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Supplementary Figures 1-2 from Elevated Frequencies of Self-reactive CD8+ T Cells following Immunization with a Xenoantigen Are Due to the Presence of a Heteroclitic CD4+ T-Cell Helper Epitope

Immunization of mice with human dopachrome tautomerase (hDCT) provides greater protection against melanoma than immunization with the murine homologue (mDCT). We mapped the CD8+ and CD4+ T-cell epitopes in both proteins to better understand the mechanisms of the enhanced protection. The dominant CD8+ T-cell epitopes were fully conserved between both proteins, yet immunization with hDCT produced frequencies of CD8+ T cells that were 5- to 10-fold higher than immunization with mDCT. This difference was not intrinsic to the two proteins because comparable frequencies of CD8+ T cells were elicited by both antigens in DCT-deficient mice. Strikingly, only hDCT elicited a significant level of specific CD4+ T cells in wild-type (WT) mice. The murine protein was not devoid of CD4+ T-cell epitopes because immunization of DCT-deficient mice with mDCT resulted in robust CD4+ T-cell immunity directed against two epitopes that were not identified in WT mice. These results suggested that the reduced immunogenicity of mDCT in WT mice may be a function of insufficient CD4+ T-cell help. To address this possibility, the dominant CD4+ T-cell epitope from hDCT was introduced into mDCT. Immunization with the mutated mDCT evoked CD8+ T-cell frequencies and protective immunity comparable with hDCT. These results reveal a novel mechanism by which xenoantigens overcome tolerance. Our data also suggest that immunologic tolerance is more stringent for CD4+ T cells than CD8+ T cells, providing a mechanism of peripheral tolerance where autoreactive CD8+ T cells fail to be activated due to a lack of autoreactive CD4+ T cells specific for the same antigen. [Cancer Res 2007;67(13):6459–67]

BACKGROUND: Combining anti-PD-1/L1 antibodies and agents that restore cancer cell susceptibility to apoptosis may enhance antitumor activity. We report results from a phase 1b dose-expansion cohort of xevinapant, a first-in-class, oral, small-molecule IAP (inhibitor of apoptosis protein) inhibitor that restores cancer cell sensitivity to apoptosis, and avelumab (anti-PD-L1) in pts with advanced NSCLC. METHODS: The recommended phase 2 dose (RP2D; 28-day cycles of xevinapant 200 mg/day [days 1-10 and 15-24] + avelumab 10 mg/kg [days 1 and 15]) was previously established during the dose-escalation part of this phase 1, open-label study. In this dose-expansion cohort, pts with advanced NSCLC who progressed on first-line (1L) platinum-based chemotherapy (CTx) or anti-PD-1/L1 ± platinum-based CTx received xevinapant (at RP2D) + avelumab for 13 cycles. The primary endpoint was objective response rate (ORR) per RECIST 1.1. Secondary endpoints included duration of response (DoR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS), safety, and pharmacokinetics (PK). RESULTS: 38 pts were treated: most had squamous cell carcinoma (45%) or adenocarcinoma (42%) of the lung, 11% had prior anti-PD-L1 therapy, 71% were men, and median age was 62 years (range, 35-75). 1 pt completed 13 cycles, and 37 permanently discontinued treatment; most common reasons were progressive disease (PD; 70%) and adverse events (AEs; 27%). ORR was 10.5% (95% CI 2.9-24.8). Best overall response (BOR): 4 pts had a confirmed partial response (PR), 19 had stable disease, and 15 had PD. Median DoR in responders was 15.9 months (95% CI 3.5-29.7); DCR was 60.5% (95% CI 43.4-76.0). Median PFS was 3.5 months (95% CI 1.9-5.1); median OS was 9.4 months (95% CI 6.7-16.2). Most pts (n=37; 97.4%) had treatment-emergent AEs (TEAEs); 21 (55.3%) had grade ≥3. Most common TEAEs were decreased appetite (n=13; 34.2%) and ALT increase (n=11; 28.9%). Nine pts died due to TEAEs; none were considered treatment-related. Xevinapant and avelumab PK were comparable to monotherapy at the same doses. In exploratory biomarker analyses, plasma IL-10 levels increased during the study treatment period. In blood, activated CD4 T cells and Tregs increased during cycle 1, and activated CD8 T cells increased during the treatment period; however, these did not correlate with antitumor activity. In tumor samples, low Ki67 expression was associated with BOR of PR (n=4), and increases in macrophages, Tregs, Th1 cells, and dendritic cells were associated with disease control. CONCLUSION: Xevinapant + avelumab had tolerable safety in pts with advanced NSCLC who progressed on 1L platinum-based CTx or anti-PD-1/L1 ± platinum-based CTx; however, the study did not meet its primary endpoint as antitumor activity was comparable to historic data for avelumab second-line monotherapy. Citation Format: Quincy Chu, Tudor Ciuleanu, Rodryg Ramlau, Daniel Renouf, Rosalyn Juergens, Ewa Kalinka, Piotr Sawrycki, Jonathan Bramson, Brad Nelson, Rafael Crabbé, Daniela A. Sahlender, Philippa Crompton, Elisabeth Rouits, Dany Spaggiari, Franck Brichory, Luke Piggott, Mike Schenker, Glenwood Goss. Xevinapant plus avelumab in patients (pts) with advanced or metastatic non-small cell lung cancer (NSCLC): Phase 1b dose-expansion results and exploratory biomarker analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT202.

For the local management of pulmonary malignancies, surgical resection and stereotactic body radiation therapy (SBRT) are mutually exclusive treatments. This study aims to assess the effectiveness of SBRT on reducing tumor viability at a histologic level in the context of pulmonary metastases.This protocol describes an open-label unblinded single-arm prospective Phase 2 trial to determine the effects of dual treatment of pulmonary metastasis amenable to curative resection using neoadjuvant SBRT followed by surgical resection, the Post SBRT Pulmonary Metastasectomy (PSPM) trial. Sample size require 39 patients, with an anticipated study duration of 30-36 months. Following completion of SBRT, eligible patients will be assessed at the 4-6-week mark by the treating radiation oncologist and thoracic surgeon with a post-treatment computed tomography (CT) of the chest. Patients with no disease progression will undergo scheduled surgical resection of all metastatic tumors at 8-12 weeks post SBRT. Patients will then be evaluated postoperatively at 30 days, and every 6 months for a total of 36 months with surveillance CT scans. Patients will also undergo sequential serologic evaluation of circulating tumor DNA (ctDNA) levels throughout their respective treatment pathway. The primary outcome of this study is the rate of complete pathologic response (pCR) following SBRT, assess using the International Association for the Study of Lung Cancer (IASLC) multidisciplinary recommendations for pathologic assessment of lung cancer resection specimens after neoadjuvant therapy. Secondary outcomes include overall survival (OS), disease free survival (DFS), local recurrence rates, cancer histology effects on pCR and treatment related complications, and treatment effect on ctDNA levels. Primary and secondary outcomes will be analyzed using Fisher's Exact test and Student'sIn evaluating the effect of SBRT on pulmonary metastasis at a histologic level, this trial may increase the use of this modality in selected patients who would otherwise only undergo surgery for disease that has already metastasized. Also, the trial provides secondary benefits of evaluating the abscopal effects of radiation on pulmonary metastatic disease, and serves as a platform for more comprehensive large-scale research in this field.ClinicalTrials.gov identifier: NCT04160143 (HiREB: 7925).

Multiple myeloma (MM) remains an incurable hematological malignancy by conventional chemotherapeutic approaches. Chimeric antigen receptor T (CAR-T) cells targeting B cell maturation antigen (BCMA) on MM have shown remarkable efficacy in relapsed/refractory patients, leading to their recent FDA approval. However, many MM patients fail to achieve durable remissions leading to relapse in the months following treatment, highlighting the need to better understand mechanisms of CAR-T cell failure. Recent studies demonstrate that reducing CD28 co-stimulatory signals delivered by CARs promote CAR-T cell persistence and reduce exhaustion. Whether endogenous CD28 co-stimulation contributes to in vivo CAR-T cell transience or dysfunction is unknown. Our preliminary data indicate that genetic deletion of the endogenous CD28 receptor does not impair CAR-T cell cytotoxicity but promotes in vivo persistence while targeted mutation of CD28 reduces exhaustion marker expression. These findings suggest that targeting endogenous CD28 on CAR-T cells with CTLA4-Ig (Abatacept) may represent a viable clinical strategy to increase the durability of a potent CAR-T cell design. Importantly, in the context of MM, we have previously shown that CD28 is expressed on and delivers pro-survival signals to MM, contributing to therapy resistance. Moreover, given the abundance of CD28 ligands, CD80 and CD86, within the bone marrow, we hypothesize that CD28 signaling reduces the efficacy of BCMA CAR-T therapy by providing a survival signal to MM cells while simultaneously limiting the durability of CAR-T cells. The combination therapy of BCMA CAR-T cells and Abatacept is expected to enhance the efficacy of adoptive CAR-T cell therapy for MM patients. This work is supported by grants from NIH (R03 CA256122, R01 AI155499).

Immunosuppressive mechanisms within the tumor microenvironment have emerged as a major impediment to cancer immunotherapy. While a broad range of secreted factors, receptors/ligands, and cell populations have been described that contribute to the immunosuppression, the involvement of these processes in immune evasion by tumors is typically considered to be an intrinsic property of the tumor. Evidence is now emerging that the processes underlying immune suppression within the tumor are, in fact, triggered by immune attack and reflect a dynamic interplay between the tumor and the host's immune system. The term adaptive resistance has been coined to describe the induction of immune suppressive pathways in the tumor following active attack on the tumor. Adaptive resistance is a scalable process where the magnitude of immune suppression matches the magnitude of the immune attack; the net balance between suppression and attack determines the durability of the anti-tumor response and tumor outcome. In this chapter, we will examine the data supporting adaptive resistance and the opposing roles of T cells in simultaneously promoting both anti-tumor immunity and immune suppression within the tumor microenvironment. The clinical implications of adaptive resistance in the design and application of immunotherapeutic strategies is also discussed.

Delivery of transcription factors to cancer cells to reprogram gene expression may represent a novel strategy to augment the production of immune stimulatory cytokines and trigger a more potent antitumor response. In the present study, a bicistronic retroviral vector (AP2) was used to transduce B16-F0 melanoma cells with IFN regulatory factor (IRF)-3, which has been shown to activate type I IFN genes (IFN-beta and IFN-alpha) as well as other cytokines. Gene-modified B16 melanoma cells were inoculated s.c. into C57BL/6 syngeneic mice. In animals receiving IRF-3 B16 melanoma cells, tumors grew at a 4- to 5-fold reduced rate, and tumors that developed from these mice had a moderate-to-dense infiltration of inflammatory cells, whereas only low levels of lymphocyte infiltration were observed in mock-transduced B16 tumors. Furthermore, tumor growth was not inhibited in severe-combined immunodeficient mice after inoculation of IRF-3-expressing B16 cells, which suggested that IRF-3-mediated antitumor responses were dependent on a functional adaptive lymphocyte response. Interestingly, these in vivo effects on tumor growth correlated with higher mRNA expression of chemokines such as MIP-1beta, RANTES, and IP-10, as well as dramatic increases in vitro in the inducibility of cytokine mRNA such as IFN-beta, TNF-alpha and interleukin 6. Our results demonstrate that with weakly antigenic tumors such as B16 melanoma, IRF-3 gene transfer can mediate important antitumor responses. These findings suggest a novel role for IRF-3 as a potential molecular target for gene therapy of cancer.

T regulatory cells (Tregs) are critical for self-tolerance and are being pursued as a potential cell therapy in transplantation and autoimmunity. While polyclonal Tregs can prevent transplant rejection in mice, allo-antigen specific Tregs are more potent. A chimeric antigen receptor (CAR) may be an effective way to make ag-specific Tregs in suitable numbers for the clinic. CARs are created by combining antibody variable domains with T cell receptor signaling domains. However, CARs for Treg cell therapy must a) function within the distinct signaling network of Tregs and b) recognize a transplant-relevant antigen. CAR function in Tregs was assessed by transducing sorted cells with a highly-expressed, humanized HER2 CAR. Cells were labeled with proliferation dye and assessed for their ability to respond to target antigen. Two CARs specific for HLA-A2 were generated by cloning the variable portions of the anti-A2 antibody from a hybridoma, creating a single chain antibody and ligating it in frame in the humanized CAR. These CARs were assessed for expression and activity by flow cytometry, and cytotoxicity and proliferation assays. Preliminary data suggests that CARs can induce strong, ag-specific proliferation in human Tregs and that an ag-specific HLA-A2 CAR has been generated. Further work includes determining the effect of CAR stimulation on Treg phenotype via cytokine staining and suppression assays, and optimizing the surface expression of the HLA-A2 CAR via phage display.

Engineering T cell specificity to direct them against tumor cells utilizing chimeric antigen receptors (CARs) has emerged as an exciting technique for adoptive T cell therapy. The effectiveness of adoptive transfer therapies can be limited by a number of factors, including ability of T cells to engraft, the degree to which they infiltrate the tumor, and the immunosuppressive nature of the tumor. Oncolytic viruses (OVs) are capable of overcoming these limitations, and can serve as effective booster vaccines. In an effort to capitalize on these features, we have generated dual-specific T cells that bear TCRs specific for the OV, and CARs specific for the tumor. Using murine tumor models, we have observed significant boosting capacity of adoptively transferred dual-specific CD4+ or CD8+ T cells to tumor-bearing hosts in response to infusion with OV. Boosting with OV also dramatically influenced the distribution of the CAR-T cells, driving the cells to proliferate throughout the body. We now seek to examine the utility of dual-specific CD4+ T cells in adoptive transfer therapies. We have found that CAR-engineered CD4+ T cells are capable of mediating anti-tumor immunity, and are investigating the combination of these cells with OV to further enhance their anti-tumor efficacy. Given the promising clinical outcomes of both CAR T cells and OVs, we believe our combinatorial approach will provide a clinically feasable strategy to maximize the therapeutic potential of both therapies.

Intestinal Th2 immunity in food allergy results in the production of IgG1 and IgE, and upon antigen challenge, anaphylaxis and eosinophilic inflammation. Although allergic sensitization critically requires IL-4 to develop, the source and control of IL-4 during the initiation of Th2 immunity remains unclear. Non-intestinal and non-food allergy systems have suggested a role for innate lymphocytes such as NKT or γδ T cells as a rapid source of IL-4 required to induce Th2 polarization. In contrast, we show here that NKT-deficient IL-15 KO, β2m KO and anti-NK1.1 treated mice have completely intact Th2 food allergic responses comparable to NKT-sufficient mice, including antigen-specific IgG1 and IgE, anaphylaxis, eosinophilic inflammation and cytokine production. Likewise, γδ T cell-deficient TCRδ KO mice mount comparable Th2 immune responses to oral antigen as their WT counterparts. By restricting IL-4 expression to only CD4+ Th cells, we find that IL-4 from CD4+ Th cells themselves induce food allergy. Further, IL-4 reporter mice show that CD4+ Th cell production of IL-4 in vivo is dependent on OX40L, a costimulatory molecule that we have shown to be highly expressed on allergen-exposed dendritic cells (DCs). Although NKT and γδ T cells express IL-4, this is not upregulated after allergen priming. Together, these data show that intestinal Th2 immunity in food allergy is initiated by a CD4+ Th cell-intrinsic IL-4 program that is controlled by DC-OX40L and not NKT or γδ T cells.

As humans age, they experience a progressive loss of thymic function and a corresponding shift in the nature of the circulating CD8+ T cell population from naïve to memory phenotype. These alterations are believed to result in impaired development of CD8+ T cell immunity in older individuals; however evidence that these global changes impair antigen-specific CD8+ T cell responses in the elderly is lacking. To address this possibility, we studied CD8+ T cell memory development following primary exposure to West Nile virus (WNV) in a cohort of young (61 yrs) individuals using polychromatic flow cytometry. Although the aged cohort displayed the previously described alterations in the CD8+ T cell compartment, including a marked loss of naïve CD8+ T cells, accumulation of CD28- and CD57+ T cells, we did not observe any influence of age on either the breadth, magnitude or functionality of antigen-specific CD8+ T cells following WNV, cytomegalovirus (CMV) or Epstein-Barr virus (EBV) peptide stimulation. We did however observe virus-specific CD8+ T cell functional differences between WNV, CMV and EBV. Collectively these data fail to support the hypothesis that immunosenescence leads to defective CD8+ T cell immunity and suggest that it should be possible to develop CD8+ T cell vaccines to protect aged individuals from infections with novel and emerging viruses.

Interleukin-12 (IL12) enhances anti-tumor immunity when delivered locally within the tumor microenvironment. However, immunoregulatory elements within the tumor microenvironment are thought to dampen the effect of IL12 as an adjuvant. The objective of this study was to identify biochemical mechanisms of tumor-immune cell cross-talk that locally suppress the action of IL12. A high content in vitro assay was developed to quantify tumor-mediated suppression of the action of IL12 using a T helper type 1 cell line. Following an induction period, the melanoma cell model, B16F0, inhibited the cellular response to IL12 in vitro. This paracrine effect was not explained by induction of apoptosis or creation of a cytokine sink, despite both mechanisms at work within the co-culture assay. Secreted protein, acidic and rich in cysteine (SPARC); exosomes; and Wnt-inducible signaling protein-1 (WISP-1) were identified using a proteomics workflow and confirmed to be enriched in B16F0-conditioned media. Neutralization of WISP-1 recovered and recombinant WISP-1 dose-dependently inhibited the activity of Signal Transducer of Activated Tyrosine 4 (STAT4), a key transcription factor within the IL12 pathway, within the in vitro co-culture assay. Moreover, WISP-1 was expressed in vivo following intradermal challenge with B16F10 cells and exhibited an inverse dependence on tumor size.

Vaccination with recombinant human adenovirus serotype 5 (rHuAd5) drives a CD8+ T cell response that is dominated by effector and effector memory cells. In investigating the immunobiological mechanisms that result in this effector-skewed response, we previously showed that non-haematopoietic cells play a key role in defining the rHuAd5-elicited CD8+ T cell memory population. Furthermore, maintenance of this memory population relies upon sustained transgene expression from the adenovirus vector. While we know that following vaccination rHuAd5 drains to local lymph nodes, long-term antigen persistence occurs outside this location, since surgical removal of these nodes does not impact the nature of the memory response. Here, we show that following intramuscular vaccination, the adenovirus transgene is predominantly detected in the perinodal adipose tissue, in addition to draining lymph nodes and the muscle injection site itself. In all three locations, blood endothelial cells are the primary cell type that express the adenovirus-encoded antigen, strongly indicating that this radio-resistant cell population may have a role in presenting antigen to CD8+ T cells and ultimately in defining the memory response. Long-term antigen presentation from this cell compartment may represent a mechanism to sustain CD8+ T cell memory within the circulation and to ensure adequate protective immunity is maintained in multiple organs.

We have developed a prototypic recombinant adenovirus vaccine encoding dopachrome tautomerase (rHuAd5-hDCT) that leads to moderate suppression of B16F10 melanoma growth through a CD8+ T cell- and IFN-γ-dependent mechanism. The immunogenicity of rHuAd5-hDCT was improved by concomitant 4-1BB stimulation leading to enhanced control of tumor growth; however, most tumors ultimately relapsed. Gene expression analysis revealed a rapid adaptation of the tumor environment within 5 days of vaccination. Intratumoral IFN-γ and TNF-α expression increased rapidly following vaccination but, paradoxically, expression was almost completely attenuated at the time of highest infiltration by vaccine-induced CD8+ T cells. We also noted robust, yet transient, induction of PD-L1 and PD-L2 in the tumor following vaccination, which correlated with the attenuation of IFN-γ and TNF-α expression. Blockade of PD-1 signaling in combination with rHuAd5-hDCT enhanced effector cytokine expression in the tumor and improved growth control, but the tumors ultimately relapsed. Strikingly, the combination of PD-1 blockade and 4-1BB stimulation with rHuAd5-hDCT produced a synergistic enhancement in intratumoral T cell activity, resulting in complete regression despite no increase in the functionality or number of tumor infiltrating CD8+ T cells. These results punctuate the need to understand the dynamic changes within the tumor to optimize cancer immunotherapy.

The mechanisms that underlie the pathology of neuroinvasive West Nile virus (WNV) remain unknown. The goal of this study was to characterize the CD4+ T cell response to WNV in individuals with neuroinvasive and non-neuroinvasive symptoms. WNV-infected subjects were screened for CD4+ T cell reactivity with peptide pools derived from a library spanning the WNV polyprotein. Virus-specific CD4+ T cells were directed primarily against E, NS1, and NS3. Most WNV-specific CD4+ T cells produced IFN-γ. Surprisingly, IL-17 production was primarily associated with NS3-reactive cells. Single cell analysis revealed three distinct populations: IFN-γ+/IL-17-, IFN-γ+/IL-17+, IFN-γ-/IL-17+. Preliminary analysis suggests that elevated frequencies of virus-specific CD4+ T cells producing both IFN-γ and IL-17 are associated with protection against WNV neuroinvasive disease. This work was funded by a contract from NIAID (N01-AI-40066).

Food allergies are immune system-driven diseases that lead to reproducible adverse reactions which can be fatal. These severe systemic reactions are primary mediated by immunoglobulin E (IgE) that is derived from B cells which have been activated by T helper type 2 (Th2) cells. While much work has advanced the clinical and pharmacological management of patients with allergic diseases, much remains to be elucidated about how individuals initially acquire allergy. This Thesis details a mechanism linking initial gastrointestinal exposure to peanut (PN) allergen, to the generation of Th2 cells: PN allergen activates epithelial cell secretion of interleukin (IL)-33 and eosinophil degranulation of eosinophil peroxidase, which causes CD103+ dendritic cell (DC) activation and migration to mesenteric lymph nodes where DC OX40L engages naïve T cells to secrete IL-4 in an autocrine/paracrine manner to promote and consolidate Th2 cell differentiation. These events are followed by B cell activation and PN-specific IgE production, which sensitizes mast cells to be hypersensitive to PN re-exposure by causing immediate allergic reactions including anaphylaxis. This is later followed by eosinophilic inflammation that is partially mediated by innate lymphoid cells. As food allergy also serves as a unique model to better understand mechanisms of adaptive immunity, especially Th2 immunobiology, both basic science and clinical implications are discussed in this Thesis. Major themes include Th2 and disease heterogeneity, identification of ‘the original source of IL-4’, an unprecedented in vivo requirement for eosinophils in priming adaptive immune responses, and the need to weigh basic science findings against the human disease in natura litmus test. Looking forward, many questions remain to be answered in the field of food allergy research, but the findings of this Thesis may be one step towards the prevention, management or cure of a disease with growing public concern, potentially fatal consequences, and an unmet need in understanding its pathogenesis. Doctor of Philosophy (Medical Science)

We showed previously that histone deacetylase (HDAC) inhibition with MS-275 in the context of boosting oncolytic immunotherapy can drive heightened antitumor responses, leading to increased survival in mouse intracranial melanoma models. However, it is currently unclear how the co-administration of MS-275 directly impacts tumor growth. Here, we investigated the role of MS-275 in preventing the outgrowth of antigen-deficient tumor variants as a result of suboptimal treatment protocols. By adoptively transferring tumor antigen-specific memory T cells (Tm) that were expanded in vivo with recombinant Vesicular Stomatitis Virus (VSV-gp33), we observed complete regression of 5-day old, intradermal B16-gp33 tumors (B16-F10 overexpressing the LCMV GP33-41 epitope); however, the tumors relapsed within a month of treatment. Relapsing tumor explants were able to grow in mice that were prophylactically immunized with recombinant Adenovirus (Ad-gp33), indicating that the tumor could no longer be recognized. Strikingly however, there was zero tumor recurrence if MS-275 was co-administered with Tm and VSV-gp33, suggesting that MS-275 may prevent the emergence and/or escape of antigen loss variants. Such a benefit is lost if the administration of the drug is delayed as little as five days post VSV treatment, suggesting that its synergistic effects coincide with early immune responses and oncolytic activity. Furthermore, transplantation studies of relapsing tumor explants showed that combination treatment was unable to provide tumor protection, confirming that the mechanisms by which MS-275 prevents tumor recurrence are unlikely through direct up-regulation of antigen presentation in low- or non-antigen-expressing variants in vivo. Indeed, CD4 depletion in the absence of MS-275 resulted in sustained tumor regression, implying that immunoregulatory cells such as CD4+ Treg play a prominent role in sustaining tumor regression. Moreover, MS-275 modulates the phenotypic status of tumor-infiltrating MDSCs toward the differentiation of inflammatory macrophages. Taken together, the data suggests that combination therapy with HDACi with oncolytic immunotherapy mediates a synergized immune attack against the tumor through subversion of immunomodulatory mechanisms. Master of Science in Medical Sciences (MSMS)

Lung cancer is the leading cause of cancer related deaths in both the US and Canada and efforts still need to be made towards understanding the disease. The role of inflammation in the promotion of cancer development represents a newer avenue of research. The glycoprotein (gp)-130 cytokine interleukin-6 (IL-6) has a well established role in promoting inflammation and recent evidence suggests roles in development of certain tumors in animal models. Less is known of the related family member oncostatin M (OSM) and the functions of either IL-6 or OSM in lung cancer development is not known. Based on the hypothesis that these cytokines promote lung cancer development, IL-6 and OSM were overexpressed in the lungs of two separate mouse models for lung cancer utilizing adenovirus vectors encoding IL-6 or OSM. The first mouse model utilized a Cre-conditional oncogene KRAS G12D (developed by Tyler Jacks) in which endotracheal administration of adenovirus (Ad)-encoded Cre-recombinase resulted in increases in lung densities in a dose-dependent fashion over a period of 6 weeks that were measurable by CT scanning and histology. Increases in cytokines IL-6 and kertinocyte chemoattractant (KC) were detectable in the bronchoalveolar lavage (BAL) by week 4, as well as marked increases in alveolar macrophage numbers. Macrophages were also shown as a possible target for Cre-mediated recombination and mutant KRAS expression. Administration of either AdIL-6 or AdOSM as well as AdCre resulted in a trend toward increases in tumor burden with AdOSM based on experiments terminated at 4 weeks. The second mouse model involved endotracheal administration of the lewis lung carcinoma (LLC) cell line, which after 7 days resulted in detectable tumor burden. Administration of either AdIL-6 or AdOSM and LLC cells simultaneously was shown to increase tumor burden relative to AdDl70 co-administration. These results suggest a possible role of IL-6 or OSM in promoting lung tumor development in animal models and may ultimately reveal gp130 cytokines IL-6 or OSM as a possible therapeutic target for the treatment of lung cancer. Master of Science (MSc)

Majority of new HIV-1 infections world-wide occur via the genital tract. Therefore, achieving effective mucosal immunity will be a critical component of vaccine strategies in the prevention and control of infection during early stages of transmission itself. Rigorous efforts have been made to identify conserved epitopes and develop rational design of immunogens, in order to elicit broadly-reactive protective Abs against HIV. Newly emerging data have highlighted the significance of Ab effector functions other than classical IgG-mediated neutralization in HIV infection. In the studies contributing towards this thesis, an optimized vaccine model is described that successfully elicits potent systemic and mucosal Abs against the highly conserved epitopes of the membrane-proximal external region and the coiled coil region of gp41. Intriguing observations are reported on the IgA-inducing capacity of the coiled coil epitope, QARVLAVERY, which highlight the potential of this epitope as an attractive candidate for mucosal vaccines. Most importantly, the epitope-specific Abs proved to be functional in neutralizing HIV in a standardized assay. With particular relevance to mucosal protection, epitope-specific IgA also effectively inhibited the transcytosis of HIV in an optimized transwell assay. The effect of gp41-specific Abs was also assessed against a novel panel of HIV-1 viral clones, and exhibited significant protection. These clones selectively express envelopes from viruses that would be desired targets of prophylactic immune responses, the earliest founder population in the mucosa after virus transmission. Doctor of Philosophy (PhD)