Dissertations / Theses on the topic 'T-cell mediated immunity'

Three major questions were addressed in this dissertation: 1)Do immune abnormalities associated with autism primarily alter CD4+ T cell-mediated or humoral immune responses? 2) Are specific T cell clones expanded in autism? 3) Which, if any, infectious agents play a role in autism? CD4+ T cell-mediated (Th1) or humoral (Th2) immune responses can be distinguished on the basis of the cytokines expressed. CD4+ T-cells secrete interleukin type 2 (IL-2) and interferon-γ, whereas a Th2 response is associated with secretion of interleukin type 4(IL-4). mRNA extracted from peripheral blood mononuclear nuclear cells (PBMC) showed significantly increased levels of IL-2 and interferon-γ expression in 24 autistic subjects relative to 19 normal controls. IL-4 mRNA was undetectable in the same group of autistic subjects. These results indicate that a CD4+ T cell-mediated immune response is associated with autism. The expression of V-β chain mRNA was used as a marker or particular T cell clone expression. The expression of V-β 13 was significantly elevated in the study group of 11 autistic subjects, but not in 9 normal subjects. This suggests that T cell-mediated autoimmunity is a factor in the disease. Two types of human leukocyte antigens (HLA) alleles, DR4 and DR1, are associated with autism. The association between V-β 13 expressing T cell clones and autism was shown even more strongly in the subgroups expressing HLA DR4 or DR1. This result suggests a link between antigen presentation by HLA DR4 or DR1 and expansion of V-β 13 T cell clones. The potential involvement of pathogens suspected to trigger autism was investigated by examining T cell proliferation responses to peptide epitopes. As a group, the 24 autistic subjects did not show a decreased response to peptides derived from rubella virus, influenza A virus, herpes simplex virus type 1, cytomegalovirus, and Clostridium tetani. Another model of autism postulates that autism is induced by pathogens that possess epitopes identical to the hypervariable region 3 (HVR-3) of the HLA DR4 or DR1 alleles. Two antigens derived from the Escherichia coli dna J protein and the Epstein-Barr virus glycoprotein 110 peptides that contain sequences identical to the HVR-3 of the DR4 and DR1 alleles were examined for their ability to induce T cell proliferation in autistic and normal subjects. No effect of the DR4 or DR1 alleles on the response to these two antigens was detected. Therefore, both types of results do not support the model of immune tolerance in autism. However, average T cell proliferative activity was significantly lower in the same autistic subjects. This confirms many prior reports that reduced T-cell responses may shape susceptibility to autism. Further understanding of how immune abnormalities and infectious agents lead to autism should guide development of preventative and therapeutic strategies for this disease. (152 pages)

The molecular mechanisms governing the functional and structural decline of thymus with age, causing thymic immunosenescence, are incompletely identified. Using a bitransgenic mouse model, Dr Giangreco discovered that the over-expression of receptor tyrosine kinase ErbB2 causes reversible thymic atrophy. The over-expression of epithelial ErbB2 upon doxycycline administration in bitransgenic mice led to decreased thymus size and cellularity, loss of cortical-medullary boundary and abnormal T cell differentiation, bearing similarity to age-dependent thymic involution. This thesis set out to investigate this observation in more detail. I demonstrated that the observed atrophy in bitransgenic thymuses was because of thymus-specific ErbB2 expression, by employing foetal thymic organ cultures. In addition, I showed that over-expression of epithelial ErbB2 disrupted the thymic epithelial cells distribution. Also, an increase in Sca1+Cd49f+ epithelial cells with stem cell potential was noted, explaining why the thymic atrophy in bitransgenic mice was reversible. Exploration of the potential mechanistic pathways found that the thymic atrophy phenotype of K14-NICDER mice, in which epithelial Notch is activated upon tamoxifen administration, resembled the bitransgenic mouse thymic atrophy phenotype. However, mechanistic studies failed to establish ErbB2 acting upstream of Notch, and require further investigation. Administration of Lapatinib, an ErbB2 inhibitor improved the thymic organization and function in aged mice. Lapatinib treatment of aged mice also enhanced vaccine responses to Prevenar 13, a Streptococcus pneumoniae glycoconjugate vaccine, and increased the efficacy of vaccination to protect against subsequent pneumonia challenge. However my results showed that ErbB2 inhibition does not reverse thymic atrophy in scurfy mice, which have truncated Foxp3 protein, and an autoimmune phenotype. In conclusion, this study highlights the importance of ErbB2 in maintaining thymus homeostasis and thymus mediated immunity, and proposes a novel ErbB2 inhibition therapy for rejuvenating an aged thymus, to counter the associated immunosenescence and thereby improve vaccine responses.

The only available tuberculosis vaccine is BCG, but this requires urgent replacement or improvement for more efficacious use in the prevention of the large burden of disease in both humans and cattle. The protection provided by BCG remains valuable and in order to facilitate further vaccine development, the underlying protective mechanisms require understanding. In this thesis the putative role of the CD4 T cell response was examined. Collectively it was shown that BCG immunisation of BALB/c mice led to their colonisation by the live BCG vaccine bacilli. By six weeks postimmunisation the vaccination induced populations of antigen-specific CD4 T cells derived from the spleen and the lung which exhibited immediate effector cytokine functionality on antigen recognition. Further examinations of these cells indicated they putatively represented a canonical effector memory rather than central memory CD4 T cell population. The number of these BCG-induced antigen-specific CD4 T cells expanded on challenge infection with virulent Mycobacterium bovis, supporting their association with the protection afforded by the vaccine. In long-term vaccinates, the viable BCG bacilli continued to persist indefinitely, accompanied by the stability of both this putative effector memory CD4 T cell population and the protective capacity of the vaccine. However, elimination of the persistence of the vaccine bacilli, by antibiotic treatment, abrogated the detection of these CD4 T cells and reduced the protective capacity of the vaccine by around 50%, inferring both are dependent on live BCG persistence. The remaining independent mechanism of protection remains unclear. During this work, emphasis was also made on the potential influence and limitations of assay methodology used in analysing antigen -specific CD4 T cells. These results have important implications for our understanding of the potential mechanisms of BCG induced CD4 T cell-mediated immunity in the mouse model and its interpretation for use in vaccine development.

Tumor immunoediting is a dynamic process in which the immune response attacks tumor cells by detecting danger signals and tumor antigens. In order to survive, tumor cells develop mechanisms to avoid detection or destruction by the immune system. To counteract this, several strategies are being developed to enhance the antitumor immune response, including the depletion of immunosuppressive cells, enhancing the activation of antitumor immune cells and increasing tumor cell immunogenicity. These therapies have seen limited success individually, however, and it is likely that combination therapy with novel targets will be necessary to see reproducible beneficial responses. Epigenetic modifications are attractive therapeutic targets because they are reversible and affect gene expression in cancer cells. Within this framework, this study aimed to elucidate the role of the chromatin remodeling complex nucleosome remodeling factor (NURF) in cancer immunoediting by silencing of bromodomain PHD-finger containing transcription factor (BPTF), the largest and essential subunit of NURF. Using two syngeneic mouse models of cancer, BPTF was found to suppress T cell antitumor activity in the tumor microenvironment. In vitro, enhanced cytolytic activity was observed for individual CD8 T cell clones only from mice bearing BPTF-silenced tumors, implicating the involvement of novel antigens. Mechanistic investigations revealed that NURF directly suppresses the expression of genes encoding immunoproteasome subunits Psmb8 and Psmb9 and the antigen transporter genes Tap1 and Tap2. PSMB8 inhibition reversed the effects of BPTF ablation, consistent with a critical role for the immunoproteasome in improving tumor immunogenicity. Thus, NURF normally suppresses tumor cell antigenicity and its depletion improves CD8 T cell antitumor immunity. In a concurrent study using different tumor lines, BPTF was also found to suppress natural killer (NK) cell antitumor immunity in vivo. Enhanced NK cell cytolytic activity toward BPTF-depleted targets in vitro was dependent on the natural cytotoxicity receptors (NCR). Molecular studies revealed that BPTF directly activates heparanase (Hpse) expression, resulting in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans. Thus, NURF represses NCR co-ligand abundance and its depletion enhances NK cell cytotoxicity. Therefore, NURF emerges as a candidate therapeutic target to enhance CD8 T or NK cell antitumor immunity.

T-cells are lymphocytes that make up part of the adaptive arm of the immune system, and are essential for efficient protection from and eradication of viruses and pathogens. T-cells not only play an important role in protection from external agents, but also regulate and prevent activation towards self-peptides and detect and remove erratically growing cells. Alterations in T-cell activation and suppression contribute to auto-immunity, immunocompromised disorders, and cancer progression. The immune system, and T-cells in particular, provides daily surveillance, recognition and destruction of aberrant cells. Although the immune system is proficient at suppressing malignant progression, tumor cells acquire various methods of immune evasion. Myelodysplastic Syndrome (MDS) is a pre-malignant dysplastic disorder of the bone marrow characterized by ineffective hematopoiesis and clonality in the myeloid lineage, where lack of immune response has been implicated in the propensity for progression to acute myeloid leukemia (AML). Leukemia progression is associated with the acquisition of complex genetic abnormalities. Alterations in immune system regulation have been implicated in various stages of the disease process, although the role of the immune system in response to several therapies in MDS has not been fully discovered. Lenalidomide is a small molecule therapeutic preferentially effective in MDS patients with an interstitial chromosome 5q deletion (del(5q)). Improved erythropoiesis has also been reported to occur in 20-30% of low-risk, non-del(5q) patients. Although lenalidomide is a potent immunomodulatory drug that potentiates T-cell and NK-cell responses, the T-cell compartment in MDS is highly deregulated by aberrant repertoire skewing, decreased function and abnormal naïve and memory cell homeostasis. The presence of lymphoid infiltrates in the bone marrow of lenalidomide-responsive patients suggests that T-cells may participate in the hematopoietic response, but it is unclear if lenalidomide is capable of reversing these functional T-cell defects. We therefore assessed immunological changes in low-risk MDS patients before and after 16-weeks of lenalidomide therapy, and assessed the relationship to erythroid response. Although MDS T-cells were anergic prior to treatment, we have shown that T-cells in responders have a significant increase in antigen-induced proliferative response and T helper type-1 (Th1) cytokine production (IL-2, IFN-γ, TNF-α) compared to non-responders. The change in function positively correlated with an increase in naïve T-cells and a decrease in memory cells, indicating that lenalidomide has immunomodulatory activity to reverse anergy in MDS. Although it is known that lenalidomide may increase T-cell activation and proliferation in the absence of co-stimulatory signals, a direct mechanism of action has yet to be elucidated. Since CD28 is one of the most important co-stimulatory molecules deregulated in cancer, we therefore set out to determine if the expression of CD28 was essential for lenalidomide's mechanism in T-cells. We knocked out CD28 expression in healthy donor T-cells, and sorted on inherently deficient, CD28null, T-cells that accumulate in older healthy donors and found that lenalidomide-induced proliferation and function were completely ablated within the CD28null subset. These data indicate the immunotyrosine-based activation motifs (ITAMs) on the intracellular domain of the CD28 receptor are necessary for lenalidomide action. Interestingly, during the natural aging process, repeated exposure to antigens results in the accumulation of CD28null T-cells that are phenotypically distinct and functionally deficient due to excessive proliferative history in vivo. We therefore examined whether CD28 expression on MDS patient T-cells affected responses to lenalidomide, and if this could be used as a predictive biomarker of responsiveness. We found that patients who fail lenalidomide therapy had higher CD8+ Terminal Effector Memory (TEM), which are inherently CD28null, and that non-responders had an overall increase in CD4+ and CD8+CD28null T-cells, as well as an increase in CD28null cells within the TEM compartment compared to hematologic responders. We then sought to determine the particular protein target of lenalidomide responsible for increased CD28 receptor signaling in T-cells. Several targets in a variety of cell types have been postulated, although the direct mechanism in T-cells is unclear. Our group has previously shown that lenalidomide inhibits the activity of two haplodeficient phosphatases located within the commonly deleted region (CDR) on chromosome 5q in the MDS myeloid clone, Protein Phosphatase 2A (PP2A) and Cdc25c. PP2Ac is known to bind CD28 and is hypothesized to inhibit T-cell co-stimulation. Therefore, it is plausible that lenalidomide and other IMiDs inhibit the phosphatase activity of PP2A which leads to increased activation of T-cell proximal signals dependent on CD28 expression. We examined this hypothesis using molecular modeling and virtual screening and found that all of the IMiDs (lenalidomide, pomalidomide, and thalidomide) can theoretically interact with the catalytic pocket of the PP2A heterotrimer, potentially inhibiting PP2Ac activity. In vitro phosphatase activity assays supported these findings as lenalidomide-inhibition of PP2Ac activity was seen in both ad293 and Jurkat cell lines, and in primary T-cells. Mutations of theorized lenalidomide hydrogen-bond sites within the catalytic pocket of PP2A rendered the enzyme catalytically dead, indicating that these are important residues for enzymatic activity, but unfortunately could not be used to determine if lenalidomide activity was disrupted by mutation of those sites. These data together suggest that the ability of lenalidomide to augment immune activation in vivo in MDS patients, and potentially other diseases, is extremely important to patient response. Also, that CD28 expression on T-cells is essential for lenalidomide immune-mediated tumor eradication through CD28 downstream signaling, and potentially through inhibition of PP2A. These results are useful in designing future lenalidomide-combination therapy trials in other hematologic and solid malignancies, and could be used to help stratify patients for future therapeutic decisions in MDS and other malignancies.

The ultimate goal of antitumor vaccines is to develop memory CD8+ cytotoxic T lymphocytes (CTLs), which are critical mediators of antitumor immunity. Previous work in our lab demonstrated that the ovalbumin (OVA)-specific CD4+ T cell-based (OVA-TEXO) vaccine generated using OVA-pulsed dendritic cell (DCOVA)-released exosomes (EXOOVA) stimulates CTL responses via interleukin (IL)-2 and costimulatory CD80 signaling. To assess the potential involvement of other costimulatory pathways and to define the key constituent of costimulation for memory CTL development, we first immunized wild-type (WT) C57BL/6 and gene-knockout mice with WT CD4+ OVA-TEXO cells or OVA-TEXO cells with various molecular deficiencies. We then assessed OVA-specific primary and recall CTL responses using PE-H-2Kb/OVA257–264 tetramer and FITC-anti-CD8 antibody staining by flow cytometry. We also examined antitumor immunity against the OVA-expressing B16 melanoma cell line BL6-10OVA. We demonstrate that CD4+ OVA-TEXO cells form immunological synapses with cognate CD8+ T cells in vitro. By assessment of the pattern of ex vivo interactions between OTI CD8+ T cells and OVA-TEXO or (Kb-/-)TEXO cells lacking peptide/major histocompatibitity complex (pMHC)-I expression, we provide the first visible evidence on the critical role of exosomal pMHC-I in targeting OVA-TEXO to cognate CD8+ T cells using two-photon microscopy. By assessing primary and recall CTL responses in mice immunized with OVA-TEXO cells or with OVA-TEXO cells lacking the costimulatory molecules CD40L, 4-1BBL or OX40L, we demonstrated that these costimulatory signals are dispensable for CTL priming by OVA-TEXO cells. Interestingly, CD40L, but not 4-1BBL or OX40L, plays a crucial role in the development of functional memory CTLs against BL6-10OVA tumors. Overall, this work suggests that a novel CD4+ T cell-based vaccine that is capable of stimulating long-term functional CTL memory via CD40L signaling may represent a novel, efficient approach to antitumor vaccination. Breast cancer is the most common cancer among women in the western world. Approximately 20-30% of invasive breast carcinomas are proto-oncogene human epidermal growth factor receptor (HER)-2 positive and associated with increased metastatic potential and poor prognosis. The survival benefit of anti-HER2 driven therapies demonstrated in clinical trials indicates that HER2 is one of the most promising molecules for targeted therapy to date. Above results prompt us to assess whether CD4+ T-cell-based vaccine can stimulate efficient HER2-specific CD8+ CTL responses and antitumor immunity in transgenic mice with HER2-specific self-immune tolerance. We prepared HER2-specific HER2-TEXO using ConA-stimulated CD4+ T cells with uptake of exosomes released from HER2-expressing AdVHER2-transfected DCs. We found that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2/HER2-expressing B16 melanoma BL6-10HLA-A2/HER2 in 2/8 double transgenic HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2+ breast cancer. In contrast to CD4+CD25+ regulatory T cells (Tregs), mechanisms of CD8+CD25+ Treg-mediated immunosuppression are not well understood. In this study, we purified polyclonal CD8+CD25+ Tregs from C57BL/6 mouse splenocytes and expanded them in culture medium containing CD3/CD28 microbeads. By using these amplified CD8+CD25+ Tregs, we demonstrated that CD8+CD25+ Tregs inhibit naive CD4+ T-cell proliferation and induce naive T-cell anergy by up-regulating T-cell anergy-associated early growth response 2 (EGR2), and by decreasing T-cell proliferation and IL-2-secretion upon stimulation. They also impact the expression of perforin on effector CTLs and directly induce perforin-mediated CTL apoptosis. CD8+CD25+ Tregs, when pulsed with OVA323-339 peptide, exert an enhanced inhibition. Interestingly, CD8+CD25+ Tregs, when pulsed with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, become capable of inhibiting MOG35-55-induced experimental autoimmune encephalomyelitis (EAE). Two-photon microscopic observations suggest that OVA323-339-pulsed (armed) CD8+CD25+ Tregs reduce the interactions between DCs and cognate CD4+ T cells ex vivo by increasing velocities of T cells in mouse lymph nodes. Therefore, redirecting antigen-specificity to nonspecific CD8+CD25+ Tregs can be achieved for enhanced immunosuppression through their arming with the antigen-specific pMHC-II complexes. This approach may have great impact on improvement of endogenous polyclonal Treg-mediated immunotherapy for autoimmune diseases. Taken together, our studies demonstrate that nonspecific polyclonal CD4+ T cells and CD8+CD25+ Tregs, when armed with HER2 and MOG antigen-specific pMHC-I and -II complexes, become capable of stimulating enhanced HER2-specific CTL responses and antitumor immunity in double transgenic HLA-A2/HER2 mice and inducing enhanced MOG-specific immunosuppression in MOG-induced EAE mice, respectively. Therefore, redirecting antigen specificity to nonspecific CD4+ T and CD8+CD25+ Tregs by pMHC complex arming may have great impact in development of novel T cell-based vaccines for treatment of cancer and autoimmune diseases.

Indiana University-Purdue University Indianapolis (IUPUI)
Immunotherapy for cancer has held much promise as a potent modality of cancer treatment. The ability to selectively destroy diseased cells and leave healthy cells unharmed has been the goal of cancer immunotherapy for the past thirty years. However, the full capabilities of cancer immunotherapies have been elusive. Cancer immunotherapies have been consistently hampered by limited immune reactivity, a diminishing immune response over time, and a failure to overcome self-tolerance. Many of these deficiencies have been borne-out by immunotherapies that have focused on the adoptive transfer of activated or genetically modified mature CD8+ T cells. The limitations inherent in therapies involving terminally differentiated mature lymphocytes include limited duration, lack of involvement of other components of the immune system, and limited clinical efficacy. We sought to overcome these limitations by altering and enhancing long-term host immunity by genetically modifying then transplanting HSCs. To study these questions and test the efficiency of gene transfer, we cloned a tumor reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen TRP-1, then constructed both a high expression lentiviral delivery system and a TCR Tg expressing the same TCR genes. We demonstrate with both mouse and human HSCs durable, high-efficiency TCR gene transfer, following long-term transplantation. We demonstrate the induction of spontaneous autoimmune vitiligo and a TCR-specific TH1 polarized memory effector CD4+ T cell population. Most importantly, we demonstrate the destruction of subcutaneous melanoma without the aid of vaccination, immune modulation, or cytokine administration. Overall, these results demonstrate the creation of a novel translational model of durable lentiviral gene transfer, the induction of spontaneous CD4+ T cell immunity, the breaking of self-tolerance, and the induction of anti-tumor immunity.

The receptor-recognized form of α₂-macroglobulin (α₂M*) targets antigens (Ag) to professional Ag-presenting cells (APCs) for rapid internalization, processing, and presentation. When employed as an Ag delivery vehicle, α₂M* amplifies major histocompatibility complex (MHC) class II presentation as demonstrated by increased antibody (Ab) titers. Recent evidence, however, suggests that α₂M*-encapsulation may also enhance Ag-specific cytotoxic T lymphocyte (CTL) immunity. In these studies, we demonstrate that α₂M*-delivered Ag (ovalbumin, OVA) enhances the production of specific in vitro and in vivo CTL responses.

Murine splenocytes expressing a transgenic T cell receptor (TCR) specific for CTL peptide OVA_257-264 (SIINFEKL) demonstrated up to 25-fold greater IFN-γ and IL-2 secretion when treated in vitro with α₂M*-OVA compared to soluble OVA. The frequency of IFN-γ -producing cells was increased ~15-fold as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cells, as assayed by tetramer binding and [3H]thymidine incorporation, and cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also significantly enhanced by α₂M*-OVA. Furthermore, CTL responses were observed at Ag doses tenfold lower than those required with OVA alone.

We also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with α₂M*-OVA. These α₂M*-OVA-immunized mice displayed increased protection against a subcutaneously implanted OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The anti-tumor response observed with α₂M*-mediated Ag delivery was comparable to that of an accepted vaccine adjuvant (CpG 1826) and appeared superior to a cell-based vaccine technique.

To further understand the mechanism underlying this enhanced CTL immunity, the subsets of professional APCs capable of cross-presenting α₂M*-encapsulated Ag were investigated. Although both dendritic cells (DCs) and macrophages appear to stimulate some degree of cross-priming in response to α₂M*-encapsulated Ag, CD8+CD4- and CD8-CD4+ DCs appear to do so with the greatest efficiency. The implications of this finding to the ongoing debate regarding the relative contributions of APC subsets to Ag cross-presentation and the determinants of which cells cross-present with high efficiency are discussed.

These observations demonstrate that α₂M*-mediated Ag delivery promotes cross-presentation resulting in enhanced Ag-specific CTL immunity. Considered in the context of previous work, these results support α₂M* as an effective Ag delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.

Dissertation