Implications For Cancer Immunotherapy Research Articles

Pharmacological DNA demethylation: Implications for cancer immunotherapy

ABSTRACTRecent studies have demonstrated that DNA demethylation agents can mimic a viral infection by induction of dsRNAs. This viral mimicry leads to an antiviral response mediated by the cytosolic pattern recognition receptor MDA5, followed by MAVS (IPS1) activation, IRF7 nuclear translocation and upregulation of type III Interferon and interferon-stimulated genes.

Suppression of intratumoral CCL22 by type i interferon inhibits migration of regulatory T cells and blocks cancer progression.

The chemokine CCL22 is abundantly expressed in many types of cancer and is instrumental for intratumoral recruitment of regulatory T cells (Treg), an important subset of immunosuppressive and tumor-promoting lymphocytes. In this study, we offer evidence for a generalized strategy to blunt Treg activity that can limit immune escape and promote tumor rejection. Activation of innate immunity with Toll-like receptor (TLR) or RIG-I-like receptor (RLR) ligands prevented accumulation of Treg in tumors by blocking their immigration. Mechanistic investigations indicated that Treg blockade was a consequence of reduced intratumoral CCL22 levels caused by type I IFN. Notably, stable expression of CCL22 abrogated the antitumor effects of treatment with RLR or TLR ligands. Taken together, our findings argue that type I IFN blocks the Treg-attracting chemokine CCL22 and thus helps limit the recruitment of Treg to tumors, a finding with implications for cancer immunotherapy.

Abstract B03: Molecular profiling of immune activation associated with melanoma regression induced by diphencyprone

Abstract Topical diphencyprone (DPCP), a hapten that causes delayed-type hypersensitivity reactions, has been used to treat cutaneous metastases in melanoma patients, with an 84% regression rate and minimal side effects observed in a 50-patient case series. However, the immunological mechanisms underlying these cases of regression are not well understood. Here we report the case of a 91-year-old female with a history of primary melanoma treated by excision, followed by several recurrences initially responsive to imiquimod, presenting to us with two unresponsive, unresectable cutaneous melanoma metastases on her left lower leg. After 14 weeks of topical DPCP application twice weekly, the patient's melanoma lesions first became very inflamed and then were no longer clinically visible, and we confirmed the absence of melanoma by analysis of biopsy tissue. Immunohistochemistry for Melan-A as well as qRT-PCR analysis of various melanocyte/melanoma markers both demonstrated high levels in the pre-treatment melanoma biopsy, which returned to peri-lesional (non-melanoma) skin levels following DPCP treatment. The patient remains well five months after finishing her DPCP treatment regimen with no cutaneous metastases visible. To better ascertain the mechanisms involved in immune-mediated tumor regression, we studied biopsy tissues at various time points. Previous work from our group has demonstrated that gene expression markers of all major T cell subsets (Th1, Th2, Th9, Th17, Th22, and regulatory T cells) increase at 3 days after a single application of DPCP in healthy volunteers. However, this previous report did not examine the effects of repeated applications of DPCP, as is traditionally required when DPCP is used as a treatment (for clinical conditions that include alopecia areata and warts in addition to melanoma). In our current study, we noted that DPCP led to extensive immune cell infiltrates (by H&E staining as well as immunohistochemistry for CD3, CD11c, and CD163), both after a single and repeated applications of DPCP. We found that the qRT-PCR measures of various Th1-related genes (IFNG, IL12p35, IL12p40, IL12RB1, CXCL9, CXCL10, and CXCL11) increased over time whereas Th2 T cell (IL4, IL5, and IL13) and regulatory T cell (Foxp3) markers increased initially but then decreased. Other T cell subsets [Th9 (IL9), Th17 (IL17A), and Th22 (IL22)] were also increased following a single application of DPCP but decreased with chronic application. In contrast, IL24 increased further with chronic DPCP application. This overall shift towards Th1 polarization and IL24 induction would be expected to promote anti-neoplastic effects, and reaffirms the utility of repeated DPCP applications in melanoma treatment. Another potential anti-neoplastic effector we found to be increased upon DPCP application was granulysin, which co-localized with NKp46+ natural killer cells more than CD8+ cytotoxic T cells by two-color immunofluorescence. To further examine the differences between a single and repeated applications of DPCP, we compared our microarray data from the peri-lesional site 3 days after the first application of DPCP with the peri-lesional site on the last day of DPCP treatment (day 98, after 14 weeks of twice weekly applications). The genes uniquely upregulated at day 98 included natural killer cell markers such as granzyme H, and we histologically confirmed the presence of increased natural killer cells at this time point compared to day 3. Overall, here we provide comprehensive histological and gene expression profiling of a case of successful immune-mediated melanoma metastasis regression induced by DPCP. Our study of several biopsies over time revealed shifts in T cell polarization that could have important implications for cancer immunotherapy. Future study of additional patients (both responders and non-responders to DPCP) will likely provide valuable insight into the roles played by different immune cell subsets in promoting tumor regression. Citation Format: Nicholas Gulati, Daniel G. Coit, Judilyn Fuentes-Duculan, Patricia Gilleaudeau, Mary Sullivan-Whalen, Mayte Suárez-Fariñas, James G. Krueger. Molecular profiling of immune activation associated with melanoma regression induced by diphencyprone. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B03.

The emerging Protumor role of γδ T lymphocytes: implications for cancer immunotherapy.

Tumor-infiltrating lymphocytes are key mediators of tumor immune surveillance and are important prognostic indicators in cancer progression. Among the various lymphocyte subsets implicated in protection against cancer are γδ T lymphocytes, which can kill tumor cells and secrete potent antitumor cytokines. By contrast, recent reports have revealed an unexpected series of protumor functions of γδ T cells in mouse models and human patients. In particular, specific γδ T-cell subsets are capable of recruiting immunosuppressive myeloid populations, inhibiting antitumor responses, and enhancing angiogenesis, thus promoting cancer progression. A common mediator of such functions appears to be the cytokine IL17, whose pathogenic effects can override the antitumor immune response orchestrated by IFNγ. Here, we review these studies and discuss their implications for the manipulation of γδ T cells in cancer immunotherapy.

Gene Expression Profile of Dendritic Cell-Tumor Cell Hybrids Determined by Microarrays and Its Implications for Cancer Immunotherapy.

Background. Dendritic cell- (DC-) tumor fusion cells stimulate effective in vivo antitumor responses. However, therapeutic approaches are dependent upon the coadministration of exogenous 3rd signals. The purpose of this study was to determine the mechanisms for inadequate 3rd signaling by electrofused DC-tumor cell hybrids. Methods. Murine melanoma cells were fused with DCs derived from C57BL/6 mice. Quantitative real-time PCR (qPCR) was used to determine relative changes in Th (T helper) 1 and Th2 cytokine gene expression. In addition, changes in gene expression of fusion cells were determined by microarray. Last, cytokine secretion by fusion cells upon inhibition of signaling pathways was analyzed by ELISA. Results. qPCR analyses revealed that fusion cells exhibited a downregulation of Th1 associated cytokines IL-12 and IL-15 and an upregulation of the Th2 cytokine IL-4. Microarray studies further showed that the expression of chemokines, costimulatory molecules, and matrix-metalloproteinases was deregulated in fusion cells. Lastly, inhibitor studies demonstrate that inhibition of the PI3K/Akt/mTOR signaling pathway could restore the secretion of bioactive IL-12p70 by fusion cells. Conclusion. Our results suggest that combining fusion cell-based vaccination with administration of inhibitors of the PI3K/Akt/mTOR signaling pathway may enhance antitumor responses in patients.

Tumor-induced myeloid dysfunction and its implications for cancer immunotherapy.

Immune function relies on an appropriate balance of the lymphoid and myeloid responses. In the case of neoplasia, this balance is readily perturbed by the dramatic expansion of immature or dysfunctional myeloid cells accompanied by a reciprocal decline in the quantity/quality of the lymphoid response. In this review, we seek to: (1) define the nature of the atypical myelopoiesis observed in cancer patients and the impact of this perturbation on clinical outcomes; (2) examine the potential mechanisms underlying these clinical manifestations; and (3) explore potential strategies to restore normal myeloid cell differentiation to improve activation of the host antitumor immune response. We posit that fundamental alterations in myeloid homeostasis triggered by the neoplastic process represent critical checkpoints that govern therapeutic efficacy, as well as offer novel cellular-based biomarkers for tracking changes in disease status or relapse.

γδ T Lymphocytes as a First Line of Immune Defense: Old and New Ways of Antigen Recognition and Implications for Cancer Immunotherapy.

Among γδT cells, the Vδ1 subset, resident in epithelial tissues, is implied in the defense against viruses, fungi, and certain hematological malignancies, while the circulating Vδ2 subpopulation mainly respond to mycobacteria and solid tumors. Both subsets can be activated by stress-induced molecules (MIC-A, MIC-B, ULBPs) to produce pro-inflammatory cytokines and lytic enzymes and destroy bacteria or damaged cells. γδT lymphocytes can also recognize lipids, as those associated to M. tuberculosis, presented by the CD1 molecule, or phosphoantigens (P-Ag), either autologous, which accumulates in virus-infected cells, or microbial produced by prokaryotes and parasites. In cancer cells, P-Ag accumulate due to alterations in the mevalonate pathway; recently, butyrophilin 3A1 has been shown to be the presenting molecule for P-Ag. Of interest, aminobisphosphonates indirectly activate Vδ2 T cells inducing the accumulation of P-Ag. Based on these data, γδT lymphocytes are attractive effectors for cancer immunotherapy. However, the results obtained in clinical trials so far have been disappointing: this review will focus on the possible reasons of this failure as well as on suggestions for implementation of the therapeutic strategies.

Early Carcinogenesis Involves the Establishment of Immune Privilege via Intrinsic and Extrinsic Regulation of Indoleamine 2,3-dioxygenase-1: Translational Implications in Cancer Immunotherapy.

Although prolonged genetic pressure has been conjectured to be necessary for the eventual development of tumor immune evasion mechanisms, recent work is demonstrating that early genetic mutations are capable of moonlighting as both intrinsic and extrinsic modulators of the tumor immune microenvironment. The indoleamine 2,3-dioxygenase-1 (IDO) immunoregulatory enzyme is emerging as a key player in tumor-mediated immune tolerance. While loss of the tumor suppressor, BIN-1, and the over-expression of cyclooxygenase-2 have been implicated in intrinsic regulation of IDO, recent findings have demonstrated the loss of TβRIII and the upregulation of Wnt5a by developing cancers to play a role in the extrinsic control of IDO activity by local dendritic cell populations residing within tumor and tumor-draining lymph node tissues. Together, these genetic changes are capable of modulating paracrine signaling pathways in the early stages of carcinogenesis to establish a site of immune privilege by promoting the differentiation and activation of local regulatory T cells. Additional investigation of these immune evasion pathways promises to provide opportunities for the development of novel strategies to synergistically enhance the efficacy of the evolving class of T cell-targeted “checkpoint” inhibitors.

Abstract 2564: Histone deacetylase 6 is a novel target for enhancing T-cell function during anti-tumor response and tumor-peptide vaccination

Abstract Histone deacetylases (HDACs) are a family of epigenetic regulators with emerging roles in both tumor and immune system biology. In particular, HDAC6 has been reported to modulate the acetylation HSP90, a regulator of regulatory T-cell (Treg) suppressive activity (de Zoeten, Mol Cel Biol, 2011). Here we report a novel role of HDAC6 in T-cell activation and response against tumor or peptide vaccine. Initially the relative expression of HDAC6 was evaluated in mouse and human T-cells, revealing decreased expression of this HDAC following CD3/CD28 stimulation. To further investigate the role(s) of HDAC6 in T-cell function, an HDAC6KO mouse model was utilized. Characterization of the T-cell compartments of the HDAC6KO mouse model showed a slight, but statistically significant increase in CD4+ T-cell population in the lymph nodes at the expense of a decreased percentage of CD8+ T-cells. Further investigation using isotype-specific HDAC6 inhibitors showed similar results when WT T-cells were activated and treated with an HDAC6 inhibitor. In addition to this population skewing, HDAC6 inhibition led to an enhanced expression of CD69 in WT CD4+ T-cells. This result was reproduced in human CD4+ T-cells, indicating a role of HDAC6 in regulating T-cell activation. To evaluate advantages of HDAC6 inhibition for tumor immunotherapy, melanoma-bearing mice were adoptively transferred with T-cells activated and treated with an HDAC6 inhibitor. Ex vivo analysis of memory T-cell distribution in the lymph nodes demonstrated an increased percent of CD44+CD62L+ T-cells upon HDAC6 inhibition, suggesting a role of HDAC6 in the formation or distribution of memory CD4+ and CD8+ T-cells. In a separate experiment, HDAC6KO mice inoculated with melanoma presented significantly delayed tumor growth. Additionally, in a tumor-peptide vaccination model, HDAC6KO mice displayed a remarkably reduced contraction phase in the antigen-specific CD8+ T-cell compartment when compared to WT mice after peptide vaccine administration. To further evaluate whether these in vivo responses were dependent on T-cells, WT or lympho-deficient, lymphoma-bearing mice were treated with an HDAC6 inhibitor. HDAC6 inhibition led to reduced tumor burden in WT mice, which was absent in lympho-deficient mice, highlighting the importance of lymphocytes in the aforementioned in vivo results. Thus, the role of HDAC6 in regulating immune cells during anti-tumor response appears to be more important than the previously reported role as a regulator of Treg function. The data presented demonstrates a positive effect of HDAC6 in T-cell activation, maintenance of anti-tumor response in vivo and persistence of reactive T-cells following peptide vaccination. The results herein described have revealed an unexplored role of HDAC6 with positive implications for cancer immunotherapy, warranting further investigation. Citation Format: Andressa S. Laino, David M. Woods, Fengdong Cheng, HongWei Wang, Eduardo M. Sotomayor. Histone deacetylase 6 is a novel target for enhancing T-cell function during anti-tumor response and tumor-peptide vaccination. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2564. doi:10.1158/1538-7445.AM2014-2564

The role of CD40 expression in dendritic cells in cancer biology; a systematic review.

CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor superfamily and is essential in activation of dendritic cells. Dendritic cells (DCs) are antigen-presenting cells capable of initiating cytotoxic T-lymphocyte immune response against cancer cells. However, there are few studies on the characterization of DCs in cancer, specifically their expression of CD40, despite its implication in cancer immunotherapy. We reviewed available data on the expression of CD40 on DCs in various cancers, and its implications for cancer immunotherapy. A systematic review on CD40 expression on DCs in cancer was performed with reference to preferred reporting items for systematic reviews and meta-analyses (PRISMA). Studies that satisfied the inclusion and exclusion criteria were 21 out of 927. Variations in type and status of the cancers, source of DCs and methodology for detecting CD40 expression amongst the studies resulted in contrasting results. DCs generally expressed low CD40 in tumor infiltrating DCs (tiDCs), in DCs derived by in vitro culture from blood monocytes using cytokine stimulation (MoDCs) and in DCs exposed in vitro to tumor cells lines; the studies suggested that CD40 expression in DCs is impaired in cancer particularly in metastatic disease. However, DCs identified in fresh peripheral blood mononuclear cells (PBMC) expressed higher numbers of CD40 positive cells in some cancer patients, which could be due to tumor-derived factors leading to partially-stimulated DCs. The results provide evidence that some cancer patients may show partial systemic DC activation and expression of increased CD40 in response to the presence of tumor but that such activity may become abortive in the presence of factors produced by the tumor. This review has thus identified key papers on CD40 expression on DCs in various cancers and discusses the limitations and contrasting results of these studies in relation to variations in methodology. The results highlight the need for further studies on the role of CD40-CD40 ligand pathway to inform cancer treatment.

CTLA-4 Is Expressed by Activated Mouse NK Cells and Inhibits NK Cell IFN-γ Production in Response to Mature Dendritic Cells

NK cells express an array of activating and inhibitory receptors that determine NK cell responses upon triggering by cognate ligands. Although activating NK cell receptors recognize mainly ligands expressed by stressed, virus-infected, or transformed cells, most inhibitory receptors engage MHC class I, preventing NK cell activation in response to healthy cells. In this study, we provide insight into the regulation and function of additional receptors involved in mouse NK cell responses: CTLA-4 and CD28. CTLA-4 and CD28 engage the same ligands, B7-1 and B7-2, which are primarily expressed by APCs, such as dendritic cells. Our data demonstrate that activation of mouse NK cells with IL-2 induces the expression of CTLA-4 and upregulates CD28. CTLA-4 expression in IL-2-expanded NK cells was further up- or downregulated by IL-12 or TGF-β, respectively. Using gene-deficient NK cells, we show that CD28 induces, and CTLA-4 inhibits, IFN-γ release by NK cells upon engagement by the recombinant ligand, B7-1, or upon coculture with mature dendritic cells. Notably, we show that mouse NK cells infiltrating solid tumors express CD28 and CTLA-4 and respond to stimulation with recombinant B7-1, suggesting that the NK cell responses mediated by the CD28/CTLA-4:B7-1/B7-2 system could be of importance during malignant disease. Accordingly, our study might have implications for immunotherapy of cancer based on blocking anti-CTLA-4 mAbs.

IL-17 and IL-4 producing CD8+ T cells in tumor draining lymph nodes of breast cancer patients: positive association with tumor progression.

CD8+ cytotoxic T lymphocytes have been recently divided based on their cytokine expression profile. To evaluate the percentages of CD8+ lymphocytes and their effector subsets including Tc1, Tc2 and Tc17 in the tumor draining lymph nodes (TDLNs) of patients with breast cancer. Single cell suspensions were obtained from TDLNs of 42 patients with breast cancer. Staining of the cell surface markers and intracellular cytokines was performed using appropriate fluorochrome-conjugated antibodies. The data was acquired on a four-color flow cytometer and was analyzed by CellQuestPro software package. The percentages of different CD8+ cell subtypes (Tc1, Tc2 and Tc17) were quantified in CD8+ T lymphocytes. The comparison was made between LN+ versus LN- patients, as well as patients in different clinico-pathological status. The percentage of Tc1, Tc2 and Tc17 subsets were not significantly different between LN+ and LN- patients. Despite no difference in the percentages of Tc1 cells in LN+ patients with infiltrative ductal carcinoma (IDC), the mean expression of IFN-γ by Tc1 cells decreased significantly in comparison to LN- patients. On the other hand, the percentages of Tc2 and Tc17 effector subsets were increased in advanced stages (p=0.018 and p=0.009, respectively). As the first study to investigate various effector subtypes of CD8+ lymphocytes in TDLNs of patients with breast cancer, our data collectively suggests a positive association between IL-17- and IL-4-producing CD8+ T cell percentages (Tc2 and Tc17) in TDLNs with breast cancer progression. Although the number of Tc1 cells seems not to be affected by cancer progression, down-regulation of IFN-γ by these cells seems to be associated with tumor metastasis to TDLNs. These findings may have implications in cancer immunotherapy based on CD8+ effector subsets.

Tumor-induced suppressive CD8+ T cells: implications for cancer immunotherapy

Meeting abstracts The immune system has the potential to be a powerful tool to destroy tumors; however despite ample evidence of endogenous anti-tumor immune responses in many patients, as well as years of immunotherapy development, truly effective immune-based therapies remain out of reach. We

Comparison of Vaccine-Induced Effector CD8 T Cell Responses Directed against Self- and Non–Self-Tumor Antigens: Implications for Cancer Immunotherapy

It is generally accepted that CD8 T cells play a major role in tumor control, yet vaccination aimed at eliciting potent CD8 T cell responses are rarely efficient in clinical trials. To try and understand why this is so, we have generated potent adenoviral vectors encoding the endogenous tumor Ags (TA) tyrosinase-related protein-2 (TRP-2) and glycoprotein 100 (GP100) tethered to the invariant chain (Ii). Using these vectors, we sought to characterize the self-TA-specific CD8 T cell response and compare it to that induced against non-self-Ags expressed from a similar vector platform. Prophylactic vaccination with adenoviral vectors expressing either TRP-2 (Ad-Ii-TRP-2) or GP100 (Ad-Ii-GP100) had little or no effect on the growth of s.c. B16 melanomas, and only Ad-Ii-TRP-2 was able to induce a marginal reduction of B16 lung metastasis. In contrast, vaccination with a similar vector construct expressing a foreign (viral) TA induced efficient tumor control. Analyzing the self-TA-specific CD8 T cells, we observed that these could be activated to produce IFN-γ and TNF-α. In addition, surface expression of phenotypic markers and inhibitory receptors, as well as in vivo cytotoxicity and degranulation capacity matched that of non-self-Ag-specific CD8 T cells. However, the CD8 T cells specific for self-TAs had a lower functional avidity, and this impacted on their in vivo performance. On the basis of these results and a low expression of the targeted TA epitopes on the tumor cells, we suggest that low avidity of the self-TA-specific CD8 T cells may represent a major obstacle for efficient immunotherapy of cancer.

Molecular Pathways: Coexpression of Immune Checkpoint Molecules: Signaling Pathways and Implications for Cancer Immunotherapy

The expression of immune checkpoint molecules on T cells represents an important mechanism that the immune system uses to regulate responses to self-proteins. Checkpoint molecules include cytotoxic T lymphocyte antigen-4, programmed death-1, lymphocyte activation gene-3, T-cell immunoglobulin and mucin protein-3, and several others. Previous studies have identified individual roles for each of these molecules, but more recent data show that coexpression of checkpoint molecules occurs frequently on cancer-specific T cells as well as on pathogen-specific T cells in chronic infections. As the signaling pathways associated with each checkpoint molecule have not been fully elucidated, blocking multiple checkpoints with specific monoclonal antibodies results in improved outcomes in several chronic viral infections as well as in a wide array of preclinical models of cancer. Recent clinical data suggest similar effects in patients with metastatic melanoma. These findings support the concept that individual immune checkpoint molecules may function through nonoverlapping molecular mechanisms. Here, we review current data regarding immune checkpoint molecule signaling and coexpression, both in cancer and infectious disease, as well as the results of preclinical and clinical manipulations of checkpoint proteins.

Activation of complement by monoclonal antibodies that target cell-associated β2-microglobulin: Implications for cancer immunotherapy

β2-Microglobulin (β2M), the light chain of the class I major histocompatibilty complex (MHC-I), is a promising tumor target for monoclonal antibodies (mAbs) in cancer immunotherapy. Several reports indicate that chelation of cell-associated β2M by specific mouse mAbs promotes tumor cell destruction by inducing apoptosis or other cytotoxic signaling pathways. Human mAbs employed in cancer therapy are usually IgG1, which mediates cell-killing by effector mechanisms including complement dependent cytotoxicity (CDC). The analogous mouse IgG2a and IgG2b isotypes are similarly effective in activating complement. Therefore, we examined the complement-activating properties of anti-β2M mouse mAbs 1B749 (IgG2a) and HB28 (IgG2b) when either mAb was bound to tumor cell lines or normal cells; we compared these β2M-specific mAbs with mouse mAb W6/32 (IgG2a), specific for human leukocyte antigens in the MHC-I heavy chain. All three mAbs bind to most human cell lines and normal cells in approximately equal amounts, consistent with a 1:1 stoichiometry for the HLA heavy chain in association with β2M. The three mAbs promote rapid C3b deposition and substantial CDC of human cell lines, and mAbs 1B749 and W6/32 have robust cytotoxic activity on reaction with normal mononuclear cells and platelets. Curiously, mAb HB28 induces modest C3b deposition and little CDC of normal cells, and its weaker complement-fixing activity was confirmed by ELISA. Based on these findings, we suggest that human IgG mAbs that target β2M for cancer immunotherapy be selected or engineered so as not to activate complement, thus eliminating the potential adverse effects of complement-mediated lysis of normal cells.

Identification of Special AT-Rich Sequence Binding Protein 1 as a Novel Tumor Antigen Recognized by CD8+ T Cells: Implication for Cancer Immunotherapy

BackgroundA large number of human tumor-associated antigens that are recognized by CD8+ T cells in a human leukocyte antigen class I (HLA-I)-restricted fashion have been identified. Special AT-rich sequence binding protein 1 (SATB1) is highly expressed in many types of human cancers as part of their neoplastic phenotype, and up-regulation of SATB1 expression is essential for tumor survival and metastasis, thus this protein may serve as a rational target for cancer vaccines.Methodology/Principal FindingsTwelve SATB1-derived peptides were predicted by an immuno-informatics approach based on the HLA-A*02 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from HLA-A*02+ healthy donors and/or HLA-A*02+ cancer patients. The recognition of HLA-A*02+ SATB1-expressing cancer cells was also tested. Among the twelve SATB1-derived peptides, SATB1565–574 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and cancer patients. Importantly, SATB1565–574-specific T cells recognized and killed HLA-A*02+ SATB1+ cancer cells in an HLA-I-restricted manner.Conclusions/SignificanceWe have identified a novel HLA-A*02-restricted SATB1-derived peptide epitope recognized by CD8+ T cells, which, in turn, recognizes and kills HLA-A*02+ SATB1+ tumor cells. The SATB1-derived epitope identified may be used as a diagnostic marker as well as an immune target for development of cancer vaccines.

Abstract B97: The role of JNK isoforms in adenovirus-induced autophagy: Implications for cancer immunotherapy using oncolytic adenoviruses.

Abstract Glioblastoma multiforme is the most common and lethal form of primary brain tumor in adults. Targeted immunotherapy using recombinant oncolytic viruses is a promising strategy for the treatment of brain tumors. Delta-24-RGD is a highly selective and potent oncolytic adenovirus that induces autophagy in malignant gliomas. Data from our ongoing clinical trial suggested Delta-24-RGD initiated an anti-tumor immune response in patients. Importantly, autophagy promotes the presentation of viral antigens for activation of immune cells; therefore, we sought to elucidate the molecular mechanisms by which adenoviruses induce autophagy. Adenoviral replication induced phosphorylation of JNK that was required for autophagy. Our results indicated that JNK regulates autophagy through Bcl-2 phosphorylation and consequent dissociation of Bcl-2 and beclin 1. Contrary to starvation-induced autophagy, adenovirus-mediated autophagy was facilitated by both JNK1 and JNK2 isoforms of JNK. Moreover, our data indicated that adenoviral antigens were presented at the cell surface, which was ablated upon down-regulation of JNK or autophagy. We propose a JNK/autophagy-dependent mechanism for the immune presentation of viral antigens that can be leveraged when developing recombinant adenoviruses for the advancement of anti-tumor immunotherapy. Thus, we developed a genetically-modified Delta-24 adenovirus expressing an EGFRvIII epitope that is aberrantly expressed in gliomas. The majority (>80%) of cells infected with Delta-24-FvIII presented the fiber-bound EGFRvIII epitope on their surface, an effect that was diminished upon inhibition of autophagy. Our data demonstrated that oncolytic adenoviruses can be modified to present cancer antigens for an anti-tumor immune response that may be enhanced by positively regulating autophagy. Citation Format: Sarah R. Klein, Hong Jiang, Sujan Piya, Zhimin Lu, Candelaria Gomez-Manzano, Juan Fueyo. The role of JNK isoforms in adenovirus-induced autophagy: Implications for cancer immunotherapy using oncolytic adenoviruses. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B97.

Advanced Non Small Cell Lung Cancer Patients with Fewer Numbers of Blood Myeloid Dendritic Cells and CD4 Cells Prior to Treatment Have Inferior Progression-Free Survival: Implications for Cancer Immunotherapy

AbstractAbstract 4837 Background:The tumor microenvironment includes tumor cells, and host-derived endothelial cells, fibroblasts, innate and adaptive immune cells. Tumors may induce neo-vascularization that supports local tumor growth or immune suppression and tolerance that facilitates tumor metastasis. We hypothesized that the patients with higher numbers of circulating CD34+ endothelial progenitor cells (CD34+/CD146+/CD45-, CEC), a cellular bio-marker for vasculogenesis, would have worse post-treatment outcomes and patients with more hematopoietic progenitor cells (CD34+/CD45+/CD45dim/CD133+, HPC) and Immune cells including T-cells would have better outcomes. Methods:We analyzed blood samples from sixty-two patients with advanced NSCLC at 3 time points: before chemotherapy, after cycle one, and at completion of treatment or progression of disease, in an IRB-approved protocol. CEC, HPC, and immune subsets were measured by high throughput multi-parameter flow cytometry, 2.5,000,000 events were acquired using a lyse, no-wash method optimized for rare event detection. Primary outcomes were progression free survival(PFS) and Overall Survival(OS) from the time of study entry. The patient population was stratified into groups based on optimum cut-off point for each cell subset of interest. Statistical analysis was done with log-rank test and Cox regression. Results:Mean age at diagnosis was 64 (37–87 years), 30 events (death) occurred with median follow-up of 9.3 months. Forty-six patients (74%) had disease progression with a median follow-up of 4.7 months. At baseline lower numbers of WBC, Neutrophil lymphocyte ratio(NLR), CEC, HPC were associated with better PFS, while only WBC and Neutrophil lymphocyte ratio (NLR) were associated with a favorable OS. While lower numbers of Immune cells were associated with worse PFS and OS (increased HR death or relapse) in univariate analysis as noted in the Table. Only covariates that were significant and non collinear were entered in the Multivariable model adjusted for age, gender, smoking, race, TNM stage, pathology, and performance status at diagnosis. This showed that baseline numbers of CD4+ T-cell (HR 0.46; 95% CI 0.33–0.98; p= 0.045), Myeloid DC (HR 0.38; 95% CI 0.39–0.81; p=0.012), HPC (continuous variable) (HR 0.78; 95% CI 0.64–0.93; p= 0.008) were significant for disease progression, while NLR was significant for death after study entry (Figure; HR 3; 95% CI 1.45–6.25; p=0 0.003). Conclusions:In patients with advanced NSCLC, lower numbers of HPC and NLR were associated with improved PFS and OS respectively. Lower numbers of immune subsets at diagnosis were associated with inferior outcomes to treatment, supporting the role for immune-mediated disease control.Table 1Univariate AnalysisProgression Free Survival (HR death or relapse)Overall Survival (HR death)CovariateMedian numberHR (95% CI)P-valueHR (95% CI)P-valueMetastasis2.44 (1.26–4.76)0.007Initial PS1.96 (1.0–4.0)0.049CEC, cells/uL (by 0.02058)0.084 (0.95–16.6)0.042HPC, cells/uL (continuous variable)0.920.75 (0.61–0.93)0.005CD4 Cell Count, cells/ml (by 480)7460.34 (0.17–0.68)0.0020.34 (0.16–0.74)0.004WBC, cells/ml (by 6.2)8.832.22 (0.97–5)0.0545 (1.16–25)0.02NLR (by 3.8283)5.513.12 (1.61–6.25)<0.0013.03 (1.44–6.25)0.002Gamma delta T cells, cells/ml (by 17)610.52 (0.27–0.99)0.040.39 (0.18–0.84)0.014Myeloid dendritic cells, cells/ml (by 2.912)5.940.28 (0.13–0.56)<0.0010.40 (0.18–0.88)0.019Plasmacytoid dendritic cells/ml (by 2.3667)2.370.4 (0.16–1.0)0.04B cells, cells/ml (by 70.86)187.790.37 (0.18–0.75)0.0040.42 (0.19–0.90)0.023B regulatory cells, cells/ml (by 3.948)22.70.41 (0.20–0.81)0.0090.28 (0.13–0.62)<0.001 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract LB-132: The potential role of the multienzyme aminoacyl-tRNA synthetase complex in Th-1 immunity: implications for cancer immunotherapy

Abstract Recent advances in dendritic cell (DC)-based cancer vaccines have highlighted the potential utility of arming the immune system to target neoplastic self. A strong, yet controlled, TH1 response has typically been the goal of DC-based protocols, as it is cell-based adaptive immunity that possesses the ability to most-efficiently destroy the nascent tumor or occult micrometastases. Nonetheless, many DC-intrinsic factors have impeded the development of a vaccine strong enough to impart meaningful clinical results, and there remains a need for further study of the parameters required for maximal, targeted DC activity. Previously, we have shown that contemporaneous loading of DC with overlapping MHC class I and MHC class II peptide antigens leads to an enhanced TH1 response when compared to canonical DC stimulation as assayed by release of IL-12, sCTLA-4, proliferation of CD8+ CD25+, INF-γ release, and specific killing of target both in vitro and in vivo. These and other data have lead to the hypothesis that DC possess a sensor mechanism capable of discerning amino acid sequence similarities between peptides bound by MHC class I and class II and that tRNA synthetases may serve as critical components of the putative recognition machinery due to their inherent ability to recognize and distinguish between individual amino acid side chains. Here we report that the aminoacyl-tRNA synthetase complex (MACS), a large molecular complex comprised of at least nine aminoacyl-tRNA synthetases, possess interesting characteristics that might link it to this phenomenon. Components of the MACS complex, including the p43 structural subunit, were found in DC exosomes, the contents of which mimic those of the late endosome, a known cross-presentation compartment. MARS (methionyl-tRNA synthetase), a component of the MACS, as well as non-MACS components VARS (valyl-tRNA synthetase) and GARS (glycyl-tRNA synthetase) were found within exosomes as well. UVC cross-linking of exosomal extracts resulted in the incorporation of individual tRNA-synthetase isoforms into a very large complex of indeterminate size. p43 was differentially released from the MACS in response to the loading of DC only with matched class I (GFP mRNA) and class II (recombinant GFP protein) determinants. siRNA knockdown of MACS structural component p38 was able to ablate the ability of doubly-loaded DC to enhance the generation of CD8+CD25+ cells as well as the ability of these cells to secrete IFN-γ. Further, siRNA knockdown of p38 abrogated the release of p43 from DC loaded with matched class I and II determinants. With an enhanced understanding of these regulatory phenomena, the development of small molecule TH1 agonists and/or immunotherapeutic protocols with enhanced clinical efficacy become realistic and achievable goals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-132. doi:1538-7445.AM2012-LB-132