Browsing by Subject "CD8-Positive T-Lymphocytes"
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Item Autoregulatory CD8 T-Cells Modulate CNS Autoimmune Disease by Targeting Encephalitogenic CD4 T-Cells(2013-01-16) Ortega, Sterling Bolivar; Karandikar, Nitin J.Multiple Sclerosis (MS) is a disease, which presents with neurological dysfunction and is believed to have an immunological etiology. Lesions in the central nervous system (CNS) are characterized by an inflammatory cellular infiltration and demyelination of neuronal axons. It is believed that myelin sheath-targeting CD4 T-cells are important mediators of this disease. While it is known that CD8 T-cells are present, oligoclonally expanded and are the predominant T-cell population in the MS CNS lesion, their antigen specificity and function remains to be elucidated. Using a murine model of MS, experimental autoimmune encephalomyelitis (EAE), we are currently evaluating the role of these poorly studied myelin antigen-specific CD8 T-cells in the context of this autoimmune disease. We have observed that myelin oligodendrocyte glycoprotein peptide (MOG₃₅₋₅₅)-specific CD8 T-cells do not mediate EAE, but in fact are capable of suppressing both de novo and established clinical disease. Corroborating these data, CD8-/- C57BL/6 mice are now shown to exhibit a more severe EAE. However, the characterization, mechanism of action and cellular targets of these autoreactive regulatory CD8 T-cells are still unknown. Initial observations revealed that disease ameliorating CD8 T-cells are not unique to MOG₃₅₋₅₅-induced EAE, as proteolipid protein peptide (PLP₁₇₈₋₁₉₁)-induced EAE in B6 and SJL mice strains were capable of generating disease ameliorating CNS antigen-specific CD8 T-cells. Autoreactive regulatory (autoregulatory) CD8 T-cells exhibit a central memory phenotype (CCR7+CD62L+CD44-) and produce IFN-γ and perforin. Disease suppression by these cells is dependent on recognition of cognate antigen in vivo within the context of MHC Class Ia. These cells do not traffic to the CNS upon transfer into naïve mice; however, they gain CNS access following induction of CNS inflammation, suggesting both a peripheral immune compartment and CNS mechanism of action. Interestingly, autoregulatory CD8 T-cell-mediated suppression is IFN-γ and perforin-dependent and can be augmented by IL-12 in vitro modulation. Next, we asked whether autoregulatory CD8 T-cells could directly target encephalitogenic myelin-specific CD4 T-cells. We now report that treatment with myelinspecific CD8 T-cells results in significantly attenuated adoptive (CD4 T-cell mediated) EAE. Moreover, increased disease severity in CD8-/- mice correlated with an increase in autoreactivity and inflammatory cytokine production by myelin-specific CD4 T-cells. This is reversible upon adoptive transfer of MOG₃₅₋₅₅-specific CD8 T-cells. Targeting of encephalitogenic CD4 T-cells by myelin-specific CD8 T-cells is sufficient, as induction of wildtype adoptive EAE in KᵇDᵇ-/- recipient mice could be suppressed. In vivo proliferation assays revealed a global suppression/cytotoxicity of MOG-specific CD4 T-cells. These studies define the immune regulatory function of autoreactive CD8 T-cells in EAE. Our results demonstrate that autoregulatory CD8 T-cells have an important disease ameliorating role in EAE, which is a disease of perturbed immune regulation. Understanding this arm of the adaptive immune system offers a promising strategy for immunotherapeutic intervention of MS.Item AXL Targeting Restores PD-1 Blockade Sensitivity of STK11/LKB1 Mutant NSCLC Through Expansion of TCF1+ CD8 T Cells(December 2021) Li, Huiyu; Akbay, Esra A.; DeBerardinis, Ralph J.; Fu, Yang-Xin; Brekken, Rolf A.; Minna, John D.; Aguilera, Todd A.Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB) for unknown reasons. We found that introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically, this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype that was recapitulated in human STK11/LKB1 mutant NSCLCs. We found that systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expands tumor-associated TCF1+ PD-1+ CD8 T cells, restoring therapeutic response to PD-1 ICB for KPL tumors. This effect was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. Anecdotal NSCLC patients with STK11/LKB1 mutant tumors also demonstrated responses to the combination of AXL inhibitor bemcentinib and pembrolizumab. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.Item CD8+ T Cells Are Required for the Therapeutic Action of Glatiramer Acetate in Autoimmune Demyelinating Disease(2013-06-28) Tyler, Andrew Farley; Niederkorn, Jerry Y.; Eagar, Todd N.; Pasare, Chandrashekhar; Street, Nancy E.; Karandikar, Nitin J.Glatiramer acetate (GA, Copaxone®) is an FDA-approved immunomodulatory therapy for multiple sclerosis (MS), an immune-mediated demyelinating disease of the central nervous system. Our group has previously shown that GA therapy induces CD8+ T cell responses responsible for suppression of CD4+ T cell responses in MS patients. Using a murine model of MS, experimental autoimmune encephalomyelitis (EAE), we also demonstrated that CD8+ T cells are necessary in mediating the therapeutic effects of GA, and adoptive transfer of GA-induced CD8+ T cells resulted in amelioration of EAE, establishing a role as a viable immunotherapy in demyelinating disease. Here, we show that GA treatment, as well as the suppressive function of GA CD8+ T cells, requires IFNγ and perforin, but not IL-10, expression and activation by non-classical MHC class I molecules both in vitro and in vivo. GA-induced regulatory myeloid cells, previously shown to activate CD4+ regulatory T cells in an antigen-independent manner, depend on CD8+ T cells and MHC class I expression to suppress disease in vivo, an effect mediated by MHC class I-mediated induction of CD8+, but not CD4+, T cell responses. GA induces an anti-inflammatory “type 2” phenotype in monocytes in vivo that is unnecessary for the suppression of disease. The drug also inhibits the expression of surface markers of maturation such as CD11c and MHC class II. GA CD8+ T cells reduce the proliferative potential of autoimmune, neuroantigen-specific CD4+ T cells and induce CD4+CD25+Foxp3+ regulatory T cells in vivo. Additionally, several MHC class I-binding peptide epitopes associated with GA treatment of dendritic cells were identified by LC/MS-MS and tested for disease suppression and ability to activate GA CD8+ T cells. These studies demonstrate an essential role for CD8+ T cells in GA therapy and identify their potential as an adoptive immunotherapeutic agent.Item CNS-Specific, Autoreactive CD8+ T Cells Have a Regulatory Role in Autoimmune Demyelination(2010-05-14) York, Nathan Ronald; Karandikar, Nitin J.Multiple Sclerosis (MS) is an inflammatory disorder characterized by the destruction of myelin sheaths, which encase neurons of the central nervous system. A great deal of our current knowledge about the immune pathogenesis of MS derives from work in its murine model, experimental autoimmune encephalomyelitis (EAE), which can be induced by inoculation with a specific neuroantigen or by adoptive transfer of CNS-specific activated T cells. The vast majority of studies in MS and EAE have focused on the role of CD4+ T cells in this disease, with the underlying assumption that MS, like EAE, is a CD4+ Th1-mediated autoimmune disease. However, several reports have implicated both CD4+ and CD8+ T cells in the pathogenesis and regulation of these diseases. In this study, we show the presence of antigen-specific, autoreactive CD8+ T cells in several models of EAE. Furthermore, through series of adoptive transfer studies, we show that these cells play a regulatory role in the pathogenesis of autoimmune demyelination. Using novel in vivo killing assays, we show that these cells retain their killing capacity and that they target both activated APC and CD4+ T cells that have been loaded with the specific antigen. These cells are also shown to produce cytokines that may be involved in disease regulation. We also show that these cells modify antigen-presenting capacity of APC. In conclusion, our studies provide strong evidence that antigen-specific CD8+ T cells are involved in regulatory processes in the context of autoimmune demyelinationItem A Cytokine Receptor Masked IL-2 Prodrug Selectively Activates Tumor-Infiltrating Lymphocytes for Potent Antitumor Therapy(August 2021) Hsu, Eric Jonathan; Zhang, Chengcheng "Alec"; Farrar, J. David; Malladi, Srinivas; Yan, Nan; Fu, Yang-XinCancers are very difficult to treat, and many cancer patients fail to respond to numerous standard of care therapies. Many of these tumors have been observed to lack functional CD8 T cells, which have been observed to be correlated with improved patient prognosis. One of the main strategies to combat the lack of functional tumor infiltrating immune cells is to treat patients with immune stimulating cytokines such as interleukin-2 (IL-2). As a potent lymphocyte activator, IL-2 is an FDA approved treatment for multiple metastatic cancers. However, its clinical use is limited by short half-life, low potency, and severe in vivo toxicity. Current IL-2 engineering strategies exhibit evidence of peripheral cytotoxicity. Here, limitations of both recombinant IL-2 and these next generation IL-2 variants are addressed through the engineering of a novel IL-2 prodrug (ProIL2). Numerous designs of ProIL2 were designed, engineered, and tested until a final optimal construct was synthesized. The activity of a CD8 T cell-preferential IL-2 mutein/Fc fusion protein is masked with IL2 receptor beta linked to a tumor-associated protease substrate. ProIL2 restores activity after cleavage by tumor-associated enzymes, and preferentially activates inside tumors, where it expands antigen-specific CD8 T cells. This significantly reduces IL-2 toxicity and mortality without compromising antitumor efficacy. ProIL2 also overcomes resistance of cancers to immune checkpoint blockade. Furthermore, neoadjuvant ProIL2 treatment can eliminate metastatic cancer through an abscopal effect. Lastly, ProIL2 can also synergize with radiation therapy to more effectively control both primary and metastatic cancer. Further protein engineering strategies are being implemented to overcome potential limitations of ProIL2. Taken together, this approach presents an effective tumor targeting therapy with reduced toxicity.Item Dendritic Cells Suppress Pathogen-Induced Inflammasome Activation to Prime Naïve T Cells(2021-05-01T05:00:00.000Z) McDaniel, Margaret Mae; van Oers, Nicolai S. C.; Pasare, Chandrashekhar; Fu, Yang-Xin; Alto, Neal; Satterthwaite, Anne B.Activation of inflammasome leads to pyroptotic cell death thereby eliminating the replicative niche of virulent pathogens, a process integral to innate immunity. While inflammasome-associated cytokines such as IL-1β and IL-18 have an established role in T cell function, whether inflammasome activation in dendritic cells (DCs) is critical for T cell priming is not clear. Here, we find that lymphoid organ resident conventional DCs (cDCs) actively suppress inflammasome activation to prevent pyroptotic cell death. This protection from inflammasome-induced cell death preserves the ability of cDCs to prime both CD4 and CD8 T cells. Transcription factors IRF8 and IRF4, in cDC1s and cDC2s respectively, mediate this suppression of inflammasome activation by limiting the expression of inflammasome-associated genes. Additionally, overexpression of either of IRF4 or IRF8 is sufficient to inhibit inflammasome activation in macrophages, cells that are normally permissive to inflammasome activation. Furthermore, we find that reduced expression of IRF8 leads to aberrant inflammasome activation in cDC1s which hampers their ability to prime CD8 T cells. These results uncover the importance as well as the molecular mechanism of inflammasome suppression in cDCs and ascribe a novel post-developmental role for IRF4 and IRF8 in cDC function.Item Development of an IL12 Prodrug to Treat Solid Tumors with Minimal Toxicity(2022-05) Moon, Benjamin Ik; Gao, Jinming; Hammers, Hans; Li, Bo; Fu, Yang-Xin; Qiao, JianCytokines are secreted molecules that guide the immune system to respond correctly to various challenges. Among all cytokines, IL12 is perhaps the most powerful at polarizing the immune response into a Type 1, cell-mediated phenotype. Cell-mediated immunity plays a critical role in cancer immunoediting, allowing CTLs to recognize and kill aberrant cells. Because of this, IL12 has been tested in many different preclinical and clinical studies for its potential use as an anti-tumor therapeutic agent. However, IL12 also causes severe, dose-limiting systemic toxicity due to on-target, off-tumor activation of peripheral immune cells. Newer attempts at IL12-mediated delivery focus on restricting IL12 activity to within the tumor as much as possible, but they each have their own limitations. To address these problems, we developed a novel IL12 prodrug, pro-IL12, that is actively blocked until it is preferentially activated within the TME. We achieved this by using portions of the IL12 receptor attached with a flexible linker to sterically block the active site of IL12. The linker contains a substrate site that can be cleaved by tumor-specific proteinases, thereby releasing the blocker and activating the prodrug. Pro-IL12 successfully maintained anti-tumor efficacy with reduced toxicity compared to its non-prodrug counterpart. We determined that the mechanism of anti-tumor immunity was predominantly through pre-existing, intratumor CD8+ T cells that produce IFNγ after direct binding of the prodrug to cell surface IL12 receptor complexes. Pro-IL12 also worked in combination with TKI and ICB to achieve even more potent tumor control. In a follow up study, I propose that a higher dose of pro-IL12 might use distinct cellular and molecular mechanisms. Indeed, high dose pro-IL12 more effectively controls large tumors at the cost of reintroducing systemic toxicity. Mechanistically, this dose used a broader, T cell-dependent mechanism that was independent of IFNγ. Further analysis determined that IFNγ was responsible for all manifestations of toxicity and that IFNγ blockade given concurrently with pro-IL12 could limit toxicity with no effect on efficacy. Additionally, the absence of IFNγ signaling on T cells had no effect on their phenotype or ability to control the tumor. As a whole, these studies document the development of a next generation, IL12 immunotherapy for the treatment of solid tumors with an emphasis on its mechanisms of tumor control that are distinct from toxicity.Item Disease Exacerbation in Multiple Sclerosis Patients is Characterized by Loss of Terminally Differentiated CD8+ T Regulatory Cells(2014-02-04) Cunnusamy, Khrishen; Baughman, Ethan J.; Franco, Jorge; Ortega, Sterling B.; Greenberg, Benjamin M.; Frohman, Elliot M.; Karandikar, Nitin J.Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) that afflicts more than 400,000 people in the US. Although the etiology of the disease is unknown, pathogenic T cells are thought to underlie MS immune pathology. In contrast to the current paradigm, we recently showed that MS patients harbor CNS-specific CD8+ T regulatory cells (CD8 Tregs) that are deficient during disease relapse. In the current study, we demonstrate that the neuroantigen-specific CD8 Tregs were cytolytic and eliminated pathogenic CD4+ T cells. Sorting of CD8+ T cells using an array of surface cellular markers revealed that the CD8 Tregs were terminally differentiated (CD27-, CD45RO-). The CD8 Treg-mediated suppression was perforin, granzyme B, and interferon-γ-dependent. Interestingly, we found that MS patients with acute disease exacerbation displayed a significant loss (averaging 25%) in the terminally differentiated CD8+ T cells, with a concurrent loss in perforin and granzyme B expression. In order to restore the regulatory potential of impaired CD8 Tregs during exacerbation, we pre-treated exacerbation-derived bulk CD8+ T cells with the cytokine IL-12 and significantly increased the suppressive capability of the cells by ~48% through upregulation of granzyme B and perforin. Our studies uncover the immune suppressive mechanism of neuroantigen-specific CD8 Tregs, and may contribute to the design of clinically relevant immune therapies for MS patients.Item Dysregulated B Cells in Relapsing Remitting Multiple Sclerosis and Their Impact on T-Cell Function(2015-03-10) Ireland, Sara Jean; Monson, Nancy L.; Farrar, J. David; Davis, Laurie; van Oers, Nicolai S. C.; Kasper, LloydRelapsing-remitting multiple sclerosis (MS) is an autoimmune mediated inflammatory demyelinating disorder of the central nervous system. The role of B cells in the pathoetiology of MS is substantiated by cell depletion therapies but is not well understood. We hypothesized that B cells from MS patients would secrete more pro-inflammatory cytokines and support T cell responses to self-antigens and that the therapeutic agent glatiramer acetate (GA) could modulate these aspects of B cell function. To test this hypothesis we interrogated the ability of memory and naive B cells from healthy donors (HD), MS patients, and GA-treated MS patients (GA-MS) to proliferate and secrete cytokines in vitro. We identified a remarkable loss of IL-10 secretion by B cells from MS patients, but a marked increase in the production of IL-6, particularly from naïve B cells. We also found that memory B cells from MS patients exhibited hyperactive proliferation compared to healthy donors and naïve B cells from MS patients. GA had no measurable impact on any of the B cell functions we tested; however, B cells from GA-treated MS patients had a restored ability to produce IL-10, greatly enhanced immunoglobulin production, altered proliferation capacity and a transient diminishment of IL-6 production for patients on therapy for less than 32 months. To address whether memory or naïve B cells from MS patients supported neuro-antigen specific T cell responses, we co-cultured B and T cells in the absence or presence of foreign or neuro-antigens. We found that memory and naïve B cells from MS patients support more CD4+ T cell proliferation and TH1 and TH17 responses to neuro-antigens despite a similar frequency of neuroantigen specific T cells. Together, these data reveal that B cells from MS patients exhibit dysregulated proliferation and cytokine secretion that can be modulated by GA. Furthermore B cells from MS patients support neuroantigen-specific T cell proliferation and pro-inflammatory cytokine production in response to self neuro-antigens.Item The Effects of Imatinib Mesylate on Antigen-Specific Cd8+ T Cell Responses(2006-12-20) Sinai, Parisa; Forman, JamesImatinib mesylate (IM) is a chemical compound designed to inhibit the constitutive tyrosine kinase activity of the Bcr-Abl oncogene in Chronic Myelogenous Leukemia (CML). While IM is very potent in treating CML, little is understood concerning the effects of IM on the immune response. In this study, I have examined the influence of IM on antigen specific CD8 T cells. Mature OT-1 TCR transgenic T cells were transferred into B6.Thy1.1 recipients. This transfer was followed by an infection with Listeria monocytogenes (LM) expressing the cognate epitope OVA 257-264 (LM-OVA) to assess whether IM affects the specific CD8 T cell response generated to this intracellular pathogen. In vitro studies revealed that IM had no effect on proliferation, apoptosis, or IFN-gamma secretion of na?or memory OT-1 T cells at doses of =5muM, although at higher doses inhibition was observed. Adoptive transfer and in vivo infection studies demonstrated that in the presence of IM the primary response of OT-1 T cells in vivo to LM-OVA infection was unaltered as measured by OT-1 T cell percentages in spleen and blood, their expression of IFN-gamma and their proliferation. However, IM influenced the primary OT-1 response by decreasing the expression of the IL-7Ralpha memory marker on OT-1 cells. IM treatment for >28 days resulted in a decreased percentage of OT-1 memory T cells before recall in blood. Furthermore, the response of memory OT-1 cells after LM-OVA rechallenge was diminished as measured by OT-1 T cell percentages in blood. In addition, IM treatment reduced the expression of IL-7Ralpha , a receptor required for memory cell survival, on effector and memory OT-1 cells. The function of memory OT-1 cells as measured by IFN-gamma expression was unaltered as was their proliferation as measured by incorporation of Brdu. Infection assays revealed that clearance of LM-OVA by memory mice is not altered by IM. In addition, the in vivo proliferation of memory OT-1 cells was not altered by IM. While IM did not alter the percentage of CD4 T cells in spleen and blood, IFN-gamma expression by CD4 T cells after recall was decreased. Together, these data demonstrate that IM reduces the number of OT-1 cells following a secondary challenge to an LM-OVA infection, and this may be a result of decreased IL-7Ralpha expression on effector and memory OT-1 cells.Item Modulation of CD8+ T Cell Function by Beta2-Adrenergic Receptor Signaling(2017-05-31) Estrada, Leonardo Daniel; Malter, James; Farrar, J. David; Pfeiffer, Julie K.; Stowe, Ann; D'Orso, IvánThere is intimate crosstalk between the nervous system and the immune system. The nervous system can respond to immune system cues through afferent nerve endings. One effect of this response is the secretion of neurotransmitters in peripheral organs through efferent nerve endings. Primary and secondary immune organs are innervated predominantly by sympathetic nerve endings. The nerve termini are adrenergic and secrete norepinephrine upon stimulation, which can modulate immune cell function primarily through the beta2-adrenergic receptor (ADRB2). Our previous work has demonstrated that various subsets of CD8+ T cells have differential expression of the ADRB2 transcript. The role of ADRB2 signaling on the differentiation and acute effector functions of CD8+ T cells remains poorly understood. More importantly, its effect on CD8+ T cell memory development remains elusive. Therefore, I comprehensively addressed the role of ADRB2 signaling on different aspects of CD8+ T cell function. In the current body of work I demonstrate that intrinsic ADRB2 signaling downregulates CD8+ T cell acute effector functions, without affecting differentiation into effector cells. This is true with various forms of T cell receptor activation and with endogenous as well as pharmacological ADRB2 ligands. Furthermore, CD8+ T cell interferon-gamma secretion induced by innate cytokines was not affected. Finally, ADRB2-deficient CD8+ T cells fail to develop into memory cells after an in vivo viral infection. This effect is intrinsic to the ability of CD8+ T cells to signal through the ADRB2 as shown by bone marrow chimera experiments. The inability of ADRB2-deficient CD8+ T cells to develop into memory cells is accompanied by higher expression of several activation-related genes, as well as decreased expression of CD25 five days post-infection. However, restoration of CD25 expression in ADRB2-deficient cells with IL-2/anti-IL-2 treatment was unable to rescue memory development. Taken together, my results demonstrate for the first time that the sympathetic nervous system controls CD8+ T cell memory development through ADRB2 signaling on CD8+ T cells.Item Neuroantigen-Specific CD8+ Regulatory T-Cell Function Is Deficient During Acute Exacerbation of Multiple Sclerosis(2013-05-31) Baughman, Ethan James; Karandikar, Nitin J.; Street, Nancy E.; Thiele, Dwain L.; Forman, James; Greenberg, Benjamin M.Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). MS is thought to be T-cell-mediated, with prior research predominantly focusing on CD4+ T-cells. There is a high prevalence of CNS-specific CD8+ T-cell responses in MS patients and healthy subjects. However, the role of neuroantigen-specific CD8+ T-cells in MS is poorly understood, with the prevalent notion that these may represent pathogenic T-cells. We show here that healthy subjects and MS patients demonstrate similar magnitudes of CD8+ and CD4+ T-cell responses to various antigenic stimuli. Interestingly, CD8+ T-cells specific for CNS autoantigens, but not those specific for control foreign antigens, exhibit immune regulatory ability, suppressing proliferation of CD4+CD25- T-cells when stimulated by their cognate antigen. While CD8+ T-cell-mediated immune suppression is similar between healthy subjects and clinically quiescent treatment-naïve MS patients, it is significantly deficient during acute exacerbation of MS. Of note, the recovery of neuroantigen-specific CD8+ T-cell suppression correlates with disease recovery post-relapse. In healthy adult subjects, we observed that the CD62L- subset of CD8+ T cells harbored increased CNS- and Copaxone-specific suppressive ability, when compared to the CD62L+ subset and bulk CD8+ T cells, and that the CD28+ subset of CD8+ T cells harbored increased global suppressive ability, when compared to the CD28- subset. In contrast, we observed CD8+ T cells from neonates harbored increased global suppressive ability in the CD28- subset. The mechanism of neuroantigen-specific suppression by CD8+ T cells was dependent upon HLA class I, IFN gamma, with possible partial involvement by NKG2D, PD-1, and IL-10. These studies reveal a novel immune suppressor function for neuroantigen-specific CD8+ T-cells that is clinically relevant in the maintenance of peripheral tolerance and the intrinsic regulation of MS immune pathology.Item Reciprocal Regulation of CD4+ and CD8+ T Lymphocyte Effector and Memory Fates by Interleukin 12 and Type 1 Interferon(2009-06-18) Ramos, Hilario Jose; Farrar, J. DavidCytokine signaling networks play an important role in bridging the innate and adaptive immune responses. For example, the innate cytokines Interleukin-12 (IL-12) and type I interferon (IFN-a/b) are induced to high levels by intracellular bacterial and viral infections and have been shown to promote adaptive T lymphocyte responses to infection. While the role for IL-12 on the development of T lymphocyte effector responses has been well characterized, the exact role for IFN-a/b on these responses has been controversial. Therefore, the present study set forth to characterize the distinct roles for IL-12 and IFN-a/b on the development of effector and memory responses in human CD4+ and CD8+ T cells. My work has found that IL-12 drives the development of effector CD4+ and CD8+ T cells. In contrast, IFN-a/b was incapable of promoting these responses and this was due to a difference in the kinetics of activation of two downstream transcription factors STAT4 and T-bet. Further examination of CD8+ T cells revealed a distinct role for IFN-a/b in the development of a population of central memory T cells (TCM). Alternatively, IL-12 drove the development of effector memory cells (TEM). The variegated development of TCM and TEM was dictated by differential cytokine receptor expression and further, the strength of primary T cell receptor (TCR) activation determined the responsiveness to cytokine polarization. Finally, these studies uncovered a novel role for CD8+ T cell licensing of CTL activity through the costimulatory CD27/CD70 pathway. Therefore, taken together, these findings support a novel model in which TCR activation and costimulation act to shape the ability for IL-12 and IFN-a/b to differentially program the development of distinct classes of effector and memory CD8+ T lymphocytes. These studies have direct bearing on the design and development of effective therapeutics and vaccines and demonstrate a new understanding on the modulation of the adaptive immune response to intracellular infection.Item Regulation of Human Cytotoxic T Lymphocyte Functions by IL-12(2013-07-30) Chowdhury, Fatema Zahra; Pasare, Chandrashekhar; Farrar, J. David; Forman, James; Cobb, Melanie H.CD8+ T cells or cytotoxic T lymphocytes (CTLs) play a major role in our defense against intracellular pathogens by secreting effector cytokines and directly killing infected cells. Upon sensing of pathogen, antigen presenting cells secrete innate cytokines such as IL-12 and IFN-α. CTL functions are specified by both antigen recognition and innate cytokines yielding a diverse population comprised of short-term effectors and long-lived memory cells. We have demonstrated that IL-12, in particular, programs effector CTL differentiation in human. Our objective was to elucidate the key pathways programmed by IL-12 that leads to effector function. Using whole transcriptome analyses, we have identified a serine/threonine protein kinase MAP3K8 or Tpl2 to be selectively induced by IL-12. Furthermore, the functionally identified effector memory CTL population expressed higher levels of MAP3K8 mRNA ex vivo compared to the naïve/central memory CTL population. MAP3K8 or Tpl2 has been shown to play an important role in activating the MAP kinase pathways in innate immune cells such as macrophages. MAP kinase pathways are three-tiered kinase cascades that coordinate many different cellular responses by relaying the instructions from extracellular signals. Using specific small molecule inhibitors, we have demonstrated that MAP3K8/Tpl2 in human CTLs functions upstream of MEK/ERK MAP kinase pathway, leading to effector functions. An inhibitor for MAP3K8/Tpl2 blocked IFN-γ and TNF-α secretion as well as cytolysis of target cells in a dose-dependent manner. However, MAP3K8/Tpl2 deficient murine CTLs did not exhibit any functional deficiency either in vitro or in vivo. Furthermore, we have found that the Tpl2 inhibitor did not block IFN-γ and TNF-α secretion from murine effector CTLs. In summary, we found that IL-12, not IFN-α, programs effector function in human CTLs at the genetic level. Additionally, MAP3K8/Tpl2, a member of the MAP kinase pathway, is regulated by IL-12 during CTL priming in human and is a critical regulator of antigen receptor-mediated effector functions. Taken together, we have discovered a species-specific role for IL-12 regulated MAP3K8/Tpl2 in effector function of human CTLs, which plays a major role in adaptive immune responses to intracellular pathogens and tumors.Item The Role of Therapy-Induced CD8+ T Cells in a Mouse Model of Multiple Sclerosis(2009-06-18) Mendoza, Jason Paul; Karandikar, Nitin J.Murine experimental autoimmune encephalomyelitis (EAE) is an induced, autoimmune, demyelinating disease model for human multiple sclerosis (MS). Glatiramer acetate (GA) is an approved immunomodulatory therapy for MS that was discovered through studies in EAE. It is thought that the main mode of action of GA is the induction of a Th1 to Th2 shift in CD4+ T cells. We have shown that, in human MS, GA therapy induces an upregulation of cytotoxic, suppressor CD8+ T cell responses, suggesting that these cells are integrally involved in mediating the immune effects of this drug. In this study, we show that GA induces robust CD8+ T cell responses even in mice. Whereas GA immunization ameliorates the clinical and histologic severity of EAE in wild type C57BL/6 mice, such protection is not observed in CD8(-/-) mice, indicating that CD8+ T cells are required in mediating the effects of GA. Moreover, adoptive transfer of CD8+ T cells from GA-immunized (but not OVA-immunized) mice results in amelioration of EAE in CD8(-/-) and wildtype mice, suggesting therapeutic potential for GA-specific CD8+ T cells. These cells appear to mediate their effects through a cytotoxic/suppressor mechanism, similar to our findings in human MS. In addition, GA treatment results in decreased myelin oligodendrocyte glycoprotein (MOG)-specific CD4+ T cell responses. In conclusion, our studies provide strong evidence that the in vivo induction of immune modulatory CD8+ T cells is an essential step in mediating therapeutic protection during autoimmune demyelination.Item Sympathetic Neural Control of Inflammation by ADRB2-Mediated IL-10 Secretion(2018-04-16) Agac, Didem; Hooper, Lora V.; Farrar, J. David; Gill, Michelle A.; Shiloh, MichaelThe nervous and immune systems reciprocally regulate their functions through the release of chemical messengers. Norepinephrine (NE), a neurotransmitter released by catecholaminergic nerve endings, allows the sympathetic nervous system to communicate with immune cells through adrenergic receptors (ADR). Although, the effects of adrenergic signaling has been studied in multiple cell types, its role in modulation of innate immune cells is relatively unknown. Here, I demonstrate a novel role for the beta2-ADR (ADRB2) in controlling inflammation. NE suppresses pro-inflammatory cytokine secretion from primary macrophages in response to multiple TLR agonists, and ADRB2 signaling enhances early induction of IL-10. In addition to its in vitro affects, I have shown that ADRB2 signaling controls inflammation in vivo. The in vivo role of this pathway was assessed by using an infection model, experimental colitis and LPS endotoxemia model. ADRB2-/- animals presented with splenomegaly and greater weight loss in infection and colitis, compared to ADRB2 sufficient animals, respectively. ADRB2-/- animals rapidly succumbed to a sub-lethal LPS challenge, which correlated with elevated serum levels of TNFα and reduced IL-10. Administration of exogenous IL-10 increased the survival of the ADRB2-/-. Additionally, the ADRB2-specific agonist salmeterol rescued wild-type animals from a lethal LPS challenge, which was reversed by neutralizing anti-IL-10 antibody. These observations suggest that ADRB2 signaling is critical for controlling inflammation through the rapid induction of IL-10. Transcriptome analysis revealed that the NR4A nuclear orphan family members were induced by NE. The presence of several putative NR4A binding sites within the IL-10 promoter suggests that these factors may directly regulate IL-10 expression in response to ADRB2 signaling. Additionally, mice that deficient in NR4A1 are susceptible in LPS endotoxemia model. These results suggest a novel pathway for control of inflammation via neuroendocrine cues. Understanding this pathway will provide new insights into how the nervous and immune systems communicate through ADRB2 signaling.