IL-23R+ innate lymphoid cells induce colitis via interleukin-22-dependent mechanism.

Polymorphisms of interleukin (IL)-23R and signaling components are associated with several autoimmune diseases, including inflammatory bowel diseases (IBD). Similar to T helper type 17 (Th17) lineage, type 3 innate lymphoid cells (ILCs) express retinoic acid-related orphan receptor γt (Rorγt) and IL-23R and hence, produce Th17-type cytokines. Recent reports implicated type 3 ILCs in IBD; however, how IL-23R signaling in these cells contributes to pathogenesis is unknown. IL-22, produced in copious amounts by type 3 ILCs, was reported to have both beneficial and pathogenic effects in adaptive, yet only a protective role in innate colitis models. Herein, by employing chronic CD45RB(high) CD4(+) T-cell transfer and anti-CD40 antibody-induced acute innate colitis models in Rag1(-/-) mice, we demonstrated opposite roles for IL-23R in colitogenesis: in the former a protective, and in the latter a pathogenic role. Furthermore, we show that IL-23R signaling promotes innate colitis via IL-22 as neutralization of IL-22 protected mice from colitis and adding back of IL-22 to IL-23R-deficient animals restored the disease. Collectively, our results reveal that similar to its controversial role during chronic or adaptive colitis, IL-22 may also have opposite roles in innate colitis pathogenesis in a context and insult-dependent manner.

Th17 cells in immunity and autoimmunity.

The primary function of Th17 cells appears to be the clearance of extracellular pathogens during infections. However, Th17 cells also promote inflammation and have been implicated in the pathogenesis of many experimental autoimmune diseases and human inflammatory conditions. Transforming growth factor beta (TGFbeta) is a critical differentiation factor for the generation of regulatory T (T-reg) cells, whereas the combination of interleukin 6 (IL6) and TGFbeta induces the differentiation of pathogenic Th17 cells. Therefore, it is proposed that at the steady state (in the absence of any inflammatory stimuli), TGFbeta, which is produced by various cells types including naturally occurring T-reg (nT-reg) cells, encourages the generation of induced T-reg (iT-reg) cells, which together with nT-reg cells keep autoreactive T cells under check. IL6, an acute phase protein produced by the activated immune system, inhibits the function of T-reg cells and instead promotes the differentiation of Th17 cells. Thus, IL6 plays a pivotal role in dictating the balance between the generation of T-reg and Th17 cells. This reciprocal relationship between T-reg and Th17 cells is further supported by the results obtained in IL6(-/-) mice, which show a severe defect in the generation of Th17 cells and increased numbers of T-reg cells in the peripheral repertoire.

Contrasting effects of cyclosporine and rapamycin in de novo generation of alloantigen-specific regulatory T cells.

The outcome of T-cell-mediated responses, immunity or tolerance, critically depends on the balance of cytopathic versus regulatory T (T(reg)) cells. In the creation of stable tolerance to MHC incompatible allografts, reducing the unusually large mass of donor-reactive cytopathic T effector (T(eff)) cells via apoptosis is often required. Cyclosporine (CsA) blocks activation-induced cell death (AICD) of T(eff) cells, and is detrimental to tolerance induction by costimulation blockade, whereas Rapamycin (RPM) preserves AICD, and augments the potential of costimulation blockade to create tolerance. While differences between CsA and RPM in influencing apoptosis of activated graft-destructive T(eff) cells are apparent, their effects on graft-protective T(reg) cells remain enigmatic. Moreover, it is unclear whether tolerizing regimens foster conversion of naïve peripheral T cells into alloantigen-specific T(reg) cells for graft protection. Here we show, using reporter mice for T(reg) marker Foxp3, that RPM promotes de novo conversion of alloantigen-specific T(reg) cells, whereas CsA completely inhibits this process. Upon transfer, in vivo converted T(reg) cells potently suppress the rejection of donor but not third party skin grafts. Thus, the differential effects of RPM and CsA on T(eff) and T(reg) cells favor the use of RPM in shifting the balance of aggressive to protective type alloimmunity.

Extraction of human Langerhans cells: a method for isolation of epidermis-resident dendritic cells.

Langerhans cells (LCs) are immature dendritic cells in the epidermis that play a central role in T-lymphocyte mediated skin immunity. Upon activation with antigenic stimuli, they differentiate drastically into mature dendritic cells while migrating from the epidermis to regional lymph nodes. Thus, in order to study biological details of immature LCs, it is crucial to isolate epidermis-resident, immature LCs without dermal dendritic cell contamination. Methods for extracting LCs from human skin as well as in vitro derivation of LC-like cells from hematopoietic progenitor cells have been described previously, but the cell preparations can potentially contain a significant number of dendritic cells that are not identical to epidermal LCs. Here, we describe a technique by which purely epidermis-resident LCs are extracted from human skin. Following digestion of human skin with dispase, the epidermis was separated mechanically without any attached dermal component. The trypsinized epidermal cells were then fractionated by centrifugation with a discontinuous density gradient composed of bovine albumin and sodium metrizoate. The LC-enriched preparation thus obtained contained 80% to >90% CD1a+, E-cadherin+ cells that expressed Birbeck granules and the Lag protein. Consistent with their being at an immature stage, the freshly isolated LCs lacked the expression of CD83, a marker for mature dendritic cells. The purified LCs were able to activate allogeneic T cells, indicating that the cells retained T-cell stimulation ability even after extraction. Thus, the present work offers an opportunity for precise in vitro studies of epidermal LCs.

Role of astrocytes in antigen presentation and naive T-cell activation.

Astrocytes may have a role in antigen presentation in inflammatory diseases of the central nervous system (CNS) such as MS and EAE. In this study, we have assessed whether purified astrocyte cultures could stimulate naive CD4(+) or CD8(+) T-cells from TCR transgenic mice. As previously described, astrocytes sustained antigen-specific CD4(+) T-cell proliferation only in the presence of IFN-gamma, which promotes expression of both MHC class II and B7 molecules on astrocytes. In addition, we show that astrocytes also have the capacity to present antigens to naive CD8(+) T-cell and promote their proliferation. In one system, this CD8(+) T-cell proliferation was dependent on IFN-gamma-induced upregulation of B7 molecules on astrocytes. However, in a second TCR transgenic system, astrocytes could induce naive CD8(+) T-cell proliferation even in the absence of IFN-gamma. The possible implications of these findings for the pathophysiology of CNS inflammatory diseases are discussed.

Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development.

Nuclear factor of activated T cells (NFAT) is a critical regulator of early gene transcription in response to TCR-mediated signals. Here, we show that mice lacking both NFATp and NFAT4 develop a profound lymphoproliferative disorder likely due to a lowered threshold for TCR signaling coupled with increased resistance to apoptosis secondary to defective FasL expression. NFAT mutant mice also have allergic blepharitis, interstitial pneumonitis, and a 10(3) to 10(4) fold increase in serum IgG1 and IgE levels, secondary to a dramatic and selective increase in Th2 cytokines. This phenotype may be ascribed to unopposed occupancy of the IL-4 promoter by NFATc. Our data demonstrate that lymphoid homeostasis and Th2 activation require a critical balance among NFAT family members.

The transcription factor NFAT4 is involved in the generation and survival of T cells.

Nuclear factor of activated T cells (NFAT) is a family of four related transcription factors implicated in cytokine and early response gene expression in activated lymphocytes. Here we report that NFAT4, in contrast to NFATp and NFATc, is preferentially expressed in DP thymocytes. Mice lacking NFAT4 have impaired development of CD4 and CD8 SP thymocytes and peripheral T cells as well as hyperactivation of peripheral T cells. The thymic defect is characterized by increased apoptosis of DP thymocytes. The increased apoptosis and hyperactivation may reflect heightened sensitivity to TcR-mediated signaling. Further, mice lacking NFAT4 have impaired production of Bcl-2 mRNA and protein. NFAT4 thus plays an important role in the successful generation and survival of T cells.

Delayed lymphoid repopulation with defects in IL-4-driven responses produced by inactivation of NF-ATc.

The NF-AT family of transcription factors activates early immune response genes such as cytokines. In the adult, NF-ATc is expressed exclusively in the lymphoid system and is induced upon lymphocyte activation. NF-ATc null mutant mice die in utero of cardiac failure, precluding analysis of the role of NF-ATc in lymphocyte activation. By using RAG-2-deficient blastocyst complementation, we now demonstrate that young, highly chimeric mice lacking NF-ATc have impaired repopulation of both thymus and peripheral lymphoid organs. Furthermore, NF-ATc deficiency impaired T lymphocyte activation and secretion of IL-4. B lymphocytes displayed reduced proliferation and a selective loss of IL-4-driven immunoglobulin isotypes both in vivo and in vitro. Our data demonstrate that NF-ATc is essential for the optimal generation and function of mature T and B lineage cells, with an especially profound effect on IL-4-driven responses.

Chimeric peptides: a new approach to enhancing the immunogenicity of peptides with low MHC class I affinity: application in antiviral vaccination.

Recruitment of the CTL repertoire specific for subdominant epitopes that have a low MHC class I-binding affinity could be the way to achieve an efficient protective immunity against spontaneous tumors and viruses with high mutation rate. However, we have reported recently that subdominant peptides of influenza A Puerto Rico/8/34 (flu PR8) nucleoprotein (NP) with low Db affinity are only partially able to protect mice against lethal influenza infection. This seems to be due to their inability to recruit the specific CTL repertoire, and suggests that subdominant peptides could be used for vaccination only if they become highly immunogenic. In this work, we describe an approach that allows enhancement of the immunogenicity of every low affinity peptide presented by the Db molecule. It consists in producing chimeric peptides composed by amino acids from a high Db affinity peptide (NP366) in positions that interact with the MHC, and amino acids from low Db affinity nonimmunogenic influenza NP-derived peptides (NP17, NP97, NP330, and NP469) in positions that are exposed to the TCR. All chimeric peptides tested exhibited a high Db affinity and efficiently recruited the CTL repertoire specific for the corresponding low Db affinity peptide. Furthermore, vaccination with chimeric peptides that corresponded to subdominant NP17 and NP97 peptides induced a very potent anti-flu PR8 protective immunity.

Selectivity of the major histocompatibility complex class II presentation pathway of cortical thymic epithelial cell lines.

Major histocompatibility complex (MHC) restriction of the immune response is established during positive selection of T cells in the thymus. This occurs mainly through interactions of T cell receptor of developing thymocytes with MHC/peptide ligands on cortical thymic epithelial cells (TEC). An ongoing controversy concerns the origin and the role of peptides involved in the positive selection of thymocytes. Evidence provided here shows that processing of MHC class II complexes in cortical TEC differs from that of medullary TEC. Removal of the invariant chain associated with MHC class II complexes was rapid and complete in medullary TEC which present peptides from both exogenous and cytosolic origin. In cortical TEC, a large fraction of class II dimers remained associated with a 10-12-kDa fragment of invariant chain (Ii). Incomplete removal of Ii correlated with the inability of cortical TEC to present peptides from exogenous origin. However, presentation of peptides from cytosolic proteins by cortical TEC remained possible. Thus, most peptides from exogenous proteins may be excluded from participating in positive selection of CD4+ T cells by a mechanism limiting Ii breakdown.

Major histocompatibility complex class I presentation of exogenously acquired minor alloantigens initiates skin allograft rejection.

Major histocompatibility complex (MHC) class I molecules present peptides from endogenous proteins. However, in some cases class I-restricted peptides can also derive from exogenous antigens. This MHC class I exogenous presentation could be involved in minor histocompatibility antigen (mHAg)-disparate allograft rejection when donor alloantigens are not expressed in graft antigen-presenting cells (APC) that initiate the rejection mechanism. Here we addressed this question by using a skin graft experimental model where donors (H-2b or H-2d Tg beta-gal mice) expressed the mHAg like beta-galactosidase (beta-gal) in keratinocytes but not in Langerhans' cells (LC) which have an APC function. Rejection of Tg beta-gal skin by a beta-gal-specific CD8 cytotoxic T lymphocyte (CTL) effector mechanism should require presentation by donor and/or recipient LC of MHC class I-restricted peptides of exogenous beta-gal shed by keratinocytes. Indeed, our results showed that 1) H-2b Tg beta-gal skin was rejected by H-2bxs and H-2bxd recipients; 2) rejection was mediated by beta-gal-specific CD8+ CTL effectors; and 3) H-2bxd mice having rejected H-2b Tg beta-gal skin generated beta-gal-specific CTL restricted by H-2b and H-2d class I molecules and rejected subsequently grafted H-2d Tg beta-gal skin in an accelerated fashion, demonstrating that recipient LC have presented exogenous beta-gal-derived MHC class I epitopes. These results lead to the conclusion that MHC class I exogenous presentation of donor mHAg can initiate allograft rejection.

Protection against lethal viral infection by vaccination with nonimmunodominant peptides.

CTL response of H-2b mice to influenza PR8 virus is directed against the nucleoprotein (NP)-derived immunodominant 366-374 (NP366PR8) peptide presented by the Db molecule. However, NP has three nonimmunodominant peptides corresponding to the 17-25 (NP17), 55-63 (NP55), and 97-105 (NP97) sequences that have the Db consensus motifs and bind to the Db molecule with an intermediate (NP55) or low (NP17 and NP97) affinity. In a previous report, we have shown that NP55 peptide is naturally processed by infected cells. In the present work, we studied whether nonimmunodominant peptides can protect mice against viral infection. Antiviral protection was evaluated by measuring three parameters: survival after inoculation of a lethal dose of mouse-adapted PR8 virus, percentage of pulmonary lesions in surviving mice, and virus clearance from lungs of infected mice. Our results showed that immunization of B6 mice with nonimmunodominant peptides protected from PR8 virus infection, although less efficiently than immunization with the immunodominant NP366PR8 peptide. Protection was mediated by CD8 T cells. The efficacy of nonimmunodominant peptides correlated with their Db binding affinity; the low affinity binders NP17 and NP97 induced a weaker protection than the intermediate affinity binder NP55. A mixture of NP366PR8 and nonimmunodominant peptides gave a higher protection than NP366PR8 peptide alone. In conclusion, nonimmunodominant peptides protect against a viral infection with an efficacy that is proportional to their affinity for the restricting class I molecule.

CD4 T cell tolerance to nuclear proteins induced by medullary thymic epithelium.

Thymic epithelium is involved in negative selection, but its precise role in selecting the CD4 T cell repertoire remains elusive. By using two transgenic mice, we have investigated how medullary thymic epithelium (mTE) and bone marrow (BM)-derived cells contribute to tolerance of CD4 T cells to nuclear beta-galactosidase (beta-gal). CD4 T cells were not tolerant when beta-gal was expressed in thymic BM-derived cells. In contrast, CD4 T cells of mice expressing beta-gal in mTE were tolerized. Tolerance resulted from presentation of endogenous beta-gal by mTE cells but not from cross-priming. mTE cells presented nuclear beta-gal to a Th clone in vitro, while thymic dendritic cells did not. The data indicate that mTE but not thymic BM-derived cells can use a MHC class II endogenous presentation pathway to induce tolerance to nuclear proteins.

Medullary thymic epithelial cells induce tolerance to intracellular proteins.

The role of the medullary thymic epithelial cells in tolerance induction to MHC class I restricted self peptides has been analyzed by studying the beta-galactosidase (beta-gal)-specific cytotoxic T cell response of a transgenic mouse expressing beta-gal in the thymus, skin, and central nervous system (Tg beta-gal mouse). Our results showed that: 1) beta-gal expression in the thymus was limited in a subpopulation of medullary epithelial cells, and bone marrow-derived thymic cells were beta-gal-1; 2) Tg beta-gal mice did not mount an anti-beta-gal CTL response even in the presence of exogenous IL-2, while Tg beta-gal-->B6 chimeras responded to beta-gal as strongly as NTg beta-gal mice; 3) Tg beta-gal mice did not generate CTL against the immunodominant Kb-restricted beta-gal 497-504 peptide; 4) tolerance was due to the thymic epithelial cells that expressed beta-gal because nude mice grafted with thymus from Tg beta-gal mice were also unable to respond to beta-gal; 5) the Tg beta-gal mouse-derived beta-gal+ medullary epithelial TEC.X10 line presented the Kb-restricted beta-gal 497-504 epitope. In conclusion, these results demonstrate that medullary thymic epithelial cells induce a complete tolerance towards class I-restricted self peptides presented on their own surface.

The J alpha segment contributes to the affinity of V beta 6+ cells for vSAG-7 (Mls-1a) presented by I-A molecules.

Recognition of superantigens (SAG) by T cells is major histocompatibility complex (MHC) dependent but not MHC restricted. In the case of vSAG-7 (Mls-1a), encoded by the Mtv-7 provirus, I-E molecules play a dominant role in the vSAG-7-MHC-T-cell receptor (TCR) interaction, the I-A molecule being less important. vSAG-7 is recognized predominantly by T cells bearing the V beta 6 element, which are deleted in Mtv-7+ mice; this deletion is nearly complete in mice expressing I-E molecules, but only partial in mice expressing exclusively the I-A molecules of permissive haplotypes. In view of these data, we hypothesized that vSAG-7-specific V beta 6+ T cells have a large spectrum of affinities for the MHC-vSAG-7 complex and that all of them, even those with a relatively low affinity, recognize the I-E-vSAG-7 complex, while only those with high affinity can recognize the I-A-vSAG-7 complex. Fourteen CD4 V beta 6+ vSAG-7-specific clones were studied and classified into three groups of avidity, depending on their interactions with different I-E- I-A(+)-vSAG-7 permissive haplotypes. Sequencing of the alpha and beta chains of their TCR suggested that the affinity for the vSAG-7 is influenced by the J alpha element. Four out of six low-affinity T-cell clones possessed the transcript for the J alpha 34 segment. Furthermore, five out of six low-affinity T-cell clones had the GGSN sequence in their CDR3 alpha, while the sixth low affinity clone had the conservative substituted SGGN sequence. These results strongly suggest that the expression of the J alpha 34 segment confers a very weak reactivity to T cells recognizing vSAG-7.

A nonimmunodominant nucleoprotein-derived peptide is presented by influenza A virus-infected H-2b cells.

Influenza A virus-infected H-2b mice mount a CTL response directed against the nucleoprotein (NP) 366-374 but not against the NP 55-63 peptide, although both peptides fulfill the prerequisites for having high binding affinity toward the Db molecule. Two hypotheses have been proposed to explain the inability of B6 mice to respond to the NP 55-63 peptide: 1) B6 mice are tolerant to the NP 55-63 peptide and 2) NP 55-63 peptide is not naturally processed by H-2b cells. Our results show that 1) B6 mice possess a NP 55-63-specific CTL repertoire because their immunization with the NP 55-63 peptide itself recruits specific CTL; 2) NP 55-63 peptide is naturally processed by the virus-infected H-2b cells but its efficient presentation by the Db molecule requires higher amounts of NP than presentation of the NP 366-374 peptide; 3) NP 55-63 peptide is naturally presented in virus infected B6 mice, however, the quantity of Db/NP 55-63 complexes at the cell surface is sufficient to tolerize but not to recruit and stimulate specific CTL; and 4) NP 55-63 peptide binds to the Db molecule with a lower affinity than NP 366-374 does and this difference could explain the inefficient presentation of the NP 55-63 peptide by B6 cells. The involvement of the self-protein-derived nonimmunodominant peptides in self-tolerance and the possibility of using nonimmunodominant peptides of viral proteins for peptide vaccination are discussed.