GNTI-122: an autologous antigen-specific engineered Treg cell therapy for type 1 diabetes.

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving ß cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.

Treg cells require Izumo1R to regulate γδT cell-driven inflammation in the skin.

Izumo1R is a pseudo-folate receptor with an essential role in mediating tight oocyte/spermatozoa contacts during fertilization. Intriguingly, it is also expressed in CD4+ T lymphocytes, in particular Treg cells under the control of Foxp3. To understand Izumo1R function in Treg cells, we analyzed mice with Treg-specific Izumo1r deficiency (Iz1rTrKO). Treg differentiation and homeostasis were largely normal, with no overt autoimmunity and only marginal increases in PD1+ and CD44hi Treg phenotypes. pTreg differentiation was also unaffected. Iz1rTrKO mice proved uniquely susceptible to imiquimod-induced, γδT cell-dependent, skin disease, contrasting with normal responses to several inflammatory or tumor challenges, including other models of skin inflammation. Analysis of Iz1rTrKO skin revealed a subclinical inflammation that presaged IMQ-induced changes, with an imbalance of Rorγ+ γδT cells. Immunostaining of normal mouse skin revealed the expression of Izumo1, the ligand for Izumo1R, electively in dermal γδT cells. We propose that Izumo1R on Tregs enables tight contacts with γδT cells, thereby controlling a particular path of skin inflammation.

Interferon-α2b Treatment for COVID-19 Is Associated with Improvements in Lung Abnormalities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes coronavirus disease 2019 (COVID-19), a lung disease that may progress to systemic organ involvement and in some cases, death. The identification of the earliest predictors of progressive lung disease would allow for therapeutic intervention in those cases. In an earlier clinical study, individuals with moderate COVID-19 were treated with either arbidol (ARB) or inhaled interferon (IFN)-α2b +/-ARB. IFN treatment resulted in accelerated viral clearance from the upper airways and in a reduction in the circulating levels of the inflammatory biomarkers IL-6 and C-reactive protein (CRP). We have extended the analysis of this study cohort to determine whether IFN treatment had a direct effect on virus-induced lung abnormalities and also to ascertain whether any clinical or immune parameters are associated with worsening of lung abnormalities. Evidence is provided that IFN-α2b treatment limits the development of lung abnormalities associated with COVID-19, as assessed by CT images. Clinical predictors associated with worsening of lung abnormalities include low CD8+ T cell numbers, low levels of circulating albumin, high numbers of platelets, and higher levels of circulating interleukin (IL)-10, IL-6, and C-reactive protein (CRP). Notably, in this study cohort, IFN treatment resulted in a higher percentage of CD8+ T cells, lower tumor necrosis factor (TNF)-α levels and, as reported earlier, lower IL-6 levels. Independent of treatment, age and circulating levels of albumin and CRP emerged as the strongest predictors of the severity of lung abnormalities.

Integration of T-cell receptor, Notch and cytokine signals programs mouse γδ T-cell effector differentiation.

γδ T-cells perform a wide range of tissue- and disease-specific functions that are dependent on the effector cytokines produced by these cells. However, the aggregate signals required for the development of interferon-γ (IFNγ) and interleukin-17 (IL-17) producing γδ T-cells remain unknown. Here, we define the cues involved in the functional programming of γδ T-cells, by examining the roles of T-cell receptor (TCR), Notch, and cytokine-receptor signaling. KN6 γδTCR-transduced Rag2-/- T-cell progenitors were cultured on stromal cells variably expressing TCR and Notch ligands, supplemented with different cytokines. We found that distinct combinations of these signals are required to program IFNγ versus IL-17 producing γδ T-cell subsets, with Notch and weak TCR ligands optimally enabling development of γδ17 cells in the presence of IL-1ß, IL-21 and IL-23. Notably, these cytokines were also shown to be required for the intrathymic development of γδ17 cells. Together, this work provides a framework of how signals downstream of TCR, Notch and cytokine receptors integrate to program the effector function of IFNγ and IL-17 producing γδ T-cell subsets.

Role of a selecting ligand in shaping the murine γδ-TCR repertoire.

Unlike αß-T lineage cells, where the role of ligand in intrathymic selection is well established, the role of ligand in the development of γδ-T cells remains controversial. Here we provide evidence for the role of a bona fide selecting ligand in shaping the γδ-T cell-receptor (TCR) repertoire. Reactivity of the γδ-TCR with the major histocompatibility complex (MHC) Class Ib ligands, H2-T10/22, is critically dependent upon the EGYEL motif in the complementarity determining region 3 (CDR3) of TCRδ. In the absence of H2-T10/22 ligand, the commitment of H2-T10/22 reactive γδ-T cells to the γδ fate is diminished, and the specification of those γδ committed cells to the IFN-γ or interleukin-17 effector fate is altered. Furthermore, those cells that do adopt the γδ fate and mature exhibit a profound alteration in the γδTCR repertoire, including depletion of the EGYEL motif and reductions in both CDR3δ length and charge. Taken together, these data suggest that ligand plays an important role in shaping the TCR repertoire of γδ-T cells.

HEB is required for the specification of fetal IL-17-producing γδ T cells.

IL-17-producing γδ T (γδT17) cells are critical components of the innate immune system. However, the gene networks that control their development are unclear. Here we show that HEB (HeLa E-box binding protein, encoded by Tcf12) is required for the generation of a newly defined subset of fetal-derived CD73- γδT17 cells. HEB is required in immature CD24+CD73- γδ T cells for the expression of Sox4, Sox13, and Rorc, and these genes are repressed by acute expression of the HEB antagonist Id3. HEB-deficiency also affects mature CD73+ γδ T cells, which are defective in RORγt expression and IL-17 production. Additionally, the fetal TCRγ chain repertoire is altered, and peripheral Vγ4 γδ T cells are mostly restricted to the IFNγ-producing phenotype in HEB-deficient mice. Therefore, our work identifies HEB-dependent pathways for the development of CD73+ and CD73- γδT17 cells, and provides mechanistic evidence for control of the γδT17 gene network by HEB.

Induction of T Cell Development In Vitro by Delta-Like (Dll)-Expressing Stromal Cells.

Recreating the thymic microenvironment in vitro poses a great challenge to immunologists. Until recently, the only approach was to utilize the thymic tissue in its three-dimensional form and to transfer the hematopoietic progenitors into this tissue to generate de novo T cells. With the advent of OP9-DL cells (bone marrow-derived cells that are transduced to express Notch ligand, Delta-like), hematopoietic stem cells (HSC) could be induced to differentiate into T cells in culture for the first time outside of the thymic tissue on a monolayer. We, as well as others, asked whether the ability to support T cell development in vitro in a monolayer is unique to BM-derived OP9 cells, and showed that provision of Delta-like expression to thymic epithelial cells and fibroblasts also allowed for T cell development. This provides the opportunity to design an autologous coculture system where the supportive stromal and the hematopoietic components are both derived from the same individual, which has obvious clinical implications. In this chapter, we describe methods for establishing a primary murine dermal fibroblast cell population that is transduced to express Delta-like 4, and describe the conditions for its coculture with HSCs to support T cell lineage initiation and expansion, while comparing it to the now classic OP9-DL coculture.

Gamma delta T-cell differentiation and effector function programming, TCR signal strength, when and how much?

γδ T-cells boast an impressive functional repertoire that can paint them as either champions or villains depending on the environmental and immunological cues. Understanding the function of the various effector γδ subsets necessitates tracing the developmental program of these subsets, including the point of lineage bifurcation from αß T-cells. Here, we review the importance of signals from the T-cell receptor (TCR) in determining αß versus γδ lineage fate, and further discuss how the molecular components of this pathway may influence the developmental programming of γδ T-cells functional subsets. Additionally, we discuss the role of temporal windows in restricting the development of IL-17 producing γδ T-cell subtypes, and explore whether fetal and adult hematopoietic progenitors maintain the same potential for giving rise to this important subset.

Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals.

Developing thymocytes bifurcate from a bipotent precursor into αß- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) ß-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-ß and γδ-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.

Antibody class switching mediated by yeast endonuclease-generated DNA breaks.

Antibody class switching in activated B cells uses class switch recombination (CSR), which joins activation-induced cytidine deaminase (AID)-dependent double-strand breaks (DSBs) within two large immunoglobulin heavy chain (IgH) locus switch (S) regions that lie up to 200 kilobases apart. To test postulated roles of S regions and AID in CSR, we generated mutant B cells in which donor Smu and accepter Sgamma1 regions were replaced with yeast I-SceI endonuclease sites. We found that site-specific I-SceI DSBs mediate recombinational IgH locus class switching from IgM to IgG1 without S regions or AID. We propose that CSR evolved to exploit a general DNA repair process that promotes joining of widely separated DSBs within a chromosome.