David Y. Oh

Tumor Suppressor NM23-H1 Is a Granzyme A-Activated DNase during CTL-Mediated Apoptosis, and the Nucleosome Assembly Protein SET Is Its Inhibitor

Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.

Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment.

The three-dimensional thymic microenvironment and calcium signaling pathways are essential for driving positive selection of developing T cells. However, the nature of calcium signals and the diversity of their effects in the thymus are unknown. We describe here a thymic slice preparation for visualizing thymocyte motility and signaling in real time with two-photon microscopy. Naive thymocytes were highly motile at low intracellular calcium concentrations, but during positive selection cells became immobile and showed sustained calcium concentration oscillations. Increased intracellular calcium was necessary and sufficient to arrest thymocyte motility. The calcium dependence of motility acts to prolong thymocyte interactions with antigen-bearing stromal cells, promoting sustained signaling that may enhance the expression of genes underlying positive selection.

Granzyme A Activates an Endoplasmic Reticulum-associated Caspase-independent Nuclease to Induce Single-stranded DNA Nicks

The cytotoxic T lymphocyte protease granzyme A (GzmA) initiates a novel caspase-independent cell death pathway characterized by single-stranded DNA nicking. The previously identified GzmA substrate SET is in a multimeric 270–420-kDa endoplasmic reticulum-associated complex that also contains the tumor suppressor protein pp32. GzmA cleaved the nucleosome assembly protein SET after Lys176 and disrupted its nucleosome assembly activity. The purified SET complex required only GzmA to reconstitute single-stranded DNA nicking in isolated nuclei. DNA nicking occurred independently of caspase activation. The SET complex contains a 25-kDa Mg2+-dependent nuclease that degrades calf thymus DNA and plasmid DNA. Thus, GzmA activates a DNase (GzmA-activated DNase) within the SET complex to produce a novel form of DNA damage during cytotoxic T lymphocyte-mediated death.

A Role for CD21/CD35 and CD19 in Responses to Acute Septic Peritonitis: A Potential Mechanism for Mast Cell Activation

Although it is now appreciated that mast cell-mediated release of TNF-α is critical for resolution of acute septic peritonitis, questions remain as to how mast cells are activated upon peritoneal bacterial infection. Clues to how this may occur have been derived from earlier studies by Prodeus et al. in which complement proteins C3 and C4 were shown to be required for survival following cecal ligation and puncture (CLP), a model for acute septic peritonitis. To evaluate the mechanism for mast cell activation in the CLP model, complement receptor CD21/CD35-deficient mice (Cr2null) were examined in the present study. Along with CD19-deficient (CD19null) mice, these animals exhibit decreased survival following CLP compared with wild-type littermates. Injection of IgM before CLP does not change survival rates for Cr2null mice and only partially improves survival of CD19null mice, implicating CD21/CD35 and CD19 in mast cell activation. Interestingly, early TNF-α release is also impaired in Cr2null and CD19null animals, suggesting that these molecules directly affect mast cell activation. Cr2null and CD19null mice demonstrate an impairment in neutrophil recruitment and a corresponding increase in bacterial load. Examination of peritoneal mast cells by flow cytometry and confocal microscopy reveals the expression and colocalization of CD21/CD35 and CD19. Taken together, these findings suggest that the engagement of complement receptors CD21/CD35 along with CD19 on the mast cell surface by C3 fragments may be necessary for the full expression of mast cell activation in the CLP model.

Differential Contribution of Chemotaxis and Substrate Restriction to Segregation of Immature and Mature Thymocytes

T cell development requires sequential localization of thymocyte subsets to distinct thymic microenvironments. To address mechanisms governing this segregation, we used two-photon microscopy to visualize migration of purified thymocyte subsets in defined microenvironments within thymic slices. Double-negative (CD4(-)8(-)) and double-positive (CD4(+)8(+)) thymocytes were confined to cortex where they moved slowly without directional bias. DP cells accumulated and migrated more rapidly in a specialized inner-cortical microenvironment, but were unable to migrate on medullary substrates. In contrast, CD4 single positive (SP) thymocytes migrated directionally toward the medulla, where they accumulated and moved very rapidly. Our results revealed a requisite two-step process governing CD4 SP cell medullary localization: the chemokine receptor CCR7 mediated chemotaxis of CD4 SP cells towards medulla, whereas a distinct pertussis-toxin sensitive pathway was required for medullary entry. These findings suggest that developmentally regulated responses to both chemotactic signals and specific migratory substrates guide thymocytes to specific locations in the thymus.

Immune Toxicities Elicted by CTLA-4 Blockade in Cancer Patients Are Associated with Early Diversification of the T-cell Repertoire

While immune checkpoint blockade elicits efficacious responses in many patients with cancer, it also produces a diverse and unpredictable number of immune-related adverse events (IRAE). Mechanisms driving IRAEs are generally unknown. Because CTLA-4 blockade leads to proliferation of circulating T cells, we examined in this study whether ipilimumab treatment leads to clonal expansion of tissue-reactive T cells. Rather than narrowing the T-cell repertoire to a limited number of clones, ipilimumab induced greater diversification in the T-cell repertoire in IRAE patients compared with patients without IRAEs. Specifically, ipilimumab triggered increases in the numbers of clonotypes, including newly detected clones and a decline in overall T-cell clonality. Initial broadening in the repertoire occurred within 2 weeks of treatment, preceding IRAE onset. IRAE patients exhibited greater diversity of CD4+ and CD8+ T cells, but showed no differences in regulatory T-cell numbers relative to patients without IRAEs. Prostate-specific antigen responses to ipilimumab were also associated with increased T-cell diversity. Our results show how rapid diversification in the immune repertoire immediately after checkpoint blockade can be both detrimental and beneficial for patients with cancer. Cancer Res; 77(6); 1322-30. ©2016 AACR.

Neoadjuvant sipuleucel-T induces both Th1 activation and immune regulation in localized prostate cancer

ABSTRACT Sipuleucel-T is the only FDA-approved immunotherapy for metastatic castration-resistant prostate cancer. The mechanism by which this treatment improves survival is not fully understood. We have previously shown that this treatment can induce the recruitment of CD4 and CD8 T cells to the tumor microenvironment. In this study, we examined the functional state of these T cells through gene expression profiling. We found that the magnitude of T cell signatures correlated with the frequency of T cells as measured by immunohistochemistry. Sipuleucel-T treatment was associated with increased expression of Th1-associated genes, but not Th2-, Th17 – or Treg-associated genes. Post-treatment tumor tissues with high CD8+T cell infiltration was associated with high levels of CXCL10 expression. On in situ hybridization, CXCL10+ cells colocalized with CD8+T cells in post-treatment prostatectomy tumor tissue. Neoadjuvant sipuleucel-T was also associated with upregulation of immune inhibitory checkpoints, including CTLA4 and TIGIT, and downregulation of the immune activation marker, dipeptidylpeptidase, DPP4. Treatment-associated declines in serum PSA were correlated with induction of Th1 response. In contrast, rises in serum PSA while on treatment were associated with the induction of multiple immune checkpoints, including CTLA4, CEACAM6 and TIGIT. This could represent adaptive immune resistance mechanisms induced by treatment. Taken together, neoadjuvant sipuleucel-T can induce both a Th1 response and negative immune regulation in the prostate cancer microenvironment.

Abstract 4362: T cell repertoire diversification is associated with immune related toxicities following immune checkpoint inhibition in metastatic cancer patients

Immune checkpoint inhibitors can induce clinical responses to a broad range of tumor types by presumably enhancing T cell responses against the tumor. These treatments are also associated with certain side effects that are also thought to be immune mediated (termed immune-related adverse events, or IRAEs). The mechanism by which IRAEs occur, and biomarkers that may predict IRAE development, are unknown. In prior work (Cha et al. Sci Transl Med 2014), we demonstrated that treating metastatic cancer patients with checkpoint inhibitors blocking CTLA-4 can lead to T cell repertoire diversification. However, we found that prolonged survival was not associated with the induction of high frequency clonotypes, but rather the maintenance of a pre-existing T cell response. Here, we examined whether treatment-induced TCR diversification is related to another clinical outcome: the development of IRAEs. We find that IRAEs are specifically associated with a more diverse T cell repertoire and with an increase in the number of T cell clonotypes, including the generation of de novo clones. This broadening of the circulating T cell repertoire occurred early with treatment, preceding the development of IRAEs. While IRAE patients demonstrate markedly greater diversity in their CD4+ T cells, they demonstrate a greater degree of change in clonal frequencies in their CD8+ T cells after ipilimumab, which may have key implications in the pathogenic mechanism of IRAE development. Finally, clinical response to checkpoint blockade is also associated with increased diversity. These results indicate that immune repertoire diversity following immune checkpoint inhibition can be both detrimental and beneficial to patients with metastatic cancer. Citation Format: David Y. Oh, Jason Cham, Li Zhang, Grant Fong, Mark Klinger, Malek Faham, Lawrence Fong. T cell repertoire diversification is associated with immune related toxicities following immune checkpoint inhibition in metastatic cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4362.