Tania C. Felizardo

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.

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

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.

Anti-Leukemia Activity of In Vitro-Expanded Human Gamma Delta T Cells in a Xenogeneic Ph+ Leukemia Model

Gamma delta T cells (GDTc) lyse a variety of hematological and solid tumour cells in vitro and in vivo, and are thus promising candidates for cellular immunotherapy. We have developed a protocol to expand human GDTc in vitro, yielding highly cytotoxic Vgamma9/Vdelta2 CD27/CD45RA double negative effector memory cells. These cells express CD16, CD45RO, CD56, CD95 and NKG2D. Flow cytometric, clonogenic, and chromium release assays confirmed their specific cytotoxicity against Ph+ cell lines in vitro. We have generated a fluorescent and bioluminescent Ph+ cell line, EM-2eGFPluc, and established a novel xenogeneic leukemia model. Intravenous injection of EM-2eGFPluc into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice resulted in significant dose-dependent bone marrow engraftment; lower levels engrafted in blood, lung, liver and spleen. In vitro-expanded human GDTc injected intraperitoneally were found at higher levels in blood and organs compared to those injected intravenously; GDTc survived at least 33 days post-injection. In therapy experiments, we documented decreased bone marrow leukemia burden in mice treated with GDTc. Live GDTc were found in spleen and bone marrow at endpoint, suggesting the potential usefulness of this therapy.

Human Vδ1 γδ T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells

BACKGROUND AIMS There is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets. METHODS GDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr(51)-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry. RESULTS Longer initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1). CONCLUSIONS Our ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.

Potent induction of B- and T-cell immunity against human carcinoembryonic antigen-expressing tumors in human carcinoembryonic antigen transgenic mice mediated by direct lentivector injection

The applicability of immunotherapy would be dramatically broadened to a greater number of recipients if direct “off-the-shelf” products could be engineered to engender functionally potent immune responses against true “self”-tumor antigens. This would obviate the need for ex vivo culture of dendritic cells or T cells on a patient-by-patient basis, for example. The carcinoembryonic antigen (CEA) is a glycoprotein expressed in normal gut epithelium that is up-regulated in the majority of colon cancers, non-small cell lung cancers, and half of all breast cancers. Such properties make CEA an excellent and important target for cancer immunotherapy. In this study, we show stabilization of 14-day established s.c. mGC4CEA tumors in human CEA (huCEA) transgenic mice following two direct low-dose injections of 0.15 × 106 transducing units of a lentiviral vector (LV) that directs expression of huCEA (LV-huCEA). This stabilization result was reproducible and detailed analyses including antibody assays, multiplex cytokine analyses on unstimulated splenocytes, lymph node cell characterizations, tetramer staining, and immunofluorescence staining of tumor sections showed that this outcome correlated with both a cellular and humoral immune response. Similar tumor outcomes were not seen when mice were vaccinated with a control LV that engineered expression of enGFP only. The long-term potency of this vaccination strategy was also studied and revealed the requirement for maintenance of tumor antigen-specific immunity for efficient tumor control. These data support the use of direct injections of low doses of LV-huCEA for enhancement of tumor immunotherapy directed against CEA. [Mol Cancer Ther 2009;8(3):OF692–11]

Regulated Apoptosis of Genetically Modified Hematopoietic Stem and Progenitor Cells Via an Inducible Caspase-9 Suicide Gene in Rhesus Macaques

The high risk of insertional oncogenesis reported in clinical trials using integrating retroviral vectors to genetically modify hematopoietic stem and progenitor cells (HSPCs) requires the development of safety strategies to minimize risks associated with novel cell and gene therapies. The ability to ablate genetically modified cells in vivo is desirable, should an abnormal clone emerge. Inclusion of “suicide genes” in vectors to facilitate targeted ablation of vector‐containing abnormal clones in vivo is one potential safety approach. We tested whether the inclusion of the “inducible Caspase‐9” (iCasp9) suicide gene in a gamma‐retroviral vector facilitated efficient elimination of vector‐containing HSPCs and their hematopoietic progeny in vivo long‐term, in an autologous non‐human primate transplantation model. Following stable engraftment of iCasp9 expressing hematopoietic cells in rhesus macaques, administration of AP1903, a chemical inducer of dimerization able to activate iCasp9, specifically eliminated vector‐containing cells in all hematopoietic lineages long‐term, suggesting activity at the HSPC level. Between 75% and 94% of vector‐containing cells were eliminated by well‐tolerated AP1903 dosing, but lack of complete ablation was linked to lower iCasp9 expression in residual cells. Further investigation of resistance mechanisms demonstrated upregulation of Bcl‐2 in hematopoietic cell lines transduced with the vector and resistant to AP1903 ablation. These results demonstrate both the potential and the limitations of safety approaches using iCasp9 to HSPC‐targeted gene therapy settings, in a model with great relevance to clinical development. Stem Cells 2015;33:91–100

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.

Differential immune responses mediated by adenovirus- and lentivirus-transduced DCs in a HER-2/neu overexpressing tumor model.

Recent investigations have demonstrated that adenoviral and lentiviral vectors encoding HER-2 can be utilized in cancer immunotherapy. However, it is not known whether both viral systems elicit a similar immune response. Here, we compare the immune response in mice induced by dendritic cells (DCs) infected with either recombinant adenovirus or lentivirus encoding rat HER-2 (rHER-2). Both vaccine types yielded similar control of tumor growth, but we found clear differences in their immune responses 10 days after DC immunization. Adenovirus rHER-2-transduced DCs elicited locally and systemically high frequencies of CD4+ and CD8+ T cells, while lentivirus rHER-2-transduced DCs predominantly led to CD4+ T-cell infiltration at the tumor site. Splenocytes from mice immunized with lentivirus rHER-2-transduced DCs secreted higher levels of interferon (IFN)-γ, mainly by CD4+ T cells, following stimulation by RM-1-mHER-2 tumors. In contrast, the adenovirus vaccinated group exhibited CD4+ and CD8+ T cells that both contributed to IFN-γ production. Besides an established cellular immune response, the rHER-2/DC vaccine elicited a significant humoral response that was highest in the adenovirus group. DC subsets and regulatory T cells in the spleen were also differentially modulated in the two vaccine systems. Finally, adoptive transfer of splenocytes from both groups of immunized mice strongly inhibited in vivo tumor growth. Our results suggest that not only the target antigen but also the virus system may determine the nature and magnitude of antitumor immunity by DC vaccination.

Harnessing autophagy for cell fate control gene therapy

We hypothesized that rapamycin, through induction of autophagy and promotion of an antiapoptotic phenotype, would permit lentiviral (LV)-based transgene delivery to human T-Rapa cells, which are being tested in phase II clinical trials in the setting of allogeneic hematopoietic cell transplantation. Manufactured T-Rapa cells were exposed to supernatant enriched for a LV vector encoding a fusion protein consisting of truncated CD19 (for cell surface marking) and DTYMK/TMPKΔ, which provides “cell-fate control” due to its ability to phosphorylate (activate) AZT prodrug. LV-transduction in rapamycin-treated T-Rapa cells: (1) resulted in mitochondrial autophagy and a resultant antiapoptotic phenotype, which was reversed by the autophagy inhibitor 3-MA; (2) yielded changes in MAP1LC3B and SQSTM1 expression, which were reversed by 3-MA; and (3) increased T-Rapa cell expression of the CD19-DTYMKΔ fusion protein, despite their reduced proliferative status. Importantly, although the transgene-expressing T-Rapa cells expressed an antiapoptotic phenotype, they were highly susceptible to cell death via AZT exposure both in vitro and in vivo (in a human-into-mouse xenogeneic transplantation model). Therefore, rapamycin induction of T cell autophagy can be used for gene therapy applications, including the CD19-DTYMKΔ cell-fate control axis to improve the safety of T cell immuno-gene therapy.