Shoba Amarnath

The PDL1-PD1 axis converts human TH1 cells into regulatory T cells.

The inhibitory ligand PDL1 transforms immune cells from attackers into regulators. PDL1: Restoring the Peace With great power comes great responsibility. In superhero lore, special powers don’t separate the saviors from the evil villains they fight; instead, what matters is how the person behind the mask uses those powers. Immune cells are the superheroes of the body—they fight off infection and patrol the body for cancer. However, sometimes, even protective cells “go bad,” causing autoimmunity or graft-versus-host disease after transplant. Amarnath et al. now show that an inhibitory protein called programmed death ligand 1 (PDL1) can regulate renegade immune cells by converting immune response–promoting T helper type 1 (TH1) cells to regulatory T (Treg) cells—agents that selectively suppress activation of the immune system. TH1 cells secrete proinflammatory cytokines and are critical for the immune response to infection and cancer cells. In contrast to other subsets of TH cells, researchers believed TH1 cells to be relatively stable. However, PDL1 caused human TH1 cells to convert to Treg cells both in vitro and in vivo. These TH1-derived Treg cells inhibited graft-versus-host disease in mice after transplant. Moreover, inhibiting Treg differentiation by blocking the PDL1 receptor PD1 or pharmacologically inhibiting SHP1 and SHP2, which are signaling molecules that act downstream of PD1 activation, restored graft-versus-host disease in mice. These data provide the basis for future therapies: Because PDL1 is highly expressed on many cancers, inhibiting this pathway may restore T cell–mediated cancer surveillance; alternately, accentuating signaling through this pathway may prevent autoimmunity or graft-versus-host disease. With this knowledge, scientists and doctors may be able to ensure that T cells are the superheroes they are meant to be. Immune surveillance by T helper type 1 (TH1) cells is not only critical for the host response to tumors and infection, but also contributes to autoimmunity and graft-versus-host disease (GVHD) after transplantation. The inhibitory molecule programmed death ligand 1 (PDL1) has been shown to anergize human TH1 cells, but other mechanisms of PDL1-mediated TH1 inhibition such as the conversion of TH1 cells to a regulatory phenotype have not been well characterized. We hypothesized that PDL1 may cause TH1 cells to manifest differentiation plasticity. Conventional T cells or irradiated K562 myeloid tumor cells overexpressing PDL1 converted TBET+ TH1 cells into FOXP3+ regulatory T (Treg) cells in vivo, thereby preventing human-into-mouse xenogeneic GVHD (xGVHD). Either blocking PD1 expression on TH1 cells by small interfering RNA targeting or abrogation of PD1 signaling by SHP1/2 pharmacologic inhibition stabilized TH1 cell differentiation during PDL1 challenge and restored the capacity of TH1 cells to mediate lethal xGVHD. PD1 signaling therefore induces human TH1 cells to manifest in vivo plasticity, resulting in a Treg phenotype that severely impairs cell-mediated immunity. Converting human TH1 cells to a regulatory phenotype with PD1 signaling provides a potential way to block GVHD after transplantation. Moreover, because this conversion can be prevented by blocking PD1 expression or pharmacologically inhibiting SHP1/2, this pathway provides a new therapeutic direction for enhancing T cell immunity to cancer and infection.

Regulatory T Cells and Human Myeloid Dendritic Cells Promote Tolerance via Programmed Death Ligand-1

Human regulatory T cells inhibit graft-versus-host disease that can occur after tissue transplantation, in part through expression of programmed death ligand 1 and modulation of antigen-presenting cells.

Endogenous TGF-β activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4+CD25- T cells

BackgroundCD4+CD25+ T regulatory cells (Tregs) play an important role in regulating immune responses, and in influencing human immune diseases such as HIV infection. It has been shown that human CD4+CD25+ Tregs can be induced in vitro by TCR stimulation of CD4+CD25- T cells. However, the mechanism remains elusive, and intriguingly, similar treatment of murine CD4+CD25- cells did not induce CD4+CD25+Foxp3+ Tregs unless exogenous TGF-β was added during stimulation. Thus, we investigated the possible role of TGF-β in the induction of human Tregs by TCR engagement. We also explored the effects of TGF-β on HIV-1 infection mediated induction of human Tregs since recent evidence has suggested that HIV-1 infection may also impact the generation of Tregs in infected patients.ResultsWe show here that endogenous TGF-β is key to TCR induction of Foxp3 in human CD4+CD25- T cells. These events involve, first, the production of TGF-β by TCR and CD28 stimulation and the activation of latent TGF-β by reactive oxygen species generated from the activated T cells. Biologically active TGF-β then engages in the induction of Foxp3. Neutralization of active TGF-β with anti-TGF-β antibody or elimination of ROS with MnTBAP abrogated Foxp3 expression. HIV-1 infection enhanced Foxp3 expression in activated CD4+CD25- T cells; which was also abrogated by blockade of endogenous TGF-β.ConclusionSeveral conclusions can be drawn from this work: (1) TCR and CD28-induced Foxp3 expression is a late event following TCR stimulation; (2) TGF-β serves as a link in Foxp3 induction in human CD4+CD25- T cells following TCR stimulation, which induces not only latent, but also active TGF-β; (3) the activation of TGF-β requires reactive oxygen species; (4) HIV infection results in an increase in Foxp3 expression in TCR-activated CD25- T cells, which is also associated with TGF-β. Taken together, our findings reinforce a definitive role of TGF-β not only in the generation of Tregs with respect to normal immune responses, but also is critical in immune diseases such as HIV-1 infection.

Hydrogen Sulfide Is an Endogenous Potentiator of T Cell Activation

Background: T cells encounter H2S in physiological and pathophysiological settings with unknown consequences. Results: Exogenous and endogenous H2S enhances T cell activation. Conclusion: T cell activation depends on H2S signaling. Significance: H2S can act as an autocrine or paracrine T cell activator and suggests a mechanistic link to inflammatory bowel disease progression. H2S is an endogenous signaling molecule that may act via protein sulfhydrylation to regulate various physiological functions. H2S is also a byproduct of dietary sulfate metabolism by gut bacteria. Inflammatory bowel diseases such as ulcerative colitis are associated with an increase in the colonization of the intestine by sulfate reducing bacteria along with an increase in H2S production. Consistent with its increased production, H2S is implicated as a mediator of ulcerative colitis both in its genesis or maintenance. As T cells are well established mediators of inflammatory bowel disease, we investigated the effect of H2S exposure on T cell activation. Using primary mouse T lymphocytes (CD3+), OT-II CD4+ T cells, and the human Jurkat T cell line, we show that physiological levels of H2S potentiate TCR-induced activation. Nanomolar levels of H2S (50–500 nm) enhance T cell activation assessed by CD69 expression, interleukin-2 expression, and CD25 levels. Exposure of T cells to H2S dose-dependently enhances TCR-stimulated proliferation with a maximum at 300 nm (30% increase, p < 0.01). Furthermore, activation increases the capacity of T cells to make H2S via increased expression of cystathionine γ-lyase and cystathionine β-synthase. Disrupting this response by silencing these H2S producing enzymes impairs T cell activation, and proliferation and can be rescued by the addition of 300 nm H2S. Thus, H2S represents a novel autocrine immunomodulatory molecule in T cells.

Bone marrow-derived mesenchymal stromal cells harness purinergenic signaling to tolerize human Th1 cells in vivo.

The use of bone marrow‐derived mesenchymal stromal cells (BMSC) in the treatment of alloimmune and autoimmune conditions has generated much interest, yet an understanding of the therapeutic mechanism remains elusive. We therefore explored immune modulation by a clinical‐grade BMSC product in a model of human‐into‐mouse xenogeneic graft‐versus‐host disease (x‐GVHD) mediated by human CD4+ Th1 cells. BMSC reversed established, lethal x‐GVHD through marked inhibition of Th1 cell effector function. Gene marking studies indicated BMSC engraftment was limited to the lung; furthermore, there was no increase in regulatory T cells, thereby suggesting a paracrine mechanism of BMSC action. BMSC recipients had increased serum CD73 expressing exosomes that promoted adenosine accumulation ex vivo. Importantly, immune modulation mediated by BMSC was fully abrogated by pharmacologic therapy with an adenosine A2A receptor antagonist. To investigate the potential clinical relevance of these mechanistic findings, patient serum samples collected pre‐ and post‐BMSC treatment were studied for exosome content: CD73 expressing exosomes promoting adenosine accumulation were detected in post‐BMSC samples. In conclusion, BMSC effectively modulate experimental GVHD through a paracrine mechanism that promotes adenosine‐based immune suppression. Stem Cells 2015;33:1200–1212 Stem Cells 2015;33:1200–1212

REGULATORY T CELLS: WHAT ROLE DO THEY PLAY IN ANTITUMOR IMMUNITY IN PATIENTS WITH HEAD AND NECK CANCER?

Advances in the treatment modalities for head and neck squamous cell carcinoma (HNSCC) over the last 20 years involving surgery, radiotherapy, chemotherapy, and immunotherapy are not fully reflected in increases in the 5‐year survival rates, mainly due to locoregional recurrences and to a lesser extent, distant metastasis. This can, in part, be attributed to the fact that HNSCC induces severe depression of a patients immune system. Recent advances in understanding the complex host–tumor interactions have led to the identification of a distinct suppressor cell population known as regulatory T cells that play a crucial role in maintaining T‐cell tolerance to self‐antigens. Here, we present a critical review of our understanding of the involvement of regulatory T cells in controlling the T‐cell immune response in tumor occurrence and progression in HNSCC with an emphasis on current and future immunotherapeutic approaches involving regulatory T cells.

Graft rejection as a Th1-type process amenable to regulation by donor Th2-type cells through an interleukin-4/STAT6 pathway.

Graft rejection has been defined as the mirror image of graft-versus-host disease, which is biologically characterized primarily as a Th1-type process. As such, we reasoned that graft rejection would represent a Th1 response amenable to Th2 modulation. Indeed, adoptive transfer of host Th1-type cells mediated rejection of fully MHC-disparate murine bone marrow allografts more effectively than host Th2-type cells. Furthermore, STAT1-deficient host T cells did not differentiate into Th1-type cells in vivo and failed to mediate rejection. We next hypothesized that donor Th2 cell allograft augmentation would prevent rejection by modulation of the host Th1/Th2 balance. In the setting of donor Th2 cell therapy, host-anti-donor allospecific T cells acquired Th2 polarity, persisted posttransplantation, and did not mediate rejection. Abrogation of rejection required donor Th2 cell IL-4 secretion and host T-cell STAT6 signaling. In conclusion, T cell-mediated marrow graft rejection primarily resembles a Th1-type process that can be abrogated by donor Th2 cell therapy that promotes engraftment through a novel mechanism whereby cytokine polarization is transferred to host T cells.

PD-1 regulates KLRG1 + group 2 innate lymphoid cells

Group 2 innate lymphoid cells (ILC-2s) regulate immune responses to pathogens and maintain tissue homeostasis in response to cytokines. Positive regulation of ILC-2s through ICOS has been recently elucidated. We demonstrate here that PD-1 is an important negative regulator of KLRG1+ ILC-2 function in both mice and humans. Increase in KLRG1+ ILC-2 cell numbers was attributed to an intrinsic defect in PD-1 signaling, which resulted in enhanced STAT5 activation. During Nippostrongylus brasiliensis infection, a significant expansion of KLRG1+ ILC-2 subsets occurred in Pdcd1−/− mice and, upon adoptive transfer, Pdcd1−/− KLRG1+ ILC-2s significantly reduced worm burden. Furthermore, blocking PD-1 with an antibody increased KLRG1+ ILC-2 cell number and reduced disease burden. Therefore, PD-1 is required for maintaining the number, and hence function, of KLRG1+ ILC-2s.

Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD.

Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation, thereby indicating that rapamycin promotion of regulatory T cells (Tregs) is conditional. The degree to which rapamycin might inhibit human Th1/Tc1 differentiation has not been evaluated. In the presence of rapamycin, T cell costimulation and polarization with IL-12 or IFN-α permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype; activation of STAT1 and STAT4 pathways essential for Th1/Tc1 polarity was preserved during mTOR blockade but instead abrogated by PI3 kinase inhibition. Such rapamycin-resistant human Th1/Tc1 cells: (1) were generated through autophagy (increased LC3BII expression; phenotype reversion by autophagy inhibition via 3-MA or siRNA for Beclin1); (2) expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) displayed reduced apoptosis. In vivo, type I polarized and rapamycin-resistant human T cells caused increased xenogeneic graft-versus-host disease (x-GVHD). Murine recipients of rapamycin-resistant human Th1/Tc1 cells had: (1) persistent T cell engraftment; (2) increased T cell cytokine and cytolytic effector function; and (3) T cell infiltration of skin, gut, and liver. Rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, rapamycin yields an anti-apoptotic Th1/Tc1 effector phenotype by promoting autophagy.

The Pentostatin Plus Cyclophosphamide Nonmyeloablative Regimen Induces Durable Host T Cell Functional Deficits and Prevents Murine Marrow Allograft Rejection

We describe a novel animal model of nonmyeloablative bone marrow transplantation (BMT) using the purine analog pentostatin. Other cohorts of mice received another purine analog, fludarabine, which we and others have previously evaluated in nonmyeloablative murine models. We evaluated pentostatin for its ability to (1) operate synergistically with cyclophosphamide to induce host T cell depletion; (2) induce host T cell suppression, as defined by modulation of cytokine secretion in vitro and abrogation of host-versus-graft reactivity in vivo; (3) constrain host T cell recovery post-therapy; and (4) prevent the rejection of T cell-depleted, fully major histocompatibility complex-mismatched bone marrow allografts. Relative to single-agent regimens, combination regimens with pentostatin and cyclophosphamide (PC) and with fludarabine and cyclophosphamide (FC) worked synergistically to deplete host CD4(+) and CD8(+) T cells. PC and FC regimens were developed that yielded similar levels of host T cell and myeloid cell depletion. In the setting of these generally comparable states of host T cell and myeloid cell depletion, the PC regimen was found to be highly immunosuppressive, as evidenced by a reduced host T cell capacity to secrete interleukin-2 and interferon-γ in vitro, to mediate host-versus-graft reactivity in vivo, and to recover numerically and functionally during a 2-week observation period after chemotherapy. Finally, using B6 hosts treated with the 14-day chemotherapy regimens, the PC regimen more consistently prevented the rejection of BALB/c T cell-depleted allografts compared with the FC regimen (rate of alloengraftment, 14/15 [93%] of PC-treated recipients vs 8/14 [57%] of FC-treated recipients; P < .05); similar results were observed using an 8-day conditioning regimen. These data suggest that host T cell suppression, distinct from T cell depletion, may be a critical determinant of engraftment after purine analog-based regimens and also may be preferentially attained by the use of pentostatin.