Alterations in regulatory T-cells: rediscovered pathways in immunotoxicology

In addition to the effector T-cells subsets, T-cells can also differentiate into cells that play a suppressive or regulatory role in adaptive immune responses. The cell types currently identified as regulatory T-cells (T(regs)) include natural or thymic-derived T(regs), T-cells which express Foxp3(+)CD25(+)CD4(+) and can suppress immune responses to autoreactive T-cells, as well as inducible T(regs), that are generated from na\uefve T-cells in the periphery after interaction with antigens presented by dendritic cells. Inducible T(regs) include T(H)3 cells, T(r)1 cells, and Foxp3(+)-inducible T(regs). T(regs) have been shown to be critical in the maintenance of immune responses and T-cell homeostasis. These cells play an important role in suppressing responses to self-antigens and in controlling inappropriate responses to non-self-antigens, such as commensal bacteria or food in the gut. For example, depletion of CD4(+)CD25(+) T(regs) from mice resulted in the development of multi-organ autoimmune diseases. CD4(+)CD25(+) T(regs) and/or IL-10-producing T(r)1 cells are capable of suppressing or attenuating T(H)2 responses to allergens. Moreover, adoptive transfer of CD4(+)CD25(+) T(regs) from healthy to diseased animals resulted in the prevention or cure of certain autoimmune diseases, and was able to induce transplantation tolerance. Clinical improvement seen after allergen immunotherapy for allergic diseases such as rhinitis and asthma is associated with the induction of IL-10- and TGF\u3b2-producing T(r)1 cells as well as FoxP3-expressing IL-10 T-cells, with resulting suppression of the T(H)2 cytokine milieu. Activation, expansion, or suppression of CD4(+)CD25(+) T(regs) in vivo by xenobiotics, including drugs, may therefore represent a relevant mechanism underlying immunotoxicity, including immunosuppression, allergic asthma, and autoimmune diseases

Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression

The study of T regulatory cells (T reg cells) has been limited by the lack of specific surface markers and an inability to define mechanisms of suppression. We show that the expression of CD39/ENTPD1 in concert with CD73/ecto-5′-nucleotidase distinguishes CD4+/CD25+/Foxp3+ T reg cells from other T cells. These ectoenzymes generate pericellular adenosine from extracellular nucleotides. The coordinated expression of CD39/CD73 on T reg cells and the adenosine A2A receptor on activated T effector cells generates immunosuppressive loops, indicating roles in the inhibitory function of T reg cells. Consequently, T reg cells from Cd39-null mice show impaired suppressive properties in vitro and fail to block allograft rejection in vivo. We conclude that CD39 and CD73 are surface markers of T reg cells that impart a specific biochemical signature characterized by adenosine generation that has functional relevance for cellular immunoregulation

Rac1 Deletion Causes Thymic Atrophy

The thymic stroma supports T lymphocyte development and consists of an epithelium maintained by thymic epithelial progenitors. The molecular pathways that govern epithelial homeostasis are poorly understood. Here we demonstrate that deletion of Rac1 in Keratin 5/Keratin 14 expressing embryonic and adult thymic epithelial cells leads to loss of the thymic epithelial compartment. Rac1 deletion led to an increase in c-Myc expression and a generalized increase in apoptosis associated with a decrease in thymic epithelial proliferation. Our results suggest Rac1 maintains the epithelial population, and equilibrium between Rac1 and c-Myc may control proliferation, apoptosis and maturation of the thymic epithelial compartment. Understanding thymic epithelial maintenance is a step toward the dual goals of in vitro thymic epithelial cell culture and T cell differentiation, and the clinical repair of thymic damage from graft-versus-host-disease, chemotherapy or irradiation

CD4+CCR6+ T cells, but not γδ T cells, are important for the IL-23R-dependent progression of antigen-induced inflammatory arthritis in mice

IL-23 plays an important role in the development of arthritis and the IL-23 receptor (IL-23R) is expressed on different types of T cells. However, it is not fully clear which IL-23R+ T cells are critical in driving T cell-mediated synovitis. We demonstrate, using knock-in IL-23R-GFP reporter (IL-23RGFP/+) mice, that CD4+CCR6+ T cells and γδ T cells, but not CD8+ T cells, express the IL-23R(GFP). During early arthritis, IL-23R(GFP)+CD4+CCR6+ T cells, but not IL-23R(GFP)+ γδ T cells, were present in the inflamed joints. IL-23RGFP/+ mice were bred as homozygotes to obtain IL-23RGFP/GFP (IL-23R deficient/IL-23R−/−) mice, which express GFP under the IL-23R promotor. Arthritis progression and joint damage were significantly milder in IL-23R−/− mice, which revealed less IL-17A+ cells in their lymphoid tissues. Surprisingly, IL-23R−/− mice had increased numbers of IL-23R(GFP)+CD4+CCR6+ and CCR7+CD4+CCR6+ T cells in their spleen compared to WT, and IL-23 suppressed CCR7 expression in vitro. However, IL-23R(GFP)+CD4+CCR6+ T cells were present in the synovium of IL-23R−/− mice at day 4. Finally, adoptive transfer experiments revealed that CD4+CCR6+ T cells and not γδ T cells drive arthritis progression. These data suggest that IL-23R-dependent T cell-mediated synovitis is dependent on CD4+CCR6+ T cells and not on γδ T cells

Th17-Related Genes and Celiac Disease Susceptibility

Th17 cells are known to be involved in several autoimmune or inflammatory diseases. In celiac disease (CD), recent studies suggest an implication of those cells in disease pathogenesis. We aimed at studying the role of genes relevant for the Th17 immune response in CD susceptibility. A total of 101 single nucleotide polymorphisms (SNPs), mainly selected to cover most of the variability present in 16 Th17-related genes (IL23R, RORC, IL6R, IL17A, IL17F, CCR6, IL6, JAK2, TNFSF15, IL23A, IL22, STAT3, TBX21, SOCS3, IL12RB1 and IL17RA), were genotyped in 735 CD patients and 549 ethnically matched healthy controls. Case-control comparisons for each SNP and for the haplotypes resulting from the SNPs studied in each gene were performed using chi-square tests. Gene-gene interactions were also evaluated following different methodological approaches. No significant results emerged after performing the appropriate statistical corrections. Our results seem to discard a relevant role of Th17 cells on CD risk

Robust Antigen Specific Th17 T Cell Response to Group A Streptococcus Is Dependent on IL-6 and Intranasal Route of Infection

Group A streptococcus (GAS, Streptococcus pyogenes) is the cause of a variety of clinical conditions, ranging from pharyngitis to autoimmune disease. Peptide-major histocompatibility complex class II (pMHCII) tetramers have recently emerged as a highly sensitive means to quantify pMHCII-specific CD4+ helper T cells and evaluate their contribution to both protective immunity and autoimmune complications induced by specific bacterial pathogens. In lieu of identifying an immunodominant peptide expressed by GAS, a surrogate peptide (2W) was fused to the highly expressed M1 protein on the surface of GAS to allow in-depth analysis of the CD4+ helper T cell response in C57BL/6 mice that express the I-Ab MHCII molecule. Following intranasal inoculation with GAS-2W, antigen-experienced 2W:I-Ab-specific CD4+ T cells were identified in the nasal-associated lymphoid tissue (NALT) that produced IL-17A or IL-17A and IFN-γ if infection was recurrent. The dominant Th17 response was also dependent on the intranasal route of inoculation; intravenous or subcutaneous inoculations produced primarily IFN-γ+ 2W:I-Ab+ CD4+ T cells. The acquisition of IL-17A production by 2W:I-Ab-specific T cells and the capacity of mice to survive infection depended on the innate cytokine IL-6. IL-6-deficient mice that survived infection became long-term carriers despite the presence of abundant IFN-γ-producing 2W:I-Ab-specific CD4+ T cells. Our results suggest that an imbalance between IL-17- and IFN-γ-producing CD4+ T cells could contribute to GAS carriage in humans

Subdominant/Cryptic CD8 T Cell Epitopes Contribute to Resistance against Experimental Infection with a Human Protozoan Parasite

During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development

Viral Protein Fragmentation May Broaden T-Cell Responses to HIV Vaccines

High mutation rates of human immunodeficiency virus (HIV) allows escape from T cell recognition preventing development of effective T cell vaccines. Vaccines that induce diverse T cell immune responses would help overcome this problem. Using SIV gag as a model vaccine, we investigated two approaches to increase the breadth of the CD8 T cell response. Namely, fusion of vaccine genes to ubiquitin to target the proteasome and increase levels of MHC class I peptide complexes and gene fragmentation to overcome competition between epitopes for presentation and recognition.three vaccines were compared: full-length unmodified SIV-mac239 gag, full-length gag fused at the N-terminus to ubiquitin and 7 gag fragments of equal size spanning the whole of gag with ubiquitin-fused to the N-terminus of each fragment. Genes were cloned into a replication defective adenovirus vector and immunogenicity assessed in an in vitro human priming system. The breadth of the CD8 T cell response, defined by the number of distinct epitopes, was assessed by IFN-γ-ELISPOT and memory phenotype and cytokine production evaluated by flow cytometry. We observed an increase of two- to six-fold in the number of epitopes recognised in the ubiquitin-fused fragments compared to the ubiquitin-fused full-length gag. In contrast, although proteasomal targeting was achieved, there was a marked reduction in the number of epitopes recognised in the ubiquitin-fused full-length gag compared to the full-length unmodified gene, but there were no differences in the number of epitope responses induced by non-ubiquitinated full-length gag and the ubiquitin-fused mini genes. Fragmentation and ubiquitination did not affect T cell memory differentiation and polyfunctionality, though most responses were directed against the Ad5 vector.Fragmentation but not fusion with ubiquitin increases the breadth of the CD8 T vaccine response against SIV-mac239 gag. Thus gene fragmentation of HIV vaccines may maximise responses

Neoplastic Transformation of T Lymphocytes through Transgenic Expression of a Virus Host Modification Protein

Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4+CD8+CD69– lymphoma. The CD4+CD8+CD69– cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRβ-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4+CD8+CD69– thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4+CD8+ CD69– thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-)oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas