, into the periphery] when exposed to the rodent filariae Litomosoides sigmodontis. Interestingly, only a portion of the infected mice become patent, which reflects the situation in human individuals infected with Wuchereria bancrofti. Since those individuals had differing filarial‐specific profiles, this study compared differences in immune responses between Mf⁺ and Mf^– infected BALB/c mice. We demonstrate that cultures of total spleen or mediastinal lymph node cells from Mf⁺ mice produce significantly more interleukin‐5 (IL‐5) to filarial antigens but equal levels of IL‐10 when compared with Mf^– mice. However, isolated CD4⁺ T cells from Mf⁺ mice produced significantly higher amounts of all measured cytokines, including IL‐10, when compared with CD4⁺ T‐cell responses from Mf^– mice. Since adaptive immune responses are influenced by triggering the innate immune system we further studied the immune profiles and parasitology in infected Toll‐like receptor‐2‐deficient (TLR2^?/?) and TLR4^?/? BALB/c mice. Ninety‐three per cent of L. sigmodontis‐exposed TLR4^?/? BALB/c mice became patent (Mf⁺) although worm numbers remained comparable to those in Mf⁺ wild‐type controls. Lack of TLR2 had no influence on patency outcome or worm burden but infected Mf⁺ mice had significantly lower numbers of Foxp3⁺ regulatory T cells and dampened peripheral immune responses. Interestingly, in vitro culturing of CD4⁺ T cells from infected wild‐type mice with granulocyte–macrophage colony‐stimulating factor‐derived TLR2^?/? dendritic cells resulted in an overall diminished cytokine profile to filarial antigens. Hence, triggering TLR4 or TLR2 during chronic filarial infection has a significant impact on patency and efficient CD4⁺ T‐cell responses, respectively.     

Selective antigen‐specific CD4+ T‐cell,but not CD8+ T‐ or B‐cell,tolerance corrupts cancer immunotherapy

Self‐tolerance, presumably through lineage‐unbiased elimination of self‐antigen‐specific lymphocytes (CD4⁺ T, CD8⁺ T, and B cells), creates a formidable barrier to cancer immunotherapy. In contrast to this prevailing paradigm, we demonstrate that for some antigens, self‐tolerance reflects selective elimination of antigen‐specific CD4⁺ T cells, but preservation of CD8⁺ T‐ and B‐cell populations. In mice, antigen‐specific CD4⁺ T‐cell tolerance restricted CD8⁺ T‐ and B‐cell responses targeting the endogenous self‐antigen guanylyl cyclase c (GUCY2C) in colorectal cancer. Although selective CD4⁺ T‐cell tolerance blocked GUCY2C‐specific antitumor immunity and memory responses, it offered a unique solution to the inefficacy of GUCY2C vaccines through recruitment of self‐antigen‐independent CD4⁺ T‐cell help. Incorporating CD4⁺ T‐cell epitopes from foreign antigens into vaccines against GUCY2C reconstituted CD4⁺ T‐cell help, revealing the latent functional capacity of GUCY2C‐specific CD8⁺ T‐ and B‐cell pools, producing durable antitumor immunity without autoimmunity. Incorporating CD4⁺ T‐cell epitopes from foreign antigens into vaccines targeting self‐antigens in melanoma (Trp2) and breast cancer (Her2) produced similar results, suggesting selective CD4⁺ T‐cell tolerance underlies ineffective vaccination against many cancer antigens. Thus, identification of self‐antigens characterized by selective CD4⁺ T‐cell tolerance and abrogation of such tolerance through self‐antigen‐independent T‐cell help is essential for future immunotherapeutics.     

CD4+CD25+ regulatory T cells utilize FasL as a mechanism to restrict DC priming functions in cutaneous immune responses

Recent studies have suggested Fas‐mediated elimination of antigen‐presenting cells as an important mechanism down‐regulating the induction of autoimmune responses. It remains unknown whether this mechanism restricts the magnitude of immune responses to non‐self antigens. We used a mouse model of a cutaneous CD8⁺ T‐cell‐mediated immune response (contact hypersensitivity, CHS) to test if CD4⁺CD25⁺ T cells expressing FasL regulate hapten‐specific effector CD8⁺ T cell expansion through the elimination of Fas‐expressing hapten‐presenting DC. In WT mice, attenuation of CD4⁺CD25⁺ T regulatory cell activity by anti‐CD25 mAb increased hapten‐presenting DC numbers in skin‐draining LN, which led to increased effector CD8⁺ T‐cell priming for CHS responses. In contrast, CD4⁺CD25⁺ T cells did not regulate hapten‐specific CD8⁺ T‐cell priming and CHS responses initiated by Fas‐defective (lpr) DC. Thus, restricting DC priming functions through Fas–FasL interactions is a potent mechanism employed by CD4⁺CD25⁺ regulatory cells to restrict CD8⁺ T‐cell‐mediated allergic immune responses in the skin.     

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

How T cell receptor (TCR) avidity influences CD8⁺ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP^?/? mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP^?/? Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8⁺ T cell development and repertoire selection. In comparing SLAP^?/? OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP^?/? Vβ5 mice. We have found that SLAP^?/? OT-1 mice have fewer CD8⁺ thymocytes but have increased CD5 expression. SLAP^?/? OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8⁺ splenocytes upon tetramer staining. Our data demonstrate that SLAP^?/? Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8⁺ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8⁺ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8⁺ T cell development influences repertoire selection.     

IL‐7 is essential for lymphopenia‐driven turnover of colitogenic CD4+ memory T cells in chronic colitis

We previously demonstrated that IL‐7 is essential for the persistence of T‐cell‐mediated colitis, by showing that adoptive transfer of CD4⁺CD45RB^high T cells into IL‐7^?/?×RAG‐1^?/? mice did not induce colitis; and that intestinal IL‐7 is not essential for this colitis model, by showing that IL‐7^?/?×RAG‐1^?/? mice parabiosed with colitic CD4⁺CD45RB^high T‐cell‐transferred RAG‐1^?/? mice developed colitis. Here, we investigated the role of IL‐7 in the maintenance of colitogenic CD4⁺ T cells by surgically separating these parabionts. Surprisingly, the separated IL‐7^?/?×RAG‐1^?/? mice were consistently diseased after separation, although no IL‐7 mRNA was detected in the tissues of separated IL‐7^?/?×RAG‐1^?/? partners. CD4⁺ T cells isolated from the separated RAG‐1^?/? or IL‐7^?/?×RAG‐1^?/? mice were then transferred into new RAG‐1^?/? or IL‐7^?/?×RAG‐1^?/? mice. Regardless of the source of donor cells, RAG‐1^?/? recipients developed colitis, whereas IL‐7^?/?×RAG‐1^?/? recipients did not. Collectively, these results demonstrate that IL‐7 is essential for lymphopenia‐driven turnover of colitogenic CD4⁺ T cells rather than the maintenance of those cells in established colitic mice. They also provide a basis for the timing of IL‐7/IL‐7R blockade for the treatment of inflammatory bowel diseases.