Engineered Treg cells: The heir to the throne of immunotherapy.

Recently, increased interest in the use of Tregs as adoptive cell therapy for the treatment of autoimmune diseases and transplant rejection had led to several advances in the field. However, Treg cell therapies, while constantly advancing, indiscriminately suppress the immune system without the permanent stabilization of certain diseases. Genetically modified Tregs hold great promise towards solving these problems, but, challenges in identifying the most potent Treg subtype, accompanied by the ambiguity involved in identifying the optimal Treg source, along with its expansion and engineering in a clinical-grade setting remain paramount. This review highlights the recent advances in methodologies for the development of genetically engineered Treg cell-based treatments for autoimmune, inflammatory diseases, and organ rejection. Additionally, it provides a systematized guide to all the recent progress in the field and informs the readers of the feasibility and safety of engineered adoptive Treg cell therapy, with the aim to provide a framework for researchers involved in the development of engineered Tregs.

Type 1 diabetes and inborn errors of immunity: Complete strangers or 2 sides of the same coin?

Type 1 diabetes (T1D) is a polygenic disease and does not follow a mendelian pattern. Inborn errors of immunity (IEIs), on the other hand, are caused by damaging germline variants, suggesting that T1D and IEIs have nothing in common. Some IEIs, resulting from mutations in genes regulating regulatory T-cell homeostasis, are associated with elevated incidence of T1D. The genetic spectrum of IEIs is gradually being unraveled; consequently, molecular pathways underlying human monogenic autoimmunity are being identified. There is an appreciable overlap between some of these pathways and the genetic variants that determine T1D susceptibility, suggesting that after all, IEI and T1D are 2 sides of the same coin. The study of monogenic IEIs with a variable incidence of T1D has the potential to provide crucial insights into the mechanisms leading to T1D. These insights contribute to the definition of T1D endotypes and explain disease heterogeneity. In this review, we discuss the interconnected pathogenic pathways of autoimmunity, ß-cell function, and primary immunodeficiency. We also examine the role of environmental factors in disease penetrance as well as the circumstantial evidence of IEI drugs in preventing and curing T1D in individuals with IEIs, suggesting the repositioning of these drugs also for T1D therapy.

Genetic determinants of type 1 diabetes in individuals with weak evidence of islet autoimmunity at disease onset.

AIMS/HYPOTHESIS: Islet autoantibodies (AAbs) are detected in >90% of individuals with clinically suspected type 1 diabetes at disease onset. A single AAb, sometimes at low titre, is often detected in some individuals, making their diagnosis uncertain. Type 1 diabetes genetic risk scores (GRS) are a useful tool for discriminating polygenic autoimmune type 1 diabetes from other types of diabetes, particularly the monogenic forms, but testing is not routinely performed in the clinic. Here, we used a type 1 diabetes GRS to screen for monogenic diabetes in individuals with weak evidence of autoimmunity, i.e. with a single AAb at disease onset. METHODS: In a pilot study, we genetically screened 142 individuals with suspected type 1 diabetes, 42 of whom were AAb-negative, 27 of whom had a single AAb (single AAb-positive) and 73 of whom had multiple AAbs (multiple AAb-positive) at disease onset. Next-generation sequencing (NGS) was performed in 41 AAb-negative participants, 26 single AAb-positive participants and 60 multiple AAb-positive participants using an analysis pipeline of more than 200 diabetes-associated genes. RESULTS: The type 1 diabetes GRS was significantly lower in AAb-negative individuals than in those with a single and multiple AAbs. Pathogenetic class 4/5 variants in MODY or monogenic diabetes genes were identified in 15/41 (36.6%) AAb-negative individuals, while class 3 variants of unknown significance were identified in 17/41 (41.5%). Residual C-peptide levels at diagnosis were higher in individuals with mutations compared to those without pathogenetic variants. Class 3 variants of unknown significance were found in 11/26 (42.3%) single AAb-positive individuals, and pathogenetic class 4/5 variants were present in 2/26 (7.7%) single AAb-positive individuals. No pathogenetic class 4/5 variants were identified in multiple AAb-positive individuals, but class 3 variants of unknown significance were identified in 19/60 (31.7%) patients. Several patients across the three groups had more than one class 3 variant. CONCLUSIONS/INTERPRETATION: These findings provide insights into the genetic makeup of patients who show weak evidence of autoimmunity at disease onset. Absence of islet AAbs or the presence of a single AAb together with a low type 1 diabetes GRS may be indicative of a monogenic form of diabetes, and use of NGS may improve the accuracy of diagnosis.

T1D patient-derived hematopoietic stem cells are programmed to generate Tph, Tfh, and autoimmunity-associated B cell subsets in human immune system mice.

Interactions between B cells and CD4+ T cells play a central role in the development of Type 1 Diabetes (T1D). Two helper cell subsets, follicular (Tfh) and peripheral (Tph) helper T cells, are increased in patients with T1D but their role in driving B cell autoimmunity is undefined. We used a personalized immune (PI) mouse model to generate human immune systems de novo from hematopoietic stem cells (HSCs) of patients with T1D or from healthy controls (HCs). Both groups developed Tfh and Tph-like cells, and those with T1D-derived immune systems demonstrated increased numbers of Tph-like and Tfh cells compared to HC-derived PI mice. T1D-derived immune systems included increased proportions of unconventional memory CD27-IgD- B cells and reduced proportions of naïve B cells compared to HC PI mice, resembling changes reported for patients with systemic lupus erythematosus. Our findings suggest that T1D HSCs are genetically programmed to produce increased proportions of T cells that promote the development of unconventional, possibly autoreactive memory B cells. PI mice provide an avenue for further understanding of the immune abnormalities that drive autoantibody pathogenesis and T1D.

CXCR5-CXCL13 axis markers in full-term and preterm human neonates in the first weeks of life.

Term and preterm neonates have very few circulating Tfh-like cells (cTfh), and no circulating Tfr-like cells. Neonatal cTfh are CXCR5lo PD-1lo CD45RAhi , suggestive of a naive, possibly recently activated phenotype. CXCL13 is high at birth, but decreases rapidly in the first weeks of life. Overall, signs of GC activity in human neonates are weak, even in those born prematurely or after sepsis.

NFKB2 regulates human Tfh and Tfr pool formation and germinal center potential.

Mutations affecting the non-canonical pathway of NF-κB were recently identified to underlie a form of common variable immunodeficiency strongly associated with autoimmunity. Although intrinsic B-cell abnormalities explain most of the humoral defects of this disease, detailed data on the impact of NFKB2 on follicular helper (Tfh) and regulatory (Tregs) T cells are scarce. Here, we show that Tfh, CXCR5+, and CXCR5- Treg cell subsets were significantly reduced in patients heterozygous for a truncating mutation of NFKB2. Plasma CXCL13 levels were reduced, underlining an important role for NFKB2 in regulating the germinal center (GC) response. Proinflammatory IFNγ, IL-17 and IL-10 cytokine production by CD4 T cells was lower in the mutated patients, but the production of IL-4 and IL-21 was not altered. Taken together, our findings show that NFKB2 influences the quality and efficiency of human GC reaction, by affecting not only the B cells but also GC-relevant T cell subsets.

Reduced PD-1 expression on circulating follicular and conventional FOXP3+ Treg cells in children with new onset type 1 diabetes and autoantibody-positive at-risk children.

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.

A Prevalent CXCR3+ Phenotype of Circulating Follicular Helper T Cells Indicates Humoral Dysregulation in Children with Down Syndrome.

Patients with Down syndrome (DS) are characterized by increased susceptibility to autoimmunity and respiratory tract infections that are suggestive of humoral immunity impairment. Here, we sought to determine the follicular helper (Tfh) and follicular regulatory (Tfr) T cell profile in the blood of children with DS. Blood was collected from 24 children with DS, nine of which had autoimmune diseases. Children with DS showed skewed Tfh differentiation towards the CXCR3+ phenotype: Tfh1 and Tfh1/17 subsets were increased, while Tfh2 and Tfh17 subsets were reduced. While no differences in the percentage of Tfr cells were seen, the ratio of Tfh1 and CXCR3+PD-1+ subsets to Tfr cells was significantly increased in the affected children. The excessive polarization towards a CXCR3+ phenotype in children with DS suggests that re-calibration of Tfh subset skewing could potentially offer new therapeutic opportunities for these patients.

Tr1 cell immunotherapy promotes transplant tolerance via de novo Tr1 cell induction in mice and is safe and effective during acute viral infection.

Tr1 cell therapy is considered an emerging approach to improve transplant tolerance and enhance allogeneic graft survival. However, it remains unclear how Tr1 cells promote transplant tolerance and whether they will be safe and stable in the face of an acute viral infection. By employing a mouse model of pancreatic islet transplantation, we report that Tr1 cell therapy promoted transplant tolerance via de novo induction of Tr1 cells in the recipients. Acute viral infection with lymphocytic choriomeningitis virus (LCMV) had no impact on Tr1 cell number and function, neither on the Tr1 cells infused nor on the ones induced, and that was reflected in the robust maintenance of the graft. Moreover, Tr1 cell immunotherapy had no detrimental effect on CD8 and CD4 anti-LCMV effector T-cell responses and viral control. Together, these data suggest that Tr1 cells did not convert to effector cells during acute infection with LCMV, maintained transplant tolerance and did not inhibit antiviral immunity.

Protein tyrosine phosphatase PTPN22 has dual roles in promoting pathogen versus homeostatic-driven CD8 T-cell responses.

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.

Lack of the protein tyrosine phosphatase PTPN22 strengthens transplant tolerance to pancreatic islets in mice.

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.

PTPN22 controls virally-induced autoimmune diabetes by modulating cytotoxic T lymphocyte responses in an epitope-specific manner.

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.

Regulatory T cells control diabetes without compromising acute anti-viral defense.

While previous reports have demonstrated the efficacy of regulatory T cell therapy in the prevention of diabetes, systemic immunocompromise and Treg instability remain key safety concerns. Here we examined the influence of induced Treg (iTreg) cell therapy on anti-viral host defense and autoimmune T cell responses during acute viral infection in a murine model of autoimmune diabetes. Protective transfers of iTregs maintained IL-10 expression, expanded in vivo and controlled diabetes, despite losing FoxP3 expression. Adoptive transfer of iTregs affected neither the primary anti-viral CD8 T cell response nor viral clearance, although a significant and sustained suppression of CD4 T cell responses was observed. Following acute viral clearance, iTregs transferred early suppressed both CD4 and CD8 T cell responses, which resulted in the reversion of diabetes. These observations indicate that iTregs suppress local autoimmune processes while preserving the immunocompetent host's ability to combat acute viral infection.

Follicular helper- and peripheral helper-like T cells drive autoimmune disease in human immune system mice.

Human immune system (HIS) mice constructed in various ways are widely used for investigations of human immune responses to pathogens, transplants and immunotherapies. In HIS mice that generate T cells de novo from hematopoietic progenitors, T cell-dependent multisystem autoimmune disease occurs, most rapidly when the human T cells develop in the native NOD.Cg- Prkdc scid Il2rg tm1Wjl (NSG) mouse thymus, where negative selection is abnormal. Disease develops very late when human T cells develop in human fetal thymus grafts, where robust negative selection is observed. We demonstrate here that PD-1 + CD4 + peripheral (Tph) helper-like and follicular (Tfh) helper-like T cells developing in HIS mice can induce autoimmune disease. Tfh-like cells were more prominent in HIS mice with a mouse thymus, in which the highest levels of IgG were detected in plasma, compared to those with a human thymus. While circulating IgG and IgM antibodies were autoreactive to multiple mouse antigens, in vivo depletion of B cells and antibodies did not delay the development of autoimmune disease. Conversely, adoptive transfer of enriched Tfh- or Tph-like cells induced disease and autoimmunity-associated B cell phenotypes in recipient mice containing autologous human APCs without T cells. T cells from mice with a human thymus expanded and induced disease more rapidly than those originating in a murine thymus, implicating HLA-restricted T cell-APC interactions in this process. Since Tfh, Tph, autoantibodies and LIP have all been implicated in various forms of human autoimmune disease, the observations here provide a platform for the further dissection of human autoimmune disease mechanisms and therapies.

Roles of the protein tyrosine phosphatase PTPN22 in immunity and autoimmunity.

PTPN22 is a protein tyrosine phosphatase expressed by the majority of cells belonging to the innate and adaptive immune systems. Polymorphisms in PTPN22 are associated with several autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis and type 1 diabetes. This review discusses the role of PTPN22 in T and B cells, and its function in innate immune cells, such as monocytes, dendritic cells and NK cells. We focus particularly on the complexity that underlies the function of PTPN22 in the biological processes of the immune system; such complexity has led various research groups to produce rather conflicting data.

Enforced IL-10 expression confers type 1 regulatory T cell (Tr1) phenotype and function to human CD4(+) T cells.

Type 1 regulatory T (Tr1) cells are an inducible subset of CD4(+) 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-producing Tr1 cell population by transducing human CD4(+) 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(+) LV-IL-10-transduced human CD4(+) T (CD4(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(LV-IL-10) T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-ß dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4(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(+) T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.

Case Report: Consistent disease manifestations with a staggered time course in two identical twins affected by adenosine deaminase 2 deficiency.

Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disease associated with a highly variable clinical presentation, including vasculitis, immunodeficiency, and hematologic manifestations, potentially progressing over time. The present study describes the long-term evolution of the immuno-hematological features and therapeutic challenge of two identical adult twin sisters affected by DADA2. The absence of plasmatic adenosine deaminase 2 (ADA2) activity in both twins suggested the diagnosis of DADA2, then confirmed by genetic analysis. Exon sequencing revealed a missense (p.Leu188Pro) mutation on the paternal ADA2 allele. While, whole genome sequencing identified an unreported deletion (IVS6_IVS7del*) on the maternal allele predicted to produce a transcript missing exon 7. The patients experienced the disease onset during childhood with early strokes (Patient 1 at two years, Patient 2 at eight years of age), subsequently followed by other shared DADA2-associated features, including neutropenia, hypogammaglobulinemia, reduced switched memory B cells, inverted CD4:CD8 ratio, increased naïve T cells, reduced follicular regulatory T cells, the almost complete absence of NK cells, T-large granular cell leukemia, and osteoporosis. Disease evolution differed: clinical manifestations presented several years earlier and were more pronounced in Patient 1 than in Patient 2. Due to G-CSF refractory life-threatening neutropenia, Patient 1 successfully underwent an urgent hematopoietic stem cell transplantation (HSCT) from a 9/10 matched unrelated donor. Patient 2 experienced a similar, although delayed, disease evolution and is currently on anti-TNF therapy and anti-infectious prophylaxis. The unique cases confirmed that heterozygous patients with null ADA2 activity deserve deep investigation for possible structural variants on a single allele. Moreover, this report emphasizes the importance of timely recognizing DADA2 at the onset to allow adequate follow-up and detection of disease progression. Finally, the therapeutic management in these identical twins raises significant concerns as they share a similar phenotype, with a delayed but almost predictable disease evolution in one of them, who could benefit from a prompt definitive treatment like elective allogeneic HSCT. Additional data are required to assess whether the absence of enzymatic activity at diagnosis is associated with hematological involvement and is also predictive of bone marrow dysfunction, encouraging early HSCT to improve functional outcomes.

Natural history of type 1 diabetes on an immunodysregulatory background with genetic alteration in B-cell activating factor receptor: A case report.

The immunological events leading to type 1 diabetes (T1D) are complex and heterogeneous, underscoring the necessity to study rare cases to improve our understanding. Here, we report the case of a 16-year-old patient who showed glycosuria during a regular checkup. Upon further evaluation, stage 2 T1D, autoimmune thrombocytopenic purpura (AITP), and common variable immunodeficiency (CVID) were diagnosed. The patient underwent low carb diet, losing > 8 kg, and was placed on Ig replacement therapy. Anti-CD20 monoclonal antibody (Rituximab, RTX) was administered 2 years after diagnosis to treat peripheral polyneuropathy, whereas an atypical mycobacteriosis manifested 4 years after diagnosis and was managed with prolonged antibiotic treatment. In the fifth year of monitoring, the patient progressed to insulin dependency despite ZnT8A autoantibody resolution and IA-2A and GADA autoantibody decline. The patient had low T1D genetic risk score (GRS = 0.22817) and absence of human leukocyte antigen (HLA) DR3/DR4-DQ8. Genetic analysis identified the monoallelic mutation H159Y in TNFRSF13C, a gene encoding B-cell activating factor receptor (BAFFR). Significant reduced blood B-cell numbers and BAFFR levels were observed in line with a dysregulation in BAFF-BAFFR signaling. The elevated frequency of PD-1+ dysfunctional Tfh cells composed predominantly by Th1 phenotype was observed at disease onset and during follow-up. This case report describes a patient progressing to T1D on a BAFFR-mediated immunodysregulatory background, suggesting a role of BAFF-BAFFR signaling in islet-specific tolerance and T1D progression.