ABSTRACT
Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.
Subject(s)
Amelogenesis Imperfecta , Autoantibodies , Celiac Disease , Polyendocrinopathies, Autoimmune , Humans , Amelogenesis Imperfecta/complications , Amelogenesis Imperfecta/immunology , Autoantibodies/immunology , Celiac Disease/complications , Celiac Disease/immunology , Immunoglobulin A/immunology , Polyendocrinopathies, Autoimmune/complications , Polyendocrinopathies, Autoimmune/immunology , Proteins/immunology , Proteins/metabolism , Ameloblasts/metabolism , Dental Enamel/immunology , Dental Enamel/metabolism , AIRE Protein/deficiency , Antigens/immunology , Antigens/metabolism , Intestines/immunology , Intestines/metabolismABSTRACT
BACKGROUND: Autoimmune polyendocrine syndrome type 1 (APS-1) is a life-threatening, autosomal recessive syndrome caused by autoimmune regulator (AIRE) deficiency. In APS-1, self-reactive T cells escape thymic negative selection, infiltrate organs, and drive autoimmune injury. The effector mechanisms governing T-cell-mediated damage in APS-1 remain poorly understood. METHODS: We examined whether APS-1 could be classified as a disease mediated by interferon-γ. We first assessed patients with APS-1 who were participating in a prospective natural history study and evaluated mRNA and protein expression in blood and tissues. We then examined the pathogenic role of interferon-γ using Aire-/-Ifng-/- mice and Aire-/- mice treated with the Janus kinase (JAK) inhibitor ruxolitinib. On the basis of our findings, we used ruxolitinib to treat five patients with APS-1 and assessed clinical, immunologic, histologic, transcriptional, and autoantibody responses. RESULTS: Patients with APS-1 had enhanced interferon-γ responses in blood and in all examined autoimmunity-affected tissues. Aire-/- mice had selectively increased interferon-γ production by T cells and enhanced interferon-γ, phosphorylated signal transducer and activator of transcription 1 (pSTAT1), and CXCL9 signals in multiple organs. Ifng ablation or ruxolitinib-induced JAK-STAT blockade in Aire-/- mice normalized interferon-γ responses and averted T-cell infiltration and damage in organs. Ruxolitinib treatment of five patients with APS-1 led to decreased levels of T-cell-derived interferon-γ, normalized interferon-γ and CXCL9 levels, and remission of alopecia, oral candidiasis, nail dystrophy, gastritis, enteritis, arthritis, Sjögren's-like syndrome, urticaria, and thyroiditis. No serious adverse effects from ruxolitinib were identified in these patients. CONCLUSIONS: Our findings indicate that APS-1, which is caused by AIRE deficiency, is characterized by excessive, multiorgan interferon-γ-mediated responses. JAK inhibition with ruxolitinib in five patients showed promising results. (Funded by the National Institute of Allergy and Infectious Diseases and others.).
Subject(s)
AIRE Protein , Interferon-gamma , Janus Kinase Inhibitors , Polyendocrinopathies, Autoimmune , Adult , Animals , Female , Humans , Male , Mice , AIRE Protein/deficiency , AIRE Protein/genetics , AIRE Protein/immunology , Autoantibodies/blood , Autoantibodies/immunology , Chemokine CXCL9/genetics , Interferon-gamma/genetics , Interferon-gamma/immunology , Janus Kinase Inhibitors/therapeutic use , Mice, Knockout , Nitriles/therapeutic use , Polyendocrinopathies, Autoimmune/genetics , Polyendocrinopathies, Autoimmune/drug therapy , Polyendocrinopathies, Autoimmune/immunology , Pyrazoles/therapeutic use , Pyrazoles/pharmacology , Pyrimidines/therapeutic use , T-Lymphocytes/immunology , Transcription Factors/genetics , Transcription Factors/immunology , Pilot Projects , Disease Models, Animal , Child , Adolescent , Middle AgedABSTRACT
BACKGROUND: Regulatory T cells (Treg) in diverse species include CD4+ and CD8+ T cells. In all species, CD8+ Treg have been only partially characterized and there is no rat model in which CD4+ and CD8+ FOXP3+ Treg are genetically tagged. RESULTS: We generated a Foxp3-EGFP rat transgenic line in which FOXP3 gene was expressed and controlled EGFP. CD4+ and CD8+ T cells were the only cells that expressed EGFP, in similar proportion as observed with anti-FOXP3 antibodies and co-labeled in the same cells. CD4+EGFP+ Treg were 5-10 times more frequent than CD8+EGFP+ Treg. The suppressive activity of CD4+ and CD8+ Treg was largely confined to EGFP+ cells. RNAseq analyses showed similarities but also differences among CD4+ and CD8+ EGFP+ cells and provided the first description of the natural FOXP3+CD8+ Treg transcriptome. In vitro culture of CD4+ and CD8+ EGFP- cells with TGFbeta and IL-2 generated induced EGFP+ Treg. CD4+ and CD8+ EGFP+ Treg were expanded upon in vivo administration of a low dose of IL-2. CONCLUSIONS: This new and unique rat line constitutes a useful model to identify and isolate viable CD4+ and CD8+ FOXP3+ Treg. Additionally, it allows to identify molecules expressed in CD8+ Treg that may allow to better define their phenotype and function not only in rats but also in other species.
Subject(s)
CD8-Positive T-Lymphocytes , T-Lymphocytes, Regulatory , Rats , Animals , T-Lymphocytes, Regulatory/metabolism , CD8-Positive T-Lymphocytes/metabolism , Interleukin-2/genetics , Interleukin-2/metabolism , Transforming Growth Factor beta/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolismABSTRACT
CD8+ Tregs have been long described and significant progresses have been made about their phenotype, their functional mechanisms, and their suppressive ability compared to conventional CD4+ Tregs. They are now at the dawn of their clinical use. In this review, we will summarize their phenotypic characteristics, their mechanisms of action, the similarities, differences and synergies between CD8+ and CD4+ Tregs, and we will discuss the biology, development and induction of CD8+ Tregs, their manufacturing for clinical use, considering open questions/uncertainties and future technically accessible improvements notably through genetic modifications.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Immune Tolerance/immunology , T-Lymphocytes, Regulatory/immunology , Animals , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Forkhead Transcription Factors/immunology , Forkhead Transcription Factors/metabolism , Humans , Immunotherapy, Adoptive/methods , Immunotherapy, Adoptive/trends , Phenotype , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes, Regulatory/metabolismABSTRACT
Autoimmune regulator (AIRE) deficiency in humans induces a life-threatening generalized autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), and no curative treatments are available. Several models of AIRE-deficient mice have been generated, and although they have been useful in understanding the role of AIRE in central tolerance, they do not reproduce accurately the APECED symptoms, and thus there is still a need for an animal model displaying APECED-like disease. We assessed, in this study, the potential of the rat as an accurate model for APECED. In this study, we demonstrate that in rat, AIRE is expressed by MHC class II (MCH-II)+ and MHC-II- medullary thymic epithelial cells in thymus and by CD4int conventional dendritic cells in periphery. To our knowledge, we generated the first AIRE-deficient rat model using zinc-finger nucleases and demonstrated that they display several of the key symptoms of APECED disease, including alopecia, skin depigmentation, and nail dystrophy, independently of the genetic background. We observed severe autoimmune lesions in a large spectrum of organs, in particular in the pancreas, and identified several autoantibodies in organs and cytokines such as type I IFNs and IL-17 at levels similar to APECED. Finally, we demonstrated a biased Ab response to IgG1, IgM, and IgA isotypes. Altogether, our data demonstrate that AIRE-deficient rat is a relevant APECED animal model, opening new opportunity to test curative therapeutic treatments.
Subject(s)
Autoimmune Diseases/immunology , Candidiasis/immunology , Immune Tolerance/immunology , Polyendocrinopathies, Autoimmune/immunology , Animals , Autoantibodies/immunology , Cytokines/immunology , Dendritic Cells/immunology , Disease Models, Animal , Epithelial Cells/immunology , Female , Genes, MHC Class II/immunology , Male , Rats , Rats, Sprague-Dawley , Thymus Gland/immunologyABSTRACT
Interleukin-34 is a cytokine with only partially understood functions, described for the first time in 2008. Although IL-34 shares very little homology with CSF-1 (CSF1, M-CSF), they share a common receptor CSF-1R (CSF-1R) and IL-34 has also two distinct receptors (PTP-ζ) and CD138 (syndecan-1). To make the situation more complex, IL-34 has also been shown as pairing with CSF-1 to form a heterodimer. Until now, studies have demonstrated that this cytokine is released by some tissues that differ to those where CSF-1 is expressed and is involved in the differentiation and survival of macrophages, monocytes, and dendritic cells in response to inflammation. The involvement of IL-34 has been shown in areas as diverse as neuronal protection, autoimmune diseases, infection, cancer, and transplantation. Our recent work has demonstrated a new and possible therapeutic role for IL-34 as a Foxp3+ Treg-secreted cytokine mediator of transplant tolerance. In this review, we recapitulate most recent findings on IL-34 and its controversial effects on immune responses and address its immunoregulatory properties and the potential of targeting this cytokine in human.
Subject(s)
Immunomodulation , Interleukins/metabolism , Animals , Clinical Trials as Topic , Humans , Macrophage Colony-Stimulating Factor/metabolism , Models, Biological , Molecular Targeted TherapyABSTRACT
In transplantation tolerance, numerous regulatory populations have the capacity to inhibit allograft rejection; however, their compensatory capacities have never been clearly evidenced. We have previously demonstrated that the tolerogenic effect mediated by CD8(+)CD45RC(low) regulatory T cells (Tregs) in a model of organ transplantation with CD40Ig could be abrogated by permanent depletion of CD8(+) cells that resulted in allograft rejection in half of the recipients. This result demonstrated that CD8(+) Tregs were essential, but also that half of the recipients still survived indefinitely. We also demonstrated that no other regulatory populations, besides CD8(+) Tregs, could induce and maintain allograft tolerance in CD40Ig-treated tolerant animals. In the current study, we analyzed the mechanisms that arose following CD8(+) Treg depletion and allowed establishment of networks of new regulatory cells to maintain allograft survival. We identified regulatory B cells (Bregs) and regulatory myeloid cells (RegMCs) as being responsible of the maintenance of the long-term allograft survival. We demonstrated that both regulatory cell subsets efficiently inhibited antidonor immune responses in adoptively transferred recipients. Although Bregs were induced, they were not essential for the maintenance of the graft as demonstrated in IgM-deficient recipients. In addition, we showed that RegMCs were the most suppressive and acted alone, whereas Bregs activity was associated with increased suppressive activity of other subsets in adoptively transferred recipients. Altogether, to our knowledge, we demonstrated in this study for the first time the emergence of both Bregs and RegMCs following Tregs depletion and highlighted the importance of regulatory cell networks and their synergistic potential in transplantation.
Subject(s)
B-Lymphocytes, Regulatory/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Graft Rejection/immunology , Heart Transplantation , Myeloid Cells/immunology , Adoptive Transfer , Animals , B-Lymphocytes, Regulatory/transplantation , Cell Communication , Cells, Cultured , Immunomodulation , Myeloid Cells/transplantation , Rats , Rats, Inbred Lew , Transplantation ToleranceABSTRACT
A reverse-genetics approach has been used to probe the mechanism underlying immune escape for influenza A virus-specific CD8(+) T cells responding to the immunodominant D(b)NP366 epitope. Engineered viruses with a substitution at a critical residue (position 6, P6M) all evaded recognition by WT D(b)NP366-specific CD8(+) T cells, but only the NPM6I and NPM6T mutants altered the topography of a key residue (His155) in the MHC class I binding site. Following infection with the engineered NPM6I and NPM6T influenza viruses, both mutations were associated with a substantial "hole" in the naïve T-cell receptor repertoire, characterized by very limited T-cell receptor diversity and minimal primary responses to the NPM6I and NPM6T epitopes. Surprisingly, following respiratory challenge with a serologically distinct influenza virus carrying the same mutation, preemptive immunization against these escape variants led to the generation of secondary CD8(+) T-cell responses that were comparable in magnitude to those found for the WT NP epitope. Consequently, it might be possible to generate broadly protective T-cell immunity against commonly occurring virus escape mutants. If this is generally true for RNA viruses (like HIV, hepatitis C virus, and influenza) that show high mutation rates, priming against predicted mutants before an initial encounter could function to prevent the emergence of escape variants in infected hosts. That process could be a step toward preserving immune control of particularly persistent RNA viruses and may be worth considering for future vaccine strategies.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/immunology , Immune Evasion/genetics , Influenza A virus/genetics , Influenza A virus/immunology , Models, Molecular , beta 2-Microglobulin/chemistry , Animals , Binding Sites/genetics , CD8-Positive T-Lymphocytes/virology , Crystallization , Epitopes, T-Lymphocyte/genetics , Flow Cytometry , Genes, MHC Class I/genetics , Mice , Mice, Inbred C57BL , Mutagenesis, Site-Directed , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , beta 2-Microglobulin/immunologyABSTRACT
T cell cross-reactivity between different strains of the same virus, between different members of the same virus group, and even between unrelated viruses is a common occurrence. We questioned here how an intervening infection with a virus containing a sub-dominant cross-reactive T cell epitope would affect protective immunity to a previously encountered virus. Pichinde virus (PV) and lymphocytic choriomeningitis virus (LCMV) encode subdominant cross-reactive NP205â212 CD8 T cell epitopes sharing 6 of 8 amino acids, differing only in the MHC anchoring regions. These pMHC epitopes induce cross-reactive but non-identical T cell receptor (TCR) repertoires, and structural studies showed that the differing anchoring amino acids altered the conformation of the MHC landscape presented to the TCR. PV-immune mice receiving an intervening infection with wild type but not NP205-mutant LCMV developed severe immunopathology in the form of acute fatty necrosis on re-challenge with PV, and this pathology could be predicted by the ratio of NP205-specific to the normally immunodominant PV NP38â45-specific T cells. Thus, cross-reactive epitopes can exert pathogenic properties that compromise protective immunity by impairing more protective T cell responses.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/immunology , Lymphocytic Choriomeningitis/immunology , Pichinde virus/immunology , Receptors, Antigen, T-Cell/immunology , Viral Proteins/immunology , Animals , CD8-Positive T-Lymphocytes/metabolism , Cross Reactions/immunology , Epitopes, T-Lymphocyte/genetics , Lymphocytic Choriomeningitis/genetics , Lymphocytic choriomeningitis virus , Male , Mice , Mutation , Pichinde virus/genetics , Receptors, Antigen, T-Cell/genetics , Viral Proteins/geneticsABSTRACT
Pathogen-specific responses are characterized by preferred profiles of peptide+class I MHC (pMHCI) glycoprotein-specific T-cell receptor (TCR) Variable (V)-region use. How TCRV-region bias impacts TCRαß heterodimer selection and resultant diversity is unclear. The D(b)PA(224)-specific TCR repertoire in influenza A virus-infected C57BL/6J (B6) mice exhibits a preferred TCRV-region bias toward the TRBV29 gene segment and an optimal complementarity determining region (CDR3) ß-length of 6 aa. Despite these restrictions, D(b)PA(224)-specific BV29(+) T cells use a wide array of unique CDR3ß sequences. Structural characterization of a single, TRBV29(+)D(b)P(A224)-specific TCRαß-pMHCI complex demonstrated that CDR3α amino acid side chains made specific peptide interactions, but the CDR3ß main chain exclusively contacted peptides. Thus, length but not amino acid sequence was key for recognition and flexibility in Vß-region use. In support of this hypothesis, retrovirus expression of the D(b)PA(224)-specific TCRVα-chain was used to constrain pairing within a naive/immune epitope-specific repertoire. The retrogenic TCRVα paired with a diversity of CDR3ßs in the context of a preferred TCRVß spectrum. Overall, these data provide an explanation for the combination of TCRV region bias and diversity within selected repertoires, even as they maintain exquisite pMHCI specificity.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Influenza A Virus, H3N2 Subtype/immunology , Orthomyxoviridae Infections/immunology , Receptors, Antigen, T-Cell/immunology , Amino Acid Sequence , Animals , Base Sequence , CD8-Positive T-Lymphocytes/metabolism , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , Flow Cytometry , Genetic Variation , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Sequence Data , Orthomyxoviridae Infections/virology , Protein Conformation , Protein Multimerization , Protein Refolding , Protein Structure, Secondary , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunologyABSTRACT
Biomarkers that could predict the evolution of the graft in transplanted patients and that could allow to adapt the care of the patients would be an invaluable tool. Additionally, certain biomarkers can be target of treatments and help to stratify patients. Potential effective biomarkers have been identified but still need to be confirmed. CD45RC, one of the splicing variants of the CD45 molecule, a tyrosine phosphatase that is critical in negatively or positively regulating the TCR and the BCR signaling, is one marker already described. The frequency of CD8+ T cells expressing high levels of CD45RC before transplantation is increased in patients with an increased risk of acute rejection. However, single biomarkers have limited predictive reliability and the correlation of the expression levels of CD45RC with other cell markers was not reported. In this study, we performed a fluorescent-based high dimensional immunophenotyping of T cells on a cohort of 69 kidney transplant patients either with stable graft function or having experienced acute transplant rejection during the first year after transplantation or at the time of rejection. We identified combinations of markers and cell subsets associated with activation/inflammation or Tregs/tolerance (HLA-DR, PD-1, IFNγ, CD28) as significant biomarkers associated to transplant outcome, and showed the importance of cell segregation based on the CD45RC marker to identify the signature of a stable graft function. Our study highlights potential reliable biomarkers in transplantation to predict and/or monitor easily graft-directed immune responses and adapt immunosuppression treatments to mitigate adverse effects.
Subject(s)
CD8-Positive T-Lymphocytes , HLA-DR Antigens , Humans , Reproducibility of Results , Leukocyte Common Antigens/metabolism , Graft Rejection , Protein Tyrosine Phosphatases , BiomarkersABSTRACT
High-avidity interactions between TCRs and peptide + class I MHC (pMHCI) epitopes drive CTL activation and expansion. Intriguing questions remain concerning the constraints determining optimal TCR/pMHCI binding. The present analysis uses the TCR transgenic OT-I model to assess how varying profiles of TCR/pMHCI avidity influence naive CTL proliferation and the acquisition of effector function following exposure to the cognate H-2K(b)/OVA(257-264) (SIINFEKL) epitope and to mutants provided as peptide or in engineered influenza A viruses. Stimulating naive OT-I CD8(+) T cells in vitro with SIINFEKL induced full CTL proliferation and differentiation that was largely independent of any need for costimulation. By contrast, in vitro activation with the low-affinity EIINFEKL or SIIGFEKL ligands depended on the provision of IL-2 and other costimulatory signals. Importantly, although they did generate potent endogenous responses, infection of mice with influenza A viruses expressing these same OVA(257) variants failed to induce the activation of adoptively transferred naive OT-I CTLps, an effect that was only partially overcome by priming with a lipopeptide vaccine. Subsequent structural and biophysical analysis of H2-K(b)OVA(257), H2-K(b)E1, and H2-K(b)G4 established that these variations introduce small changes at the pMHCI interface and decrease epitope stability in ways that would likely impact cell surface presentation and recognition. Overall, it seems that there is an activation threshold for naive CTLps, that minimal alterations in peptide sequence can have profound effects, and that the antigenic requirements for the in vitro and in vivo induction of CTL proliferation and effector function differ substantially.
Subject(s)
Lymphocyte Activation/immunology , Ovalbumin/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/immunology , Influenza A virus/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovalbumin/chemistry , Peptide Fragments/chemistry , Peptide Fragments/immunology , Polymerase Chain Reaction , Protein Binding , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolismABSTRACT
Residues within processed protein fragments bound to major histocompatibility complex class I (MHC-I) glycoproteins have been considered to function as a series of "independent pegs" that either anchor the peptide (p) to the MHC-I and/or interact with the spectrum of alphabeta-T-cell receptors (TCRs) specific for the pMHC-I epitope in question. Mining of the extensive pMHC-I structural database established that many self- and viral peptides show extensive and direct interresidue interactions, an unexpected finding that has led us to the idea of "constrained" peptides. Mutational analysis of two constrained peptides (the HLA B44 restricted self-peptide (B44DPalpha-EEFGRAFSF) and an H2-D(b) restricted influenza peptide (D(b)PA, SSLENFRAYV) demonstrated that the conformation of the prominently exposed arginine in both peptides was governed by interactions with MHC-I-orientated flanking residues from the peptide itself. Using reverse genetics in a murine influenza model, we revealed that mutation of an MHC-I-orientated residue (SSLENFRAYV --> SSLENARAYV) within the constrained PA peptide resulted in a diminished cytotoxic T lymphocyte (CTL) response and the recruitment of a limited pMHC-I specific TCR repertoire. Interactions between individual peptide positions can thus impose fine control on the conformation of pMHC-I epitopes, whereas the perturbation of such constraints can lead to a previously unappreciated mechanism of viral escape.
Subject(s)
Histocompatibility Antigens Class I/metabolism , T-Lymphocytes/immunology , Amino Acid Sequence , Amino Acid Substitution , Animals , Antigen Presentation , Epitopes/chemistry , Epitopes/genetics , Epitopes/metabolism , Female , H-2 Antigens/chemistry , H-2 Antigens/genetics , H-2 Antigens/metabolism , HLA-B Antigens/chemistry , HLA-B Antigens/genetics , HLA-B Antigens/metabolism , HLA-B44 Antigen , Histocompatibility Antigen H-2D , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/genetics , Humans , Ligands , Mice , Mice, Inbred C57BL , Models, Molecular , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/immunology , Protein Conformation , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/immunologyABSTRACT
Emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. Pre-existing CD8(+) T-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. However, mutational escape within the T cell epitopes is a substantial issue for virus control and vaccine design. Although mutations can result in a loss of T cell recognition, some variants generate cross-reactive T cell responses. In this study, we used reverse genetics to modify the influenza NP(336-374) peptide at a partially-solvent exposed residue (N->A, NPN3A mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive T cell responses. The engineered virus induced a diminished CD8(+) T cell response and selected a narrowed T cell receptor (TCR) repertoire within two V beta regions (V beta 8.3 and V beta 9). This can be partially explained by the H-2D(b)NPN3A structure that showed a loss of several contacts between the NPN3A peptide and H-2D(b), including a contact with His155, a position known to play an important role in mediating TCR-pMHC-I interactions. Despite these differences, common cross-reactive TCRs were detected in both the naïve and immune NPN3A-specific TCR repertoires. However, while the NPN3A epitope primes memory T-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. Such decreased CD8(+) responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. Furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. Thus, the protective efficacy of cross-reactive CD8(+) T cells recognising mutant viral epitopes depend on peptide-MHC-I structural interactions and functional avidity. Our study does not support vaccine strategies that include immunization against commonly selected cross-reactive variants with mutations at partially-solvent exposed residues that have characteristics comparable to NPN3A.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigens Class I/immunology , Influenza A virus/immunology , Lymphocyte Activation/immunology , Peptide Fragments/immunology , Viral Core Proteins/immunology , Animals , Antigens, Viral/immunology , Cross Reactions/immunology , Crystallography, X-Ray , Influenza A virus/genetics , Influenza Vaccines/immunology , Mice , Mice, Inbred C57BL , Mutation , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptides , Protein Structure, Secondary , Receptors, Antigen, T-Cell/immunology , Reverse Transcriptase Polymerase Chain Reaction , Viral Core Proteins/chemistry , Viral Core Proteins/geneticsABSTRACT
Despite accumulating evidence for the importance of allospecific CD8(+) regulatory T cells (Tregs) in tolerant rodents and free immunosuppression transplant recipients, mechanisms underlying CD8(+) Treg-mediated tolerance remain unclear. By using a model of transplantation tolerance mediated by CD8(+) Tregs following CD40Ig treatment in rats, in this study, we show that the accumulation of tolerogenic CD8(+) Tregs and plasmacytoid dendritic cells (pDCs) in allograft and spleen but not lymph nodes was associated with tolerance induction in vascularized allograft recipients. pDCs preferentially induced tolerogenic CD8(+) Tregs to suppress CD4(+) effector cells responses to first-donor Ags in vitro. When tolerogenic CD8(+) Tregs were not in contact with CD4(+) effector cells, suppression was mediated by IDO. Contact with CD4(+) effector cells resulted in alternative suppressive mechanisms implicating IFN-gamma and fibroleukin-2. In vivo, both IDO and IFN-gamma were involved in tolerance induction, suggesting that contact with CD4(+) effector cells is crucial to modulate CD8(+) Tregs function in vivo. In conclusion, CD8(+) Tregs and pDCs interactions were necessary for suppression of CD4(+) T cells and involved different mechanisms modulated by the presence of cell contact between CD8(+) Tregs, pDCs, and CD4(+) effector cells.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Heart Transplantation/immunology , Immune Tolerance/immunology , T-Lymphocytes, Regulatory/immunology , Adenoviridae/genetics , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/cytology , Cell Proliferation , Dendritic Cells/cytology , Dendritic Cells/immunology , Flow Cytometry , Genetic Vectors/genetics , Heart Transplantation/methods , Male , Models, Animal , Rats , Rats, Inbred Lew , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Spleen/cytology , Spleen/immunology , T-Lymphocytes, Regulatory/cytology , Transduction, Genetic , Transplantation, HomologousABSTRACT
Current influenza A virus vaccines do not generate significant immunity against serologically distinct influenza A virus subtypes and would thus be ineffective in the face of a pandemic caused by a novel variant emerging from, say, a wildlife reservoir. One possible solution would be to modify these vaccines so that they prime cross-reactive CD8(+) cytotoxic T lymphocytes (CTL) cell-mediated immunity directed at conserved viral epitopes. A further strategy is to use novel adjuvants, such as the immunomodulatory glycolipid alpha-galactosylceramide (alpha-GalCer). We show here that giving alpha-GalCer with an inactivated influenza A virus has the paradoxical effect of diminishing acute CTL immunity via natural killer T (NKT) cell-dependent expression of indoleamine 2,3-dioxygenase (IDO), an important mediator of immune suppression, while at the same time promoting the survival of long-lived memory CTL populations capable of boosting protection against heterologous influenza A virus challenge. This enhancement of memory was likely due to the alpha-GalCer-induced upregulation of prosurvival genes, such as bcl-2, and points to the potential of alpha-GalCer as an adjuvant for promoting optimal, vaccine-induced CD8(+) T cell memory.
Subject(s)
Immunization, Secondary , Immunologic Memory/immunology , Influenza A Virus, H3N2 Subtype/immunology , Influenza Vaccines/immunology , Lymphocyte Activation/immunology , Natural Killer T-Cells/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigen Presentation/immunology , Cell Differentiation/immunology , Cell Survival , Dendritic Cells/immunology , Female , Galactosylceramides/pharmacology , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Mice , Natural Killer T-Cells/cytology , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/drug effects , Up-RegulationABSTRACT
Immunotherapy using primary T cells has revolutionized medical care in some pathologies in recent years, but limitations associated to challenging cell genome edition, insufficient cell number production, the use of only autologous cells, and the lack of product standardization have limited its clinical use. The alternative use of T cells generated in vitro from human pluripotent stem cells (hPSCs) offers great advantages by providing a self-renewing source of T cells that can be readily genetically modified and facilitate the use of standardized universal off-the-shelf allogeneic cell products and rapid clinical access. However, despite their potential, a better understanding of the feasibility and functionality of T cells differentiated from hPSCs is necessary before moving into clinical settings. In this study, we generated human-induced pluripotent stem cells from T cells (T-iPSCs), allowing for the preservation of already recombined TCR, with the same properties as human embryonic stem cells (hESCs). Based on these cells, we differentiated, with high efficiency, hematopoietic progenitor stem cells (HPSCs) capable of self-renewal and differentiation into any cell blood type, in addition to DN3a thymic progenitors from several T-iPSC lines. In order to better comprehend the differentiation, we analyzed the transcriptomic profiles of the different cell types and demonstrated that HPSCs differentiated from hiPSCs had very similar profiles to cord blood hematopoietic stem cells (HSCs). Furthermore, differentiated T-cell progenitors had a similar profile to thymocytes at the DN3a stage of thymic lymphopoiesis. Therefore, utilizing this approach, we were able to regenerate precursors of therapeutic human T cells in order to potentially treat a wide range of diseases.
Subject(s)
Induced Pluripotent Stem Cells , Pluripotent Stem Cells , Humans , Thymocytes/metabolism , Pluripotent Stem Cells/metabolism , Hematopoietic Stem Cells/metabolism , Antigens, CD34/metabolism , Induced Pluripotent Stem Cells/metabolism , Cell Adhesion Molecules/metabolismABSTRACT
BACKGROUND AND OBJECTIVES: Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the CNS. Studies of immune dysfunction in MS have mostly focused on CD4+ Tregs, but the role of CD8+ Tregs remains largely unexplored. We previously evidenced the suppressive properties of rat and human CD8+CD45RClow/neg Tregs from healthy individuals, expressing Forkhead box P3 (FOXP3) and acting through interferon-gamma (IFN-γ), transforming growth factor beta (TGFß), and interleukin-34 (IL-34). secretions to regulate immune responses and control diseases such as transplant rejection. To better understand CD8+CD45RClow/neg Tregs contribution to MS pathology, we further investigated their phenotype, function, and transcriptome in patients with MS. METHODS: We enrolled adults with relapsing-remitting MS and age-matched and sex-matched healthy volunteers (HVs). CD8+ T cells were segregated based on low or lack of expression of CD45RC. First, the frequency in CSF and blood, phenotype, transcriptome, and function of CD8+CD45RClow and neg were investigated according to exacerbation status and secondarily, according to clinical severity based on the MS severity score (MSSS) in patients with nonexacerbating MS. We then induced active MOG35-55 EAE in C57Bl/6 mice and performed adoptive transfer of fresh and expanded CD8+CD45RCneg Tregs to assess their ability to mitigate neuroinflammation in vivo. RESULTS: Thirty-one untreated patients with relapsing-remitting MS were compared with 40 age-matched and sex-matched HVs. We demonstrated no difference of CSF CD8+CD45RClow and CD8+CD45RCneg proportions, but blood CD8+CD45RClow frequency was lower in patients with MS exacerbation when compared with that in HVs. CD8+CD45RCneg Tregs but not CD8+CD45RClow showed higher suppressive capacities in vitro in MS patients with exacerbation than in patients without acute inflammatory attack. In vitro functional assays showed a compromised suppression capacity of CD8+CD45RClow Tregs in patients with nonexacerbating severe MS, defined by the MSSS. We then characterized murine CD8+CD45RCneg Tregs and demonstrated the potential of CD45RCneg cells to migrate to the CNS and mitigate experimental autoimmune encephalomyelitis in vivo. DISCUSSION: Altogether, these results suggest a defect in the number and function of CD8+CD45RClow Tregs during MS relapse and an association of CD8+CD45RClow Tregs dysfunction with MS severity. Thus, CD8+CD45RClow/neg T cells might bring new insights into the pathophysiology and new therapeutic approaches of MS.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Multiple Sclerosis, Relapsing-Remitting , Multiple Sclerosis , Humans , Adult , Mice , Rats , Animals , T-Lymphocytes, Regulatory/metabolism , CD8-Positive T-Lymphocytes , Multiple Sclerosis/metabolism , Interferon-gamma/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Mice, Inbred C57BL , Forkhead Transcription Factors/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
Targeted monoclonal antibody (mAb) therapies show great promise for the treatment of transplant rejection and autoimmune diseases by inducing more specific immunomodulatory effects than broadly immunosuppressive drugs routinely used. We recently described the therapeutic advantage of targeting CD45RC, expressed at high levels by conventional T (Tconv) cells (CD45RChi), their precursors, and terminally differentiated T (TEMRA) cells, but not by regulatory T cells (Tregs; CD45RClo/-). We demonstrated efficacy of anti-CD45RC mAb treatment in transplantation, but its potential has not been examined in autoimmune diseases. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare genetic syndrome caused by loss-of-function mutations of autoimmune regulator (AIRE), a key central tolerance mediator, leading to abnormal autoreactive T cell responses and autoantibody production. Herein, we show that, in a rat model of APECED syndrome, anti-CD45RC mAb was effective for both prevention and treatment of autoimmune manifestations and inhibited autoantibody development. Anti-CD45RC mAb intervention depleted CD45RChi T cells, inhibited CD45RChi B cells, and restored the Treg/Tconv cell ratio and the altered Treg transcriptomic profile. In APECED patients, CD45RC was significantly increased in peripheral blood T cells, and lesioned organs from APECED patients were infiltrated by CD45RChi cells. Our observations highlight the potential role for CD45RChi cells in the pathogenesis of experimental and human APECED syndrome and the potential of anti-CD45RC antibody treatment.
Subject(s)
Autoimmune Diseases , Polyendocrinopathies, Autoimmune , Animals , Autoantibodies , Humans , Immunotherapy , Polyendocrinopathies, Autoimmune/genetics , Polyendocrinopathies, Autoimmune/therapy , Rats , T-Lymphocytes, RegulatoryABSTRACT
BACKGROUND: Immune homeostasis requires fully functional Tregs with a stable phenotype to control autoimmunity. Although IL-34 is a cytokine first described as mainly involved in monocyte cell survival and differentiation, we recently described its expression by CD8+ Tregs in a rat model of transplantation tolerance and by activated FOXP3+ CD4+ and CD8+ Tregs in human healthy individuals. However, its role in autoimmunity and potential in human diseases remains to be determined. METHODS: We generated Il34-/- rats and using both Il34-/- rats and mice, we investigated their phenotype under inflammatory conditions. Using Il34-/- rats, we further analyzed the impact of the absence of expression of IL-34 for CD4+ Tregs suppressive function. We investigated the potential of IL-34 in human disease to prevent xenogeneic GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, taking advantage of a biocollection, we investigated the correlation between presence of IL-34 in the serum and kidney transplant rejection. RESULTS: Here we report that the absence of expression of IL-34 in Il34-/- rats and mice leads to an unstable immune phenotype, with production of multiple auto-antibodies, exacerbated under inflammatory conditions with increased susceptibility to DSS- and TNBS-colitis in Il34-/- animals. Moreover, we revealed the striking inability of Il34-/- CD4+ Tregs to protect Il2rg-/- rats from a wasting disease induced by transfer of pathogenic cells, in contrast to Il34+/+ CD4+ Tregs. We also showed that IL-34 treatment delayed EAE in mice as well as GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, we show that presence of IL-34 in the serum is associated with a longer rejection-free period in kidney transplanted patients. CONCLUSION: Altogether, our data emphasize on the crucial necessity of IL-34 for immune homeostasis and for CD4+ Tregs suppressive function. Our data also shows the therapeutic potential of IL-34 in human transplantation and auto-immunity. HIGHLIGHTS: -Absence of expression of IL-34 in Il34-/- rats and mice leads to an unstable immune phenotype, with a production of multiple auto-antibodies and exacerbated immune pathology under inflammatory conditions. -Il34-/- CD4+ Tregs are unable to protect Il2rg-/- rats from colitis induced by transfer of pathogenic cells. -IL-34 treatment delayed EAE in mice, as well as acute GVHD and human skin allograft rejection in immune-humanized immunodeficient NSG mice.