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.     

Human CD4+ invariant NKT cells are involved in antibacterial immunity against Brucella suis through CD1d‐dependent but CD4‐independent mechanisms

Human invariant NKT (iNKT) cells are a unique subset of T cells, which recognize glycolipids presented by the CD1d. Among the iNKT cells, several functionally distinct subsets have been characterized according to CD4 and/or CD8 co‐receptor expression. The current study is focussed on the CD4⁺ iNKT cell subset and its role in an anti‐infectious response. We have examined the role of CD4⁺ iNKT cells on the intracellular Brucella suis growth. Our results indicate that CD4⁺ iNKT cells impair the intramacrophagic growth of Brucella. This inhibition is due to a combination of soluble and contact‐dependent mechanisms: IFN‐γ is weakly involved while cytotoxic activities such as the induction of the Fas pathway and the release of lytic granules are major mechanisms. The impairment of Brucella growth by CD4⁺ iNKT cells requires an interaction with CD1d on macrophage surface. Also, we have shown that although CD4 regulates several biological responses of CD4⁺ iNKT cells, it is not involved in their antibacterial activity. Here, we have shown for the first time that the CD4⁺ iNKT cell population has antibacterial activity and thus, participates directly in the elimination of bacteria and/or in the control of bacterial growth by killing infected cells.     

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.     

CD28 controls the development of innate‐like CD8+ T cells by promoting the functional maturation of NKT cells

NK T cells(NKT cells) share functional characteristics and homing properties that are distinct from conventional T cells. In this study, we investigated the contribution of CD28 in the functional development of γδ NKT and αβ NKT cells in mice. We show that CD28 promotes the thymic maturation of promyelocytic leukemia zinc finger⁺ IL‐4⁺ NKT cells and upregulation of LFA‐1 expression on NKT cells. We demonstrate that the developmental defect of γδ NKT cells in CD28‐deficient mice is cell autonomous. Moreover, we show in both wild‐type C57BL/6 mice and in downstream of tyrosine kinase‐1 transgenic mice, a mouse model with increased numbers of γδ NKT cells, that CD28‐mediated regulation of thymic IL‐4⁺ NKT cells promotes the differentiation of eomesodermin⁺ CD44^high innate‐like CD8⁺ T cells. These findings reveal a previously unappreciated mechanism by which CD28 controls NKT‐cell homeostasis and the size of the innate‐like CD8⁺ T‐cell pool.     

CD155/CD226‐interaction impacts on the generation of innate CD8+ thymocytes by regulating iNKT‐cell differentiation

The cell surface receptor CD155 influences a variety of immune processes by binding to its ligands CD226, CD96, or TIGIT. Here, we report that the interaction of CD155 with CD226 in the thymus of BALB/c mice has a dual function. It directly influences the dwell time of memory‐like CD8⁺ T cells, while it is indirectly involved in generating these cells. It was shown earlier that a massive emergence of memory‐like CD8 T cells in thymus crucially depends on abundant IL‐4, secreted in steady state by iNKT2 (where iNKT is invariant NKT) cells, a subclass of iNKT cells. Here, we show that absence of either CD155 or CD226 in BALB/c mice causes a profound shift in the iNKT subtype composition in thymus, expanding the frequency and numbers of iNKT1 cells at the expense of iNKT2 cells, as well as iNKT17 cells. This shift results in a drop of available IL‐4 and creates a scenario similar to that observed in C57BL/6 mice, where iNKT1 cells predominate and iNKT2 cells are much less frequent when compared with BALB/c mice. Yet also in C57BL/6 mice, lack of CD155 or CD226 provokes a further decline in iNKT2 cells, suggesting that the observed effects are not restricted to a particular inbred strain.     

Suppression of murine tumour growth through CD8+ cytotoxic T lymphocytes via activated DEC‐205+ dendritic cells by sequential administration of α‐galactosylceramide in vivo

Cancer immunity is mediated through the effective priming and activation of tumour‐specific class I MHC molecule‐restricted CD8⁺ cytotoxic T lymphocytes (CTLs). DEC‐205⁺ dendritic cells (DCs) can cross‐present the epitope(s) of captured tumour antigens associated with class I MHC molecules alongside co‐stimulatory molecules to prime and activate tumour‐specific CD8⁺ CTLs. Immunosuppressive tolerogenic DCs with reduced co‐stimulatory molecules may be a cause of impaired CTL induction. Hepa1‐6‐1 cells were established from the mouse hepatoma cell line Hepa1‐6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 (B6) mice and do not prime CD8⁺ CTLs. In this study, we show that the growth of ongoing tumours was suppressed by activated CD8⁺ CTLs with tumour‐specific cytotoxicity through the administration of the glycolipid α‐galactosylceramide (α‐GalCer), which is a compound known to stimulate invariant natural killer T (iNKT) cells and selectively activate DEC‐205⁺ DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation with α‐GalCer every 48 hr appeared to convert tolerogenic DEC‐205⁺ DCs into immunogenic DCs with a higher expression of co‐stimulatory molecules and a stronger cross‐presentation capacity, which primed CTL precursors and induced tumour‐specific CD8⁺ CTLs within the tumour environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC‐205⁺ DCs within tumours into immunogenic DCs through the sequential administration of an immuno‐potent lipid/glycolipid, and then activated immunogenic DCs with sufficient expression of co‐stimulatory molecules prime and activate tumour‐specific CD8⁺ CTLs within the tumour to control tumour growth.     

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.     

Thymus‐specific serine protease regulates positive selection of a subset of CD4+ thymocytes

Thymus‐specific serine protease (TSSP) was initially reported as a putative protease specifically expressed in the endosomal compartment of cortical thymic epithelial cells (cTEC). As such, TSSP is potentially involved in the presentation of the self‐peptides that are bound to MHC class II molecules expressed at the cTEC surface and are involved in the positive selection of CD4⁺ thymocytes. We tested this hypothesis by generating mutant mice deprived of Prss16, the gene encoding TSSP. TSSP‐deficient mice produced normal numbers of T cells, despite a decrease in the percentage of cTEC expressing high surface levels of MHC class II. By using sensitive transgenic models expressing MHC class II‐restricted TCR transgenes (Marilyn and OT‐II), we showed that the absence of TSSP markedly impaired the selection of Marilyn and OT‐II CD4⁺ T cells. In contrast, selection of CD8⁺ T cells expressing an MHC class I‐restricted TCR transgene (OT‐I) was unaffected. Therefore, TSSP is involved in the positive selection of some CD4⁺ T lymphocytes and likely constitutes the first serine protease to play a function in the intrathymic presentation of self‐peptides bound to MHC class II complexes.     

Lipid‐mediated presentation of MHC class II molecules guides thymocytes to the CD4 lineage

Previous studies on the MHC class‐specific differentiation of CD4⁺CD8⁺ thymocytes into CD4⁺ and CD8⁺ T cells have focused on the role of coreceptor molecules. However, CD4 and CD8 T cells develop according to their MHC class specificities even in these mice lacking coreceptors. This study investigated the possibility that lineage is determined not only by coreceptors, but is also guided by the way how MHC molecules are presented. MHC class II molecules possess a highly conserved Cys in their transmembrane domain, which is palmitoylated and thereby associates with lipid rafts, whereas neither palmitoylation nor raft association was observed with MHC class I molecules. The generation of CD4 T cells was impaired and that of CD8 T cells was augmented when the rafts on the thymic epithelial cells were disrupted. This was due to the conversion of MHC class II‐specific thymocytes from the CD4 lineage to CD8. The ability of I‐A^d molecule to associate with rafts was lost when its transmembrane Cys was replaced. The development of DO11.10 thymocytes recognizing this mutant I‐A^dm was converted from CD4 to CD8. These results suggest that the CD4 lineage commitment is directed by the raft‐associated presentation of MHC class II molecules.     

Plasmacytoid dendritic cells license regulatory T cells,upon iNKT‐cell stimulation,to prevent autoimmune diabetes

Invariant NKT (iNKT)‐cell stimulation with exogenous specific ligands prevents the development of type 1 diabetes (T1D) in NOD mice. Studies based on anti‐islet T‐cell transfer showed that iNKT cells prevent the differentiation of these T cells into effector T cells in the pancreatic lymph nodes (PLNs). We hypothesize that this defective priming could be explained by the ability of iNKT cells to induce tolerogenic dendritic cells (DCs) in the PLNs. We evaluated the effect of iNKT‐cell stimulation on T1D development by transferring naïve diabetogenic BDC2.5 T cells into proinsulin 2^?/? NOD mice treated with a long‐lasting α‐galactosylceramide regimen. In this context, iNKT cells induce the conversion of BDC2.5 T cells into Foxp3⁺ Treg cells in the PLNs accumulating in the pancreatic islets. Furthermore, tolerogenic plasmacytoid DCs (pDCs) characterized by low MHC class II molecule expression and TGF‐β production are critical in the PLNs for the recruitment of Treg cells into the pancreatic islets by inducing CXCR3 expression. Accordingly, pDC depletion in α‐galactosylceramide‐treated proinsulin 2^?/? NOD mice abrogates the protection against T1D. These findings reveal that upon repetitive iNKT‐cell stimulation, pDCs are critical for the recruitment of Treg cells in the pancreatic islets and the prevention of T1D development.     

Antigen processing and CD24 expression determine antigen presentation by splenic CD4+ and CD8+ dendritic cells

To examine heterogeneity in dendritic cell (DC) antigen presentation function, murine splenic DCs were separated into CD4⁺ and CD8⁺ populations and assessed for the ability to process and present particulate antigen to CD4⁺ and CD8⁺ T cells. CD4⁺ and CD8⁺ DCs both processed exogenous particulate antigen, but CD8⁺ DCs were much more efficient than CD4⁺ DCs for both major histocompatibility complex (MHC) class II antigen presentation and MHC class I cross-presentation. While antigen processing efficiency contributed to the superior antigen presentation function of CD8⁺ DCs, our studies also revealed an important contribution of CD24. CD8⁺ DCs were also more efficient than CD4⁺ DCs in inducing naïve T cells to acquire certain effector T-cell functions, for example generation of cytotoxic CD8⁺ T cells and interferon (IFN)-γ-producing CD4⁺ T cells. In summary, CD8⁺ DCs are particularly potent antigen-presenting cells that express critical costimulators and efficiently process exogenous antigen for presentation by both MHC class I and II molecules.     

Generation of mature T cell populations in the thymus: CD4 or CD8 down-regulation occurs at different stages of thymocyte differentiation

We have studied the differentiation and repertoire selection during the maturation of CD4⁺CD8⁺ (DP) thymocytes into CD4⁺CD8^- (CD4SP) and CD8⁺CD4^- (CD8SP) T cells, in normal mice, mice transgenic for T cell receptor (TcR)-αβ restricted by either class I or class II major histocompatibility (MHC), and in mice deficient in class I or class II MHC expression. Our data suggest that mature CD4 and CD8 T cells derive from different pathways of T cell differentiation in the thymus. Thus, interaction of DP thymocytes with MHC class II leads to the immediate down-regulation of CD8, which occurs simultaneously with an increase in TcR expression; DPTcR^loHSA^hi thymocytes mature into a CD4⁺CD8^lo TcR^hiHSA^hi intermediate population. This cell population generates CD4SP thymocytes, the majority of which are still HSA^hi. In contrast, interaction with MHC class I induces the up-regulation of TcR, which precedes the down-regulation of CD4; DPTcR^lo generate DPTcR^hi thymocytes, the majority of which are the committed precursors of CD8SP cells. Further differentiation results in CD4 down-regulation and the transition from DPTcR^hi into CD8⁺CD4^lo TcR^hiHSA^lo and CD8SPTcR^hiHSA^- T cells. Since down-regulation of CD4 and CD8 occurs at different stages of thymocyte differentiation, our results do not support a stochastic/selective model of lineage commitment in the thymus.     

CD4+CD25highFoxp3+ Treg deficiency in a Brazilian patient with Gaucher disease and lupus nephritis

Gaucher Disease (GD) is a rare autosomal recessive disorder caused by the deficient activity of beta-glucocerebrosidase. GD is one of the lysosomal storage diseases with the most remarkable alterations in the immune system, and that may manifest clinically as autoimmune disorders and malignancy. We reported the immunological evaluation of a patient with GD and lupus nephritis. Decreased absolute values of T, and NK, and an inversion of CD4⁺/CD8⁺ ratio, low levels of IgM and normal B cells were found when compared to reference values in a Brazilian population. Absence ofCD4⁺CD25^highFoxp3⁺ Treg and high levels of total NKT, iNKT cells and CD8⁺ iNKT subsets were also observed when compared to the healthy control and GD patient without lupus nephritis. Treg subset and CD8⁺ iNKT abnormalities might be involved in severe lupus nephritis in a GD patient. We conclude by emphasizing the importance of the immunological evaluation on early diagnosis of autoimmunity contributing to reduce mortality and morbidity of these patients.     

DC‐induced CD8+ T‐cell response is inhibited by MHC class II‐dependent DX5+CD4+ Treg

CD4⁺ T cells are important for CD8⁺ T‐cell priming by providing cognate signals for DC maturation. We analyzed the capacity of CD4⁺ T cells to influence CD8⁺ T‐cell responses induced by activated DC. Surprisingly, mice depleted for CD4⁺ cells were able to generate stronger antigen‐specific CD8⁺ T‐cell responses after DC vaccination than non‐depleted mice. The same observation was made when mice were vaccinated with MHC class II^?/? DC, indicating the presence of a MHC class II‐dependent CD4⁺ T‐cell population inhibiting CD8⁺ T‐cell responses. Recently we described the expansion of DX5⁺CD4⁺ T cells, a T‐cell population displaying immune regulatory properties, upon vaccination with DC. Intriguingly, we now observe an inverse correlation between CD8⁺ T‐cell induction and expansion of DX5⁺CD4⁺ T cells as the latter cells did not expand after vaccination with MHC class II^?/? DC. In vitro, DX5⁺CD4⁺ T cells were able to limit proliferation, modulate cytokine production and induce Foxp3⁺ expression in OVA‐specific CD8⁺ T cells. Together, our data show an inhibitory role of CD4⁺ T cells on the induction of CD8⁺ T‐cell responses by activated DC and indicate the involvement of DX5⁺CD4⁺, but not CD4⁺CD25⁺, T cells in this process.