A broadly applicable approach to T cell epitope identification: Application to improving tumor associated epitopes and identifying epitopes in complex pathogens

Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens

Assessing the effect of insecticide-treated cattle on tsetse abundance and trypanosome transmission at the wildlife-livestock interface in Serengeti, Tanzania

In the absence of national control programmes against Rhodesian human African trypanosomiasis, farmer-led treatment of cattle with pyrethroid-based insecticides may be an effective strategy for foci at the edges of wildlife areas, but there is limited evidence to support this. We combined data on insecticide use by farmers, tsetse abundance and trypanosome prevalence, with mathematical models, to quantify the likely impact of insecticide-treated cattle. Sixteen percent of farmers reported treating cattle with a pyrethroid, and chemical analysis indicated 18% of individual cattle had been treated, in the previous week. Treatment of cattle was estimated to increase daily mortality of tsetse by 5–14%. Trypanosome prevalence in tsetse, predominantly from wildlife areas, was 1.25% for T. brucei s.l. and 0.03% for T. b. rhodesiense. For 750 cattle sampled from 48 herds, 2.3% were PCR positive for T. brucei s.l. and none for T. b. rhodesiense. Using mathematical models, we estimated there was 8–29% increase in mortality of tsetse in farming areas and this increase can explain the relatively low prevalence of T. brucei s.l. in cattle. Farmer-led treatment of cattle with pyrethroids is likely, in part, to be limiting the spill-over of human-infective trypanosomes from wildlife areas

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Effects of Total-Body vs. Localized Radiation Exposure on the Immune System

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2018.Ionizing radiation is a form of high-energy electromagnetic radiation capable of causing double strand breaks in DNA, and killing cells. Various subsets of immune cells are particularly sensitive to radiation and can be damaged and/or killed following exposure. This becomes detrimental to tissues where immune cells form an immunological protective barrier, such as in the skin. We hypothesized that accidental radiation exposure (e.g. radiological disaster) diminishes the number of surveillent cutaneous immune cells and renders the skin more susceptible to invasion from foreign organisms. To test this, we developed a post-radiation infection model where hairless mice were exposed to a sub-lethal total-body dose of 6 Gy, followed by cutaneous inoculation of Candida albicans, an organism that causes opportunistic infections. We observed increased dissemination of C. albicans in irradiated mice, as well as reduced numbers of cutaneous CD4+ and CD8+ T cells and decreased expression of IFNγ, CXCL9, and IL-9. Administering IL-12 after radiation and prior to infection mitigated this impaired response. Interestingly, increased dissemination did not occur in mice that were irradiated 60 weeks prior to C. albicans inoculation or if they were irradiated as pups and infected as adults. Also, this impaired response did not occur in mice infected with a more virulent organism, methicillin-resistant Staphylococcus aureus, (MRSA). As these results indicate, ionizing radiation can dampen the cutaneous immune response but time of infection after exposure and type of microorganism can influence outcome. Whereas cases of accidental exposure can result in severe health complications, ionizing radiation has also been harnessed as a tool for cancer therapy when directed at tumor cells. As such, a paradigm shift has occurred in our understanding of how radiotherapy (RT) works and now suggests that the immune system is essential in mediating the anti-tumor effects of RT. Importantly, the dose and schedule of RT can dictate the magnitude of the immune response and therefore efficacy. As such, there is a concerted effort to optimize RT dose with the subsequent immune response. We hypothesize that a single ablative dose or fewer, stronger doses of radiation (hypofractionation) will result in improved tumor control when compared to a conventional scheme of smaller, more frequent doses of RT. To test this, we examined RT efficacy in a colon adenocarcinoma model between a hyperfractionated scheme that used five daily doses of 2 Gy to a hypofractionated scheme consisting of two doses of 10 Gy given on days 7 and 11 post-tumor cell injection. Overall, our data demonstrated that the hypofractionated scheme is superior to the hyperfractioned regime and resulted in enhanced long-term tumor control. We further dissected the immunologic mechanism of action in the hypofractionated scheme and determined that the first dose of 10 Gy acted as an initial immune boost by increasing the intratumoral number of CD8+ T cells. The second 10 Gy dose was essential for promoting long-term tumor control. These results suggest that later doses further boost the anti-tumor immune response, while having limited negative effects on the tumor infiltrating immune cells. How radiation affects the immune system depends on the context of the exposure. Totalbody doses impair cells of immunological barriers but the localized doses of cancer radiotherapy boosted the immune system by killing tumor cells and inducing primed tumor-specific cells to infiltrate the remaining tumor. Other factors such as risk of infection, whether the target of the immune response is an infectious organism or tumor cells, the kinetics of the immune response, and if or when subsequent radiation doses occur all affect the end result of the immune response. These factors could be the difference between a minor or serious infection, or tumor remission or outgrowth. Therefore, patient care for both accidental radiation exposure or in radiotherapy should be modified based on the situation to optimize patient outcome

Effects of Total-Body vs. Localized Radiation Exposure on the Immune System

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2018.Ionizing radiation is a form of high-energy electromagnetic radiation capable of causing double strand breaks in DNA, and killing cells. Various subsets of immune cells are particularly sensitive to radiation and can be damaged and/or killed following exposure. This becomes detrimental to tissues where immune cells form an immunological protective barrier, such as in the skin. We hypothesized that accidental radiation exposure (e.g. radiological disaster) diminishes the number of surveillent cutaneous immune cells and renders the skin more susceptible to invasion from foreign organisms. To test this, we developed a post-radiation infection model where hairless mice were exposed to a sub-lethal total-body dose of 6 Gy, followed by cutaneous inoculation of Candida albicans, an organism that causes opportunistic infections. We observed increased dissemination of C. albicans in irradiated mice, as well as reduced numbers of cutaneous CD4+ and CD8+ T cells and decreased expression of IFNγ, CXCL9, and IL-9. Administering IL-12 after radiation and prior to infection mitigated this impaired response. Interestingly, increased dissemination did not occur in mice that were irradiated 60 weeks prior to C. albicans inoculation or if they were irradiated as pups and infected as adults. Also, this impaired response did not occur in mice infected with a more virulent organism, methicillin-resistant Staphylococcus aureus, (MRSA). As these results indicate, ionizing radiation can dampen the cutaneous immune response but time of infection after exposure and type of microorganism can influence outcome. Whereas cases of accidental exposure can result in severe health complications, ionizing radiation has also been harnessed as a tool for cancer therapy when directed at tumor cells. As such, a paradigm shift has occurred in our understanding of how radiotherapy (RT) works and now suggests that the immune system is essential in mediating the anti-tumor effects of RT. Importantly, the dose and schedule of RT can dictate the magnitude of the immune response and therefore efficacy. As such, there is a concerted effort to optimize RT dose with the subsequent immune response. We hypothesize that a single ablative dose or fewer, stronger doses of radiation (hypofractionation) will result in improved tumor control when compared to a conventional scheme of smaller, more frequent doses of RT. To test this, we examined RT efficacy in a colon adenocarcinoma model between a hyperfractionated scheme that used five daily doses of 2 Gy to a hypofractionated scheme consisting of two doses of 10 Gy given on days 7 and 11 post-tumor cell injection. Overall, our data demonstrated that the hypofractionated scheme is superior to the hyperfractioned regime and resulted in enhanced long-term tumor control. We further dissected the immunologic mechanism of action in the hypofractionated scheme and determined that the first dose of 10 Gy acted as an initial immune boost by increasing the intratumoral number of CD8+ T cells. The second 10 Gy dose was essential for promoting long-term tumor control. These results suggest that later doses further boost the anti-tumor immune response, while having limited negative effects on the tumor infiltrating immune cells. How radiation affects the immune system depends on the context of the exposure. Totalbody doses impair cells of immunological barriers but the localized doses of cancer radiotherapy boosted the immune system by killing tumor cells and inducing primed tumor-specific cells to infiltrate the remaining tumor. Other factors such as risk of infection, whether the target of the immune response is an infectious organism or tumor cells, the kinetics of the immune response, and if or when subsequent radiation doses occur all affect the end result of the immune response. These factors could be the difference between a minor or serious infection, or tumor remission or outgrowth. Therefore, patient care for both accidental radiation exposure or in radiotherapy should be modified based on the situation to optimize patient outcome

Enhancing T Cell and NK Cell Mediated anti-Tumor Immune Responses Through the use of Immunotherapy

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2013.Cancer immunotherapy is a rapidly expanding field that has the potential to control not only primary tumor growth but also the growth of distant metastases. In particular, tumor metastasis to the peritoneal cavity is immunologically intriguing. Upon release into the peritoneal cavity, tumor cells initially attach to the omentum, a tissue consisting of organized immune aggregates embedded in adipose tissue. Indeed, it is to these immune aggregates that tumor cells initially bind and thrive. Despite the proximity of tumor cells and immune cells, a de novo productive immune response does not occur on the omentum. Thus, we explored various mechanisms by which to generate an immune response on the omentum. Intraperitoneal injection of particulate antigen resulted in its localization to the omentum, but only minimal cytokine and cellular responses. Immunization with lethally irradiated tumor cells however, was able to induce a more robust response, namely the expansion of potentially tumor-specific T cells. Subsequently, we challenged mice that had been immunized with lethally irradiated Colon38, a murine colon tumor cell line, to evaluate the quality of the induced anti-tumor response. Following immunization, mice demonstrated both local and peripheral immunity to specific tumor challenge, which required T cells. Interestingly, mice immunized with Colon38 also demonstrated potent non-specific anti-tumor immunity when challenged with an unrelated syngeneic tumor cell line in the peritoneal cavity, which was dependent on NK cells. Since T and NK cells are important in mediating anti-tumor immunity, we explored if IL-2, a potent stimulator of both populations, could alter tumor growth in a setting not involving pre-immunization. Mice were injected with two different clones of B16 cells that expressed different levels of IL-2. Lower levels of IL-2 were able to increase T and NK cell frequencies in tumors, but the alteration in tumor growth induced by this level of IL-2 was modest. Higher levels of IL-2 were able to prevent tumors by inducing dormancy, which was dependent on both T and NK cells. These studies highlight the potent anti-tumor capabilities of T and NK cells in both preventing and treating tumor growth and suggest new treatment strategies for malignant disease

Mechanisms of Vascular and Immune Alteration via Local Cytokine Treatment of Melanoma.

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2010.One of the earliest immunotherapies for the treatment of cancer involved the systemic administration of cytokines, and interestingly, both metastatic melanoma and renal cell carcinoma are particularly responsive to cytokine-based immunotherapies. However, although many cytokine treatments have been shown to be efficacious in animal models and in human clinical trials, the toxicity caused by high doses given systemically has been a barrier to their use in common clinical practice. The work presented in this thesis utilizes B16, a spontaneous mouse melanoma cell line, transfected to express the cytokines of interest (IL-12 or IL-2) to provide a system of constant, local production of cytokine within the tumor; a treatment modality that has been associated with increased anti-tumor response and decreased toxicity in both mice and humans. In this way, we have been able to elucidate the mechanisms by which these cytokines suppress tumor growth. IL-12 has been shown to be effective in enhancing anti-tumor responses by enhancing immunity and by limiting angiogenesis. However, how IL-12 exerts its anti-angiogenic effect is largely unknown. The majority of solid tumor vessels are chaotic and poorly formed, frequently expressing vascular endothelial growth factor receptor 3 (VEGFR3), a growth factor receptor not expressed on normal adult vessels. However, the vessels within tumors established using B16 cells transfected to express IL-12 (B16/IL-12) have a more normal morphology and do not express VEGFR3. We have shown that IFN- , is required for IL-12 to suppress the aberrant expression of VEGFR3. Further, within B16/IL-12 tumors, T cells are necessary to suppress VEGFR3 expression on tumor vessels. Finally, using IFN-, receptor knockout mice in a bone marrow chimera system, we have shown that the IFN- produced within the tumor suppresses VEGFR3 expression in two ways: 1) acting directly on tumor vessel endothelial cells, and 2) acting on the tumor infiltrating lymphocytes (TILs) to indirectly alter endothelial cells’ VEGFR3 expression. The indirect mechanism was shown to require CXCR3 expression on the immune cells themselves, indicating that a quaternary mediator, downstream of CXCR3 signaling, is required to suppress VEGFR3 on tumor vessels in the absence of direct IFN- signaling. In addition to the alteration of VEGFR3 on the tumor vessel, we found that B16/IL-12 displayed a significant delay in tumor growth and an upregulation of vascular cell adhesion molecule-1 (VCAM-1). This upregulation required IFN- produced primarily by T cells, which then acted directly on the tumor vessel endothelial cells. Indeed, the presence of intratumoral IL-12 stimulated the immune system, resulting in an increased number of IFN--producing TILs per tumor when compared to parental B16 tumors, which may have a marked effect on control of tumor growth. Our data indicate a mechanism in which tumor infiltrating immune cells regulate tumor vessel phenotype. IL-2, the other cytokine focused on in this study, is a T cell derived growth factor, which has a major role in activating many immune cell subsets, including cytotoxic T cells, natural killer cells, and lymphokine-activated killer cells. Although in clinical trials, it has led to some patients having long-lasting remissions in both melanoma and renal cell carcinoma, it may have a role in stimulating immunosuppressive T regulatory cell development, confounding its role as an anti-tumor agent. Here, we show the significant difference that treatment dose has on the anti-tumor potential of IL-2. Transfecting B16 cells to express a low dose of IL-2 led to a small delay in tumor growth and an increase in tumor infiltrating immune cells, but no long-lasting tumor control. Conversely, using a cell line transfected to produce high doses of IL-2 (B16/IL-2.4) led to lasting tumor control and no palpable tumor growth. Because the high dose IL-2 led to a more significant therapeutic benefit, we focused solely on the mechanism by which IL-2 suppresses tumor growth within B16/IL-2.4 and its effect on the tumor-infiltrating immune cells. There was a significant influx of both NK and T cells as early as day 4 post-injection, and although T cells seem to be required for tumor control, IFN- was not, indicating that the tumor cell suppression was achieved via direct cytotoxic effects or a tumor-suppressing mediator produced by tumor-infiltrating T cells. Cell proliferation experiments indicate that high doses of IL-2 suppress tumor cell division at very early time points after injection. In addition, although no tumor growth was seen unless the tumor cells stopped producing IL-2, small tumor cell foci persisted in the area of injection indicating tumor cell dormancy. These data represent a novel mechanism of dose-dependent IL-2-mediated tumor control, which may lead to insights regarding the treatment of human metastatic melanoma. This work has studied multiple mechanisms by which local cytokine treatment alters the tumor microenvironment and controls tumor growth, with the ultimate goal of understanding how immunotherapies can be better utilized to more effectively treat cancer patients

Investigation, In Situ, of the Cutaneous Dendritic Cell Response Following Ionizing Radiation Exposure

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2013.In the event of a radiological disaster, the skin would likely receive substantial ionizing radiation (IR) poisoning which could negatively impact immune-regulation within the cutaneous microenvironment. Indeed, over the past 20 years, cutaneous radiation syndrome (CRS) has been identified in victims from every major radiological accident. Secondary injuries, including radiological fallout in the form of beta-particles, incisional wounds from blast debris, or infection by opportunistic pathogens, are also likely to have deleterious effects to the overall fitness of the host and integrity of the immunological barrier, which is comprised of two cutaneous dendritic cell (cDC) populations including epidermal Langerhans cells and dermal interstitial DC (iDC). These antigen-presenting cells are critical regulators of skin homeostasis, immuno-surveillance, induction of adaptive immunity, and wound repair, however they have not been examined in the context of IR exposure. To mimic a radiological emergency, we developed a murine model of local and sub-lethal total body irradiation (TBI). Using antibody-labeled fluorescence imaging techniques, we showed in models ex vivo and in vivo that IR exposure induced cDC migration to the draining lymph node in a dose and time dependent fashion. We determined that migration was due, in part, to the up-regulation of the chemoattractant CCL21 on lymphatic vessels as well as CCR7 expressed on cDC. Secondary insults to the skin, including beta IR exposure and incisional wounding following TBI, resulted in an additive reduction of cDC. Importantly, cDC function, as examined by contact hypersensitivity assay, revealed that sites exposed to IR had muted swelling responses suggesting impaired cDC function. Indeed, local and TBI mice challenged intra-dermally in the ear with Candida albicans become moribund and were unable to control the infection resulting in systemic dissemination to the kidneys, whereas un-irradiated mice contained and cleared the pathogen. Thus, we believe that the IR-induced migration of cDC is an indiscriminate response resulting in the breakdown of the immunological barrier and increased susceptibility to opportunistic infection through irradiated skin. To mitigate the IR-induced reduction of cDC and preserve the immunological barrier, mice were treated with the immuno-stimulatory cytokine interleukin-12 which resulted in significant retention of iDC for seven days

Role of interferons and immunotherapy in enhancing radiation treatment of cancer

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2014.Radiation therapy is one of the most common treatment modalities for many different types of solid tumors. Although a lot of research has been done in the past few decades to improve the efficacy of radiation therapy, a majority of the efforts were targeted at augmenting tumor cell-killing and minimizing exposure of surrounding tissue to radiation. Recent studies have provided mounting evidence that the immune system is involved in the capacity of radiation therapy to delay tumor progression. However, due to the complexity of interactions between immune components and the tumor microenvironment, the mechanisms by which radiation mediates tumor immunity have not been completely elucidated. Our lab has previously demonstrated that interferon-gamma (IFN-γ) is an important factor that contributes to radiation-mediated anti-tumor immunity. Here, we hypothesized that radiation induces production of type I IFNs, which synergistically enhance signaling pathways downstream of IFN-γ. Further, endogenous type I IFNs are essential for tumor control and the efficacy of radiation therapy. In particular, type I IFNs are required for the recruitment, activation and cytolytic function of CD8+ T cells. Therefore, we proposed that increasing intratumoral levels of IFN-α may further improve the capacity of radiation therapy to slow tumor progression. Using B16 cell line with inducible expression of IFN-α, we demonstrated that type I IFNs can indeed result in better tumor control than radiation or IFN-α treatment alone. Besides IFN-α therapy, a Listeria monocytogenes (Lm)-based cancer vaccine has been shown to generate strong antigen-specific immune responses against cancer. Here, we evaluated whether the combinatorial therapy of radiation and administration of Lm vaccine could enhance the efficacy of radiation therapy. Using the B16-OVA mouse model, our data revealed that combining Lm vaccine and radiation therapy leads to a robust anti-tumor response that prolongs the progression-free period induced by radiation therapy. Overall, our studies provided insight on the importance of type I IFNs in the activation of radiation-mediated anti-tumor immune responses. These data also shed light on the potential of combining immunotherapy with radiation therapy in a clinical setting; hence the development of novel strategies aimed at complementing radiation-induced anti-tumor immunity should be explored