Author Correction: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1+CD38hi cells and anti-PD-1 resistance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1+CD38hi cells and anti-PD-1 resistance.

Understanding resistance to antibody to programmed cell death protein 1 (PD-1; anti-PD-1) is crucial for the development of reversal strategies. In anti-PD-1-resistant models, simultaneous anti-PD-1 and vaccine therapy reversed resistance, while PD-1 blockade before antigen priming abolished therapeutic outcomes. This was due to induction of dysfunctional PD-1+CD38hi CD8+ cells by PD-1 blockade in suboptimally primed CD8 cell conditions induced by tumors. This results in erroneous T cell receptor signaling and unresponsiveness to antigenic restimulation. On the other hand, PD-1 blockade of optimally primed CD8 cells prevented the induction of dysfunctional CD8 cells, reversing resistance. Depleting PD-1+CD38hi CD8+ cells enhanced therapeutic outcomes. Furthermore, non-responding patients showed more PD-1+CD38+CD8+ cells in tumor and blood than responders. In conclusion, the status of CD8+ T cell priming is a major contributor to anti-PD-1 therapeutic resistance. PD-1 blockade in unprimed or suboptimally primed CD8 cells induces resistance through the induction of PD-1+CD38hi CD8+ cells that is reversed by optimal priming. PD-1+CD38hi CD8+ cells serve as a predictive and therapeutic biomarker for anti-PD-1 treatment. Sequencing of anti-PD-1 and vaccine is crucial for successful therapy.

A Rationally Designed Peptide Antagonist of the PD-1 Signaling Pathway as an Immunomodulatory Agent for Cancer Therapy.

Pioneering success of antibodies targeting immune checkpoints such as PD-1 and CTLA4 has opened novel avenues for cancer immunotherapy. Along with impressive clinical activity, severe immune-related adverse events (irAE) due to the breaking of immune self-tolerance are becoming increasingly evident in antibody-based approaches. As a strategy to better manage severe adverse effects, we set out to discover an antagonist targeting PD-1 signaling pathway with a shorter pharmacokinetic profile. Herein, we describe a peptide antagonist NP-12 that displays equipotent antagonism toward PD-L1 and PD-L2 in rescue of lymphocyte proliferation and effector functions. In preclinical models of melanoma, colon cancer, and kidney cancers, NP-12 showed significant efficacy comparable with commercially available PD-1-targeting antibodies in inhibiting primary tumor growth and metastasis. Interestingly, antitumor activity of NP-12 in a preestablished CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4 and CD8 T cells, and a reduction in PD-1+ T cells (both CD4 and CD8) in tumor and blood. In addition, NP-12 also showed additive antitumor activity in preestablished tumor models when combined with tumor vaccination or a chemotherapeutic agent such as cyclophosphamide known to induce "immunologic cell death." In summary, NP-12 is the first rationally designed peptide therapeutic targeting PD-1 signaling pathways exhibiting immune activation, excellent antitumor activity, and potential for better management of irAEs.

Antigen-Specific Antitumor Responses Induced by OX40 Agonist Are Enhanced by the IDO Inhibitor Indoximod.

Although an immune response to tumors may be generated using vaccines, so far, this approach has only shown minimal clinical success. This is attributed to the tendency of cancer to escape immune surveillance via multiple immune suppressive mechanisms. Successful cancer immunotherapy requires targeting these inhibitory mechanisms along with enhancement of antigen-specific immune responses to promote sustained tumor-specific immunity. Here, we evaluated the effect of indoximod, an inhibitor of the immunosuppressive indoleamine-(2,3)-dioxygenase (IDO) pathway, on antitumor efficacy of anti-OX40 agonist in the context of vaccine in the IDO- TC-1 tumor model. We demonstrate that although the addition of anti-OX40 to the vaccine moderately enhances therapeutic efficacy, incorporation of indoximod into this treatment leads to enhanced tumor regression and cure of established tumors in 60% of treated mice. We show that the mechanisms by which the IDO inhibitor leads to this therapeutic potency include (i) an increment of vaccine-induced tumor-infiltrating effector T cells that is facilitated by anti-OX40 and (ii) a decrease of IDO enzyme activity produced by nontumor cells within the tumor microenvironment that results in enhancement of the specificity and the functionality of vaccine-induced effector T cells. Our findings suggest a translatable strategy to enhance the overall efficacy of cancer immunotherapy. Cancer Immunol Res; 6(2); 201-8. ©2018 AACR.

Concurrent PD-1 Blockade Negates the Effects of OX40 Agonist Antibody in Combination Immunotherapy through Inducing T-cell Apoptosis.

Combination therapies that depend on checkpoint inhibitor antibodies (Abs) such as for PD-1 or its ligand (PD-L1) together with immune stimulatory agonist Abs like anti-OX40 are being tested in the clinic to achieve improved antitumor effects. Here, we studied the potential therapeutic and immune effects of one such combination: Ab to PD-1 with agonist Ab to OX40/vaccine. We tested the antitumor effects of different treatment sequencing of this combination. We report that simultaneous addition of anti-PD-1 to anti-OX40 negated the antitumor effects of OX40 Ab. Antigen-specific CD8+ T-cell infiltration into the tumor was diminished, the resultant antitumor response weakened, and survival reduced. Although we observed an increase in IFNγ-producing E7-specifc CD8+ T cells in the spleens of mice treated with the combination of PD-1 blockade with anti-OX40/vaccine, these cells underwent apoptosis both in the periphery and the tumor. These results indicate that anti-PD-1 added at the initiation of therapy exhibits a detrimental effect on the positive outcome of anti-OX40 agonist Ab. These findings have important implications on the design of combination immunotherapy for cancer, demonstrating the need to test treatment combination and sequencing before moving to the clinic. Cancer Immunol Res; 5(9); 755-66. ©2017 AACR.

Agonist anti-GITR antibody significantly enhances the therapeutic efficacy of Listeria monocytogenes-based immunotherapy.

BACKGROUND: We previously demonstrated that in addition to generating an antigen-specific immune response, Listeria monocytogenes (Lm)-based immunotherapy significantly reduces the ratio of regulatory T cells (Tregs)/CD4+ and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Since Lm-based immunotherapy is able to inhibit the immune suppressive environment, we hypothesized that combining this treatment with agonist antibody to a co-stimulatory receptor that would further boost the effector arm of immunity will result in significant improvement of anti-tumor efficacy of treatment. METHODS: Here we tested the immune and therapeutic efficacy of Listeria-based immunotherapy combination with agonist antibody to glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) in TC-1 mouse tumor model. We evaluated the potency of combination on tumor growth and survival of treated animals and profiled tumor microenvironment for effector and suppressor cell populations. RESULTS: We demonstrate that combination of Listeria-based immunotherapy with agonist antibody to GITR synergizes to improve immune and therapeutic efficacy of treatment in a mouse tumor model. We show that this combinational treatment leads to significant inhibition of tumor-growth, prolongs survival and leads to complete regression of established tumors in 60% of treated animals. We determined that this therapeutic benefit of combinational treatment is due to a significant increase in tumor infiltrating effector CD4+ and CD8+ T cells along with a decrease of inhibitory cells. CONCLUSION: To our knowledge, this is the first study that exploits Lm-based immunotherapy combined with agonist anti-GITR antibody as a potent treatment strategy that simultaneously targets both the effector and suppressor arms of the immune system, leading to significantly improved anti-tumor efficacy. We believe that our findings depicted in this manuscript provide a promising and translatable strategy that can enhance the overall efficacy of cancer immunotherapy.

Enhanced Therapeutic Efficacy and Memory of Tumor-Specific CD8 T Cells by Ex Vivo PI3K-δ Inhibition.

Inhibition of specific Akt isoforms in CD8+ T cells promotes favored differentiation into memory versus effector cells, the former of which are superior in mediating antitumor immunity. In this study, we investigated the role of upstream PI3K isoforms in CD8+ T-cell differentiation and assessed the potential use of PI3K isoform-specific inhibitors to favorably condition CD8+ T cells for adoptive cell therapy. The phenotype and proliferative ability of tumor antigen-specific CD8+ T cells was assessed in the presence of PI3K-α, -ß, or -δ inhibitors. Inhibition of PI3K-δ, but not PI3K-α or PI3K-ß, delayed terminal differentiation of CD8+ T cells and maintained the memory phenotype, thus enhancing their proliferative ability and survival while maintaining their cytokine and granzyme B production ability. This effect was preserved in vivo after ex vivo PI3K-δ inhibition in CD8+ T cells destined for adoptive transfer, enhancing their survival and also the antitumor therapeutic activity of a tumor-specific peptide vaccine. Our results outline a mechanism by which inhibitions of a single PI3K isoform can enhance the proliferative potential, function, and survival of CD8+ T cells, with potential clinical implications for adoptive cell transfer and vaccine-based immunotherapies. Cancer Res; 77(15); 4135-45. ©2017 AACR.

Differential PI3Kδ Signaling in CD4+ T-cell Subsets Enables Selective Targeting of T Regulatory Cells to Enhance Cancer Immunotherapy.

To modulate T-cell function for cancer therapy, one challenge is to selectively attenuate regulatory but not conventional CD4+ T-cell subsets [regulatory T cell (Treg) and conventional T cell (Tconv)]. In this study, we show how a functional dichotomy in Class IA PI3K isoforms in these two subsets of CD4+ T cells can be exploited to target Treg while leaving Tconv intact. Studies employing isoform-specific PI3K inhibitors and a PI3Kδ-deficient mouse strain revealed that PI3Kα and PI3Kß were functionally redundant with PI3Kδ in Tconv. Conversely, PI3Kδ was functionally critical in Treg, acting there to control T-cell receptor signaling, cell proliferation, and survival. Notably, in a murine model of lung cancer, coadministration of a PI3Kδ-specific inhibitor with a tumor-specific vaccine decreased numbers of suppressive Treg and increased numbers of vaccine-induced CD8 T cells within the tumor microenvironment, eliciting potent antitumor efficacy. Overall, our results offer a mechanistic rationale to employ PI3Kδ inhibitors to selectively target Treg and improve cancer immunotherapy. Cancer Res; 77(8); 1892-904. ©2017 AACR.

Programmed death-1 & its ligands: promising targets for cancer immunotherapy.

Novel strategies for cancer treatment involving blockade of immune inhibitors have shown significant progress toward understanding the molecular mechanism of tumor immune evasion. The preclinical findings and clinical responses associated with programmed death-1 (PD-1) and PD-ligand pathway blockade seem promising, making these targets highly sought for cancer immunotherapy. In fact, the anti-PD-1 antibodies, pembrolizumab and nivolumab, were recently approved by the US FDA for the treatment of unresectable and metastatic melanoma resistant to anticytotoxic T-lymphocyte antigen-4 antibody (ipilimumab) and BRAF inhibitor. Here, we discuss strategies of combining PD-1/PD-ligand interaction inhibitors with other immune checkpoint modulators and standard-of-care therapy to break immune tolerance and induce a potent antitumor activity, which is currently a research area of key scientific pursuit.

Gene therapy using genetically modified lymphocytes targeting VEGFR-2 inhibits the growth of vascularized syngenic tumors in mice.

Immunotherapies based on adoptive cell transfer are highly effective in the treatment of metastatic melanoma, but the use of this approach in other cancer histologies has been hampered by the identification of appropriate target molecules. Immunologic approaches targeting tumor vasculature provide a means for the therapy of multiple solid tumor types. We developed a method to target tumor vasculature, using genetically redirected syngeneic or autologous T cells. Mouse and human T cells were engineered to express a chimeric antigen receptor (CAR) targeted against VEGFR-2, which is overexpressed in tumor vasculature and is responsible for VEGF-mediated tumor progression and metastasis. Mouse and human T cells expressing the relevant VEGFR-2 CARs mediated specific immune responses against VEGFR-2 protein as well as VEGFR-2-expressing cells in vitro. A single dose of VEGFR-2 CAR-engineered mouse T cells plus exogenous IL-2 significantly inhibited the growth of 5 different types of established, vascularized syngeneic tumors in 2 different strains of mice and prolonged the survival of mice. T cells transduced with VEGFR-2 CAR showed durable and increased tumor infiltration, correlating with their antitumor effect. This approach provides a potential method for the gene therapy of a variety of human cancers.

Prevention of interleukin-2 withdrawal-induced apoptosis in lymphocytes retrovirally cotransduced with genes encoding an antitumor T-cell receptor and an antiapoptotic protein.

Adoptive cell transfer using autologous tumor infiltrating lymphocytes or lymphocytes transduced with antitumor T-cell receptor (TCR) is an effective therapy for patients with metastatic melanoma. A limiting factor in the effectiveness of this treatment is the apoptosis of the transferred cells when Interleukin-2 (IL-2) administration is withdrawn. In an attempt to improve persistence of the transferred lymphocytes, we cotransduced human peripheral blood lymphocytes with retroviruses encoding Bcl-2 or Bcl-xL, antiapoptotic genes of the BCL2 family, and the MART-1 melanoma tumor antigen-specific TCR, DMF5. Lymphocytes were cotransduced with 38% to 64% cotransduction efficiency, and exhibited a marked delay in apoptosis after IL-2 withdrawal. Cotransduction with Bcl-2 or Bcl-xL did not affect cytokine secretion or lytic ability of the DMF5-transduced lymphocytes. After 5 days of IL-2 withdrawal, cotransduced lymphocytes produced similar levels of IFN-γ per cell as DMF5-alone transduced lymphocytes in response to tumor cells. Cotransduction did not alter the phenotype of lymphocytes with respect to a panel of T-cell differentiation markers. In a mouse model of melanoma, adoptively transferred T cells transduced with Bcl-2 persisted better in vivo at the site of tumor, 13 and 21 days after adoptive transfer (P=0.0064 and 0.041, respectively), with evidence of enrichment of the Bcl-2-transduced population over time (P<0.0001). Thus, by coexpressing Bcl-2 or Bcl-xL with a tumor-specific TCR, we have engineered a lymphocyte that resists apoptosis owing to IL-2 withdrawal without altering its tumor-specific function or phenotype, and thus may show improved antitumor effectiveness in vivo after cell transfer.

Antiangiogenic agents can increase lymphocyte infiltration into tumor and enhance the effectiveness of adoptive immunotherapy of cancer.

Adoptive cell transfer (ACT)-based immunotherapies can mediate objective cancer regression in animal models and in up to 70% of patients with metastatic melanoma; however, it remains unclear whether the tumor vasculature impedes the egress of tumor-specific T cells, thus hindering this immunotherapy. Disruption of the proangiogenic interaction of vascular endothelial growth factor (VEGF) with its receptor (VEGFR-2) has been reported to "normalize" tumor vasculature, enhancing the efficacy of chemotherapeutic agents by increasing their delivery to the tumor intersitium. We thus sought to determine whether disrupting VEGF/VEGFR-2 signaling could enhance the effectiveness of ACT in a murine cancer model. The administration of an antibody against mouse VEGF synergized with ACT to enhance inhibition of established, vascularized, B16 melanoma (P = 0.009) and improve survival (P = 0.003). Additive effects of an antibody against VEGFR-2 in conjunction with ACT were seen in this model (P = 0.013). Anti-VEGF, but not anti-VEGFR-2, antibody significantly increased infiltration of transferred cells into the tumor. Thus, normalization of tumor vasculature through disruption of the VEGF/VEGFR-2 axis can increase extravasation of adoptively transferred T cells into the tumor and improve ACT-based immunotherapy. These studies provide a rationale for the exploration of combining antiangiogenic agents with ACT for the treatment of patients with cancer.

Role of selenium-containing proteins in T-cell and macrophage function.

Selenium (Se) has been known for many years to have played a role in boosting the immune function, but the manner in which this element acts at the molecular level in host defence and inflammatory diseases is poorly understood. To elucidate the role of Se-containing proteins in the immune function, we knocked out the expression of this protein class in T-cells or macrophages of mice by targeting the removal of the selenocysteine tRNA gene using loxP-Cre technology. Mice with selenoprotein-less T-cells manifested reduced pools of mature and functional T-cells in lymphoid tissues and an impairment in T-cell-dependent antibody responses. Furthermore, selenoprotein deficiency in T-cells led to an inability of these cells to suppress reactive oxygen species production, which in turn affected their ability to proliferate in response to T-cell receptor stimulation. Selenoprotein-less macrophages, on the other hand, manifested mostly normal inflammatory responses, but this deficiency resulted in an altered regulation in extracellular matrix-related gene expression and a diminished migration of macrophages in a protein gel matrix. These observations provided novel insights into the role of selenoproteins in the immune function and tissue homeostasis.

The selenocysteine tRNA STAF-binding region is essential for adequate selenocysteine tRNA status, selenoprotein expression and early age survival of mice.

STAF [Sec (selenocysteine) tRNA gene transcription activating factor] is a transcription activating factor for a number of RNA Pol III- and RNA Pol II-dependent genes including the Trsp [Sec tRNA gene], which in turn controls the expression of all selenoproteins. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined. We generated transgenic mice expressing the Trsp transgene lacking the STAF-binding site and made these mice dependent on the transgene for survival by removing the wild-type Trsp. The level of Sec tRNA was unaffected or slightly elevated in heart and testis, but reduced approximately 60% in liver and kidney, approximately 70% in lung and spleen and approximately 80% in brain and muscle compared with the corresponding organs in control mice. Moreover, the ratio of the two isoforms of Sec tRNA that differ by methylation at position 34 (Um34) was altered significantly, and the Um34-containing form was substantially reduced in all tissues examined. Selenoprotein expression in these animals was most affected in tissues in which the Sec tRNA levels were most severely reduced. Importantly, mice had a neurological phenotype strikingly similar to that of mice in which the selenoprotein P gene had been removed and their life span was substantially reduced. The results indicate that STAF influences selenoprotein expression by enhancing Trsp synthesis in an organ-specific manner and by controlling Sec tRNA modification in each tissue examined.

Selenoproteins mediate T cell immunity through an antioxidant mechanism.

Selenium is an essential dietary element with antioxidant roles in immune regulation, but there is little understanding of how this element acts at the molecular level in host defense and inflammatory disease. Selenium is incorporated into the amino acid selenocysteine (Sec), which in turn is inserted into selenoproteins in a manner dependent on Sec tRNA([Ser]Sec). To investigate the molecular mechanism that links selenium to T cell immunity, we generated mice with selenoprotein-less T cells by cell type-specific ablation of the Sec tRNA([Ser]Sec) gene (trsp). Herein, we show that these mutant mice exhibit decreased pools of mature T cells and a defect in T cell-dependent antibody responses. We also demonstrate that selenoprotein deficiency leads to oxidant hyperproduction in T cells and thereby suppresses T cell proliferation in response to T cell receptor stimulation. These findings offer novel insights into immune function of selenium and physiological antioxidants.

Selective restoration of the selenoprotein population in a mouse hepatocyte selenoproteinless background with different mutant selenocysteine tRNAs lacking Um34.

Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA([Ser]Sec) gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T-->A) or 37 (37A-->G) in tRNA([Ser]Sec). The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2'-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N(6)-isopentenyladenosine. Both mutant tRNAs lacked the 2'-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with (75)Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins.

Selenoprotein expression is essential in endothelial cell development and cardiac muscle function.

LoxP-Cre technology was used to remove the selenocysteine tRNA gene, trsp, in either endothelial cells or myocytes of skeletal and heart muscle to elucidate the role of selenoproteins in cardiovascular disease. Loss of selenoprotein expression in endothelial cells was embryonic lethal. A 14.5-day-old embryo had numerous abnormalities including necrosis of the central nervous system, subcutaneous hemorrhage and erythrocyte immaturity. Loss of selenoprotein expression in myocytes manifested no apparent phenotype until about day 12 after birth. Affected mice had decreased mobility and an increased respiratory rate, which proceeded rapidly to death. Pathological analysis revealed that mice lacking trsp had moderate to severe myocarditis with inflammation extending into the mediastinitis. Thus, ablation of selenoprotein expression demonstrated an essential role of selenoproteins in endothelial cell development and in proper cardiac muscle function. The data suggest a direct connection between the loss of selenoprotein expression in these cell types and cardiovascular disease.

Selenocysteine tRNA identification in the model organisms Dictyostelium discoideum and Tetrahymena thermophila.

Characterizing Sec tRNAs that decode UGA provides one of the most direct and easiest means of determining whether an organism possesses the ability to insert selenocysteine (Sec) into protein. Herein, we used a combination of two techniques, computational to identify Sec tRNA genes and RT-PCR to sequence the gene products, to unequivocally demonstrate that two widely studied, model protozoans, Dictyostelium discoideum and Tetrahymena thermophila, encode Sec tRNA in their genomes. The advantage of using both procedures is that computationally we could easily detect potential Sec tRNA genes and then confirm by sequencing that the Sec tRNA was present in the tRNA population, and thus the identified gene was not a pseudogene. Sec tRNAs from both organisms decode UGA. T. thermophila Sec tRNA, like all other sequenced Sec tRNAs, is 90 nucleotides in length, while that from D. discoideum is 91 nucleotides long making it the longest eukaryotic sequenced to date. Evolutionary analyses of known Sec tRNAs reveal the two forms identified herein are the most divergent eukaryotic Sec tRNAs thus far sequenced.