Autoantibodies (AAbs) are a hallmark of Type 1 diabetes (T1D). Alterations in the frequency and phenotype of follicular helper (Tfh) T cells have been previously documented in patients with type 1 ...diabetes (T1D), but the contribution of follicular regulatory T (Treg) cells, which are responsible for suppressing AAb development, is less clear. Here, we investigated the frequency and activation status of follicular (CXCR5+) and conventional (CXCR5−) Treg cells in the blood of children with new-onset T1D, and children with risk for developing T1D (AAb-positive) and compared them to AAb-negative controls. Blood follicular and conventional Treg cells were higher in frequency in children with new onset T1D, but expressed reduced amounts of PD-1 as compared to AAb-negative children. Interestingly, the proportion of circulating FOXP3+ Tregs expressing PD-1 was also reduced in AAb-positive at-risk children as compared to AAb-negative controls, suggesting its potential use as a biomarker of disease progression. Follicular Treg cells were reduced in frequency in the spleens of prediabetic NOD mice as they became older and turned diabetic. Interestingly, PD-1 expression declined also on circulating follicular and conventional Treg cells in prediabetic NOD mice as they aged. Together, these findings show that the frequency of circulating follicular and conventional Treg cells and their levels of PD-1 change with disease progression in children at-risk for developing T1D and in NOD mice.
The tolerogenic anti-CD3ε monoclonal Abs (anti-CD3) are promising compounds for the treatment of type 1 diabetes. Anti-CD3 administration induces transient T cell depletion both in preclinical and in ...clinical studies. Notably, the said depletion mainly affects CD4(+) but not CD8(+) T cells. Moreover, type 1 diabetes reversal in preclinical models is accompanied by the selective expansion of CD4(+)Foxp3(+) T regulatory (Treg) cells, which are fundamental for the long-term maintenance of anti-CD3-mediated tolerance. The mechanisms that lead to this immune-shaping by affecting mainly CD4(+) T effector cells while sparing CD4(+)Foxp3(+) Treg cells have still to be fully elucidated. This study shows that CD3 expression levels differ from one T cell subset to another. CD4(+)Foxp3(-) T cells contain higher amounts of CD3 molecules than do CD4(+)Foxp3(+) and CD8(+) T cells in both mice and humans. The said differences correlate with the anti-CD3-mediated immune resetting that occurs in vivo after anti-CD3 administration in diabetic NOD mice. Additionally, transcriptome analysis demonstrates that CD4(+)Foxp3(+) Treg cells are significantly less responsive than are CD4(+)Foxp3(-) T cells to anti-CD3 treatment at a molecular level. Thus, heterogeneity in CD3 expression seems to confer to the various T cell subsets differing susceptibility to the in vivo tolerogenic anti-CD3-mediated modulation. These data shed new light on the molecular mechanism that underlies anti-CD3-mediated immune resetting and thus may open new opportunities to improve this promising treatment.
Common variable immunodeficiency (CVID) is the most frequent primary antibody deficiency whereby follicular helper T (Tfh) cells fail to establish productive responses with B cells in germinal ...centers. Here, we analyzed the frequency, phenotype, transcriptome, and function of circulating Tfh (cTfh) cells in CVID patients displaying autoimmunity as an additional phenotype. A group of patients showed a high frequency of cTfh1 cells and a prominent expression of PD‐1 and ICOS as well as a cTfh mRNA signature consistent with highly activated, but exhausted, senescent, and apoptotic cells. Plasmatic CXCL13 levels were elevated in this group and positively correlated with cTfh1 cell frequency and PD‐1 levels. Monoallelic variants in RTEL1, a telomere length‐ and DNA repair‐related gene, were identified in four patients belonging to this group. Their blood lymphocytes showed shortened telomeres, while their cTfh were more prone to apoptosis. These data point toward a novel pathogenetic mechanism in CVID, whereby alterations in DNA repair and telomere elongation might predispose to antibody deficiency. A Th1, highly activated but exhausted and apoptotic cTfh phenotype was associated with this form of CVID.
Patients with common variable immunodeficiency with no previous genetic diagnosis presenting additionally one autoimmune symptom were compared to healthy subjects. Circulating follicular helper T (Tfh) cells were analyzed for their phenotype and function by flow cytometry, while their transcriptome was determined with RNA seq. Genetic analysis was performed by WES and validated by Sanger Sequencing. An integrated pipeline led to the identification of a cluster of patients characterized by a Th1 Tfh signature, elevated plasma CXCL13 levels, shortened telomeres, and enlarged lymph nodes and spleen. Rare monoallelic mutations in RTEL1 were identified as a possible common denominator. Further analyses revealed Tfh replicative exhaustion as a possible underlying mechanism.
Aims/hypothesis
Protein tyrosine phosphatase non-receptor 22 (PTPN22) plays a central role in T cell, B cell and innate immune cell signalling. A genetic variation in
Ptpn22
is considered a major ...risk factor for the development of type 1 diabetes and has been the subject of extensive study. While several reports have addressed how
Ptpn22
might predispose to autoimmunity, its involvement in other immune-mediated diseases, such as allograft rejection, has not been explored.
Methods
To address a possible function for
Ptpn22
in allograft rejection, we used a mouse model of pancreatic islet transplantation. We performed transplant tolerance experiments and determined how PTPN22 shapes tolerance induction and maintenance.
Results
Ptpn22
−/−
recipient mice generate higher numbers of alloreactive T cells after allogeneic pancreatic islet transplantation compared with wild-type (WT) mice, but reject grafts with similar kinetics. This is not only due to their well-documented increase in forkhead box protein P3 (FOXP3)
+
T regulatory (Treg) cells but also to the expansion of T regulatory type 1 (Tr1) cells caused by the lack of PTPN22. In addition, a tolerogenic treatment known to induce transplant tolerance in WT mice via Tr1 cell generation is more effective in
Ptpn22
−/−
mice as a consequence of boosting both Tr1 and FOXP3
+
Treg cells.
Conclusions/interpretation
A lack of PTPN22 strengthens transplant tolerance to pancreatic islets by expanding both FOXP3
+
Treg and Tr1 cells. These data suggest that targeting PTPN22 could serve to boost transplant tolerance.
Abstract Ptpn22 is one of the most potent autoimmunity predisposing genes and strongly associates with type 1 diabetes (T1D). Previous studies showed that non-obese diabetic mice with reduced ...expression levels of Ptpn22 are protected from T1D due to increased number of T regulatory (Treg) cells. We report that lack of Ptpn22 exacerbates virally-induced T1D in female rat insulin promoter lymphocytic choriomeningitis virus (RIP-LCMV-GP) mice, while maintaining higher number of Treg cells throughout the antiviral response in the blood and spleen but not in the pancreatic lymph nodes. GP33–41 -specific pentamer-positive cytotoxic lymphocytes (CTLs) are numerically reduced in the absence of Ptpn22 at the expansion and contraction phase but reach wild-type levels at the memory phase. However, they show similar effector function and even a subtle increase in the production of IL-2. In contrast, NP396–404 -specific CTLs develop normally at all phases but display enhanced effector function. Lack of Ptpn22 also augments the memory proinflammatory response of GP61–80 CD4 T cells. Hence, lack of Ptpn22 largely augments antiviral effector T cell responses, suggesting that caution should be taken when targeting Ptpn22 to treat autoimmune diseases where viral infections are considered environmental triggers.
PTPN22 (protein tyrosine phosphatase non receptor 22) encodes a tyrosine phosphatase that functions as a key regulator of immune homeostasis. In particular, PTPN22 inhibits T‐cell receptor signaling ...and selectively promotes type I interferon responses in myeloid cells. To date, there is little information on the CD8 T‐cell‐intrinsic role of PTPN22 in response to a viral pathogen. We unexpectedly found that PTPN22‐deficient virus‐specific CD8 T cells failed to accumulate in wild‐type hosts after lymphocytic choriomeningitis virus infection. Lack of PTPN22 expression altered CD8 T‐cell activation and antiviral cytokine production, but did not significantly affect the composition of effector and memory cell precursors. Most significantly, in vivo, PTPN22‐deficient CD8 T cells showed a profound defect in upregulating STAT‐1 after lymphocytic choriomeningitis virus infection and considerably less phosphorylation of STAT‐1 in response to IFN‐α treatment in vitro compared with their wild‐type counterparts. In stark contrast, following transfer into lymphopenic mice, CD8 T‐cell expansion and central‐like phenotype, was considerably increased in the absence of PTPN22. Collectively, our results suggest that PTPN22 has dual roles in T‐cell clonal expansion and effector function; whereas it promotes antigen‐driven responses during acute infection by positively regulating interferon signaling in T cells, PTPN22 inhibits homeostatic‐driven proliferation.
Type 1 regulatory T (Tr1) cells are an inducible subset of CD4 super(+) Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to ...generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10-ducing Tr1 cell population by transducing human CD4 super(+) T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP super(+) LV-IL-10-transduced human CD4 super(+) T (CD4 super(LV-IL-10)) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4 super(LV-IL-10) T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)- beta dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4 super(LV-IL-10) T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4 super(+) T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.
Restoration of endogenous insulin production by islet transplantation is considered a curative option for patients with type 1 diabetes. However, recurrent autoimmunity and alloreactivity cause graft ...rejection hindering successful transplantation. Here we tested whether transplant tolerance to allogeneic islets could be achieved in non-obese diabetic (NOD) mice by simultaneously tackling autoimmunity via antigen-specific immunization, and alloreactivity via granulocyte colony stimulating factor (G-CSF) and rapamycin (RAPA) treatment. Immunization with insB9-23 peptide alone or in combination with two islet peptides (IGRP206-214 and GAD524-543) in incomplete Freund's adjuvant (IFA) were tested for promoting syngeneic pancreatic islet engraftment in spontaneously diabetic NOD mice. Treatment with G-CSF/RAPA alone or in combination with insB9-23/IFA was examined for promoting allogeneic islet engraftment in the same mouse model. InsB9-23/IFA immunization significantly prolonged syngeneic pancreatic islet survival in NOD mice by a mechanism that necessitated the presence of CD4+CD25+ T regulatory (Treg) cells, while combination of three islet epitopes was less efficacious in controlling recurrent autoimmunity. G-CSF/RAPA treatment was unable to reverse T1D or control recurrent autoimmunity but significantly prolonged islet allograft survival in NOD mice. Blockade of interleukin-10 (IL-10) during G-CSF/RAPA treatment resulted in allograft rejection suggesting that IL-10-producing cells were fundamental to achieve transplant tolerance. G-CSF/RAPA treatment combined with insB9-23/IFA did not further increase the survival of allogeneic islets. Thus, insB9-23/IFA immunization controls recurrent autoimmunity and G-CSF/RAPA treatment limits alloreactivity, however their combination does not further promote allogeneic pancreatic islet engraftment in NOD mice.
The destruction of beta cells in type 1 diabetes (T1D) results in loss of insulin production and glucose homeostasis. Treatment of non-obese diabetic (NOD) mice with immune-depleting/modulating ...agents (e.g., anti-CD3, murine anti-thymocyte-globulin (mATG)) can lead to diabetes reversal. However, for preclinical studies with these and other agents seeking to reverse disease at onset, the necessity for exogenous insulin administration is debated. Spontaneously diabetic NOD mice were treated with a short-course of mATG and insulin provided as drug therapy or by way of allogeneic islet implants. Herein we demonstrate that exogenous insulin administration is required to achieve disease reversal with mATG in NOD mice. Unexpectedly, we also observed that provision of insulin by way of allogeneic islet implantation in combination with mATG leads to a pronounced reversal of diabetes as well as restoration of tolerance to self-islets. Expansion/induction of regulatory cells was observed in NOD mice stably cured with mATG and allogeneic islets. These data suggest that transient provision of allogeneic insulin-producing islets might provide a temporary window for immune depletion to be more effective and instilling stable tolerance to endogenous beta cells. These findings support the use of a never before explored approach for preserving beta cell function in patients with recent onset T1D.
PDF
