Browsing by Department "Immunology"

Bacterial bladder infection, also known as cystitis, is one type of urinary tract infections (UTIs). These infections typically initiate when uropathogens, especially uropathogenic E. coli (UPEC), invade into the bladder through the urethra. Those bacteria could attach to the uroepithelium of the bladder and invade into bladder epithelial cells (BECs). The bladder is critical for the control of these bacteria by activating BECs and recruiting immune cells. If bacteria are not promptly cleared in the bladder, they will further invade ureters and kidneys causing pyelonephritis, which is another common form of UTIs. Nowadays, bacterial bladder infection is one of the most common bacterial infections and a big clinical burden. Its annual incidence rate among human population was estimated to be around 3%-12.6% in female and 0.5%-3% in male. Besides the high incidence rate, the recurrence rate is also very high, up to 44%. As a comparison, the recurrence rate of bacterial infections in the respiratory tract is only around 10% and that rate in the gastrointestinal tract is about 1.5% to 12%. The high incidence rate and high recurrence rate pinpoint the inefficiency of immunity in bladder. However, it is unclear which component of the bladder immunity is inefficient in clearing bacteria and preventing recurrence. By using cutting edge models and techniques, such as genetic knock-out mice, newly developed cytokine reporter mice, optimized flow cytometry and microscopy, I identified that the bladder immunity, especially CD4 T cell mediated adaptive immunity, is focusing on repairing damaged uroepithelium rather than clearing UPEC. This response is modulated by antigen presenting cells (APCs) in bladder. This abnormal bladder immunity also leads to bladder dysfunction featured by urinary frequency. By applying a vaccination strategy, I successfully improved the anti-bacteria ability of CD4 mediated bladder adaptive immunity in mouse model. In summary, this study identified that an abnormal adaptive immunity induced by the uroepithelium-APC-T cell signaling axis is responsible for the suboptimal clearance of bacteria and infection recurrence in bladder. With proper vaccination, the adaptive immunity in bladder can be tuned to be protective against UPEC infections.

LAT is a transmembrane adaptor protein that is critical for the emanation of signals downstream of the TCR. Following TCR engagement, LAT is phosphorylated on multiple tyrosine residues, allowing it to serve as a scaffold for a multi-protein signaling complex. Mutation of tyrosine 136 on LAT abrogates binding of PLC-γ1. The disruption of this interaction has severe consequences on TCR-mediated calcium signaling and MAPK activation. Mice harboring a mutation at this tyrosine, LATY136F (LATm/m) mice, have drastically impaired thymocyte development; however, CD4+ T cells in the periphery rapidly expand and instigate a fatal lymphoproliferative syndrome. In order to bypass the severe developmental defects exhibited in LATm/m mice, our laboratory previously developed a conditional knock-in mouse line in which the mutated LAT allele is expressed in mature T cells following deletion of a floxed wildtype LAT allele (ERCre+LATf/m mice). LATf/m mice develop a similar lymphoproliferative syndrome as LATm/m mice. We used both of these mouse models to analyze the contribution of two other proteins that are essential for TCR-mediated signaling, RasGRP1 and Gads, in LAT-mediated autoimmunity.

Analysis of LATm/mRasGRP1-/- mice demonstrated that the additional deletion of RasGRP1 increased the thymocyte development block and, as a result, young mice contained markedly reduced T cell populations. However, by four months of age, a lymphoproliferative disease had developed in these mice. To bypass the severe developmental block, we analyzed LATf/mRasGRP1-/- mice and observed that they developed disease similarly to LATf/m mice. We also assessed the effect of Gads deletion in both mouse models of LAT disease. LATm/mGads-/- mice had an even more dramatic block in the DN stage of thymocyte development compared to LATm/m controls, although by four months of age CD4+ T cells had expanded. Following deletion of the wildtype LAT allele, LATf/mGads-/- mice also developed disease. Our results indicated that LAT-mediated autoimmunity can occur independently of the critical T cell signaling components RasGRP1 and Gads.

In addition, we more closely examined RasGRP1-mediated Erk activation in T cells. RasGRP1 is a Ras-guanyl nucleotide exchange factor that is required for positive selection of thymocytes, activation of T cells, and control of T cell mediated-autoimmunity. While the importance of various RasGRP1 structural domains has previously been explored, RasGRP1 also contains a tail domain of unknown function. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain, RasGRP1d/d mice. Analysis of these mice demonstrated that deletion of the tail domain led to impaired T cell development but, with age, CD4+ T cells expanded and auto-antibodies were produced. RasGRP1d/d thymocytes were unable to activate Erk and underwent aberrant thymic selection processes. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, our data revealed the physiological relevance of the tail domain of RasGRP1 in the control of Erk signaling.

B cells mediate immune responses via the secretion of antibody and interactions with other immune cell populations through antigen presentation, costimulation, and cytokine secretion. Although B cells are primarily believed to promote immune responses using the mechanisms described above, some unique regulatory B cell populations that negatively influence inflammation have also been described. Among these is a rare interleukin (IL)-10-producing B lymphocyte subset termed “B10 cells.” B cell-derived IL-10 can inhibit various arms of the immune system, including polarization of Th1/Th2 cell subsets, antigen presentation and cytokine production by monocytes and macrophages, and activation of regulatory T cells. Further studies in numerous autoimmune and inflammatory models of disease have confirmed the ability of B10 cells to negatively regulate inflammation in an IL-10-dependent manner. Although IL-10 is indispensable to the effector functions of B10 cells, how this specialized B cell population is selected in vivo to produce IL-10 is unknown. Some studies have demonstrated a link between B cell receptor (BCR)-derived signals and the acquisition of IL-10 competence. Additionally, whether antigen-BCR interactions are required for B cell IL-10 production during homeostasis as well as active immune responses is a matter of debate. Therefore, the goal of this thesis is to determine the importance of antigen-driven signals during B10 cell development in vivo and during B10 cell-mediated immunosuppression.

Chapter 3 of the dissertation explored the BCR repertoire of spleen and peritoneal cavity B10 cells using single-cell sequencing to lay the foundation for studies to understand the full range of antigens that may be involved in B10 cell selection. In both the spleen and peritoneal cavity B10 cells studied, BCR gene utilization was diverse, and the expressed BCR transcripts were largely unmutated. Thus, B10 cells are likely capable of responding to a wide range of foreign and self-antigens in vivo.

Studies in Chapter 4 determined the predominant antigens that drive B cell IL-10 secretion during homeostasis. A novel in vitro B cell expansion system was used to isolate B cells actively expressing IL-10 in vivo and probe the reactivities of their secreted monoclonal antibodies. B10 cells were found to produce polyreactive antibodies that bound multiple self-antigens. Therefore, in the absence of overarching active immune responses, B cell IL-10 is secreted following interactions with self-antigens.

Chapter 5 of this dissertation investigated whether foreign antigens are capable of driving B10 cell expansion and effector activity during an active immune response. In a model of contact-induced hypersensitivity, in vitro B cell expansion was again used to isolate antigen-specific B10 clones, which were required for optimal immunosuppression.

The studies described in this dissertation shed light on the relative contributions of BCR-derived signals during B10 cell development and effector function. Furthermore, these investigations demonstrate that B10 cells respond to both foreign and self-antigens, which has important implications for the potential manipulation of B10 cells for human therapy. Therefore, B10 cells represent a polyreactive B cell population that provides antigen-specific regulation of immune responses via the production of IL-10.

Dendritic cells (DC) have been the key elements in developing cancer vaccines to induce potent T cell responses to eradicate tumors. However, the common approach adopted in clinical trials using ex vivo generated DC loaded with tumor antigens (Ag) has been challenged by its limited clinical response, complexity, and quality of the manufacturing process. Alternative efforts focused on in vivo Ag loading on endogenous primary DC have not yet been well validated in their efficacy for cancer treatment, suggesting the efficiency of in vivo Ag transfer to endogenous DC from currently available Ag-delivering vehicles needs to be further improved. Here, I aim to develop an alternative cellular vaccine platform that can circumvent the aforementioned problems. I reason that classical Ly-6Chi monocytes (i.e. monocytes hereafter) can be a promising candidate to be loaded with tumor Ag and induce effective T cell responses. With advantages including easy-purification from human peripheral blood, monocytes evidently can present antigens directly via in vivo differentiation into bona fide DC or indirectly via antigen transfer to lymphoid resident DC to induce strong Th1 or cytotoxic T lymphocyte (CTL) responses. However, whether monocytes exploit favorably direct or indirect pathway to present the same Ag they are carrying to trigger effective immune responses remains unclear. Furthermore, how exactly monocytes or monocyte-derived cells transfer antigens to lymphoid resident DC has yet to be elucidated. I hypothesized that Ag-loaded monocytes can induce strong anti-tumor immunity and began the research by investigating the immune responses that can be induced by Ag-loaded monocytes. I then went on to determine the mechanisms that mediate monocyte-induced immune responses and evaluate anti-tumor efficacy of this monocyte vaccine.

In the first part of this study, I characterized the immune responses induced by Ag-loaded monocytes. By using negative selection via magnetic-activated cell sorting (MACS) columns, I was able to purify monocytes from bone marrow (BM) cells and determined that these monocytes could be successfully loaded with Ag in the forms of proteins, peptides and mRNA. I found that intravenously (IV) injected Ag-loaded monocytes induced robust Ag-specific CD4+ and CD8+ T cell responses in mice without triggering antibody responses. This vaccine activity of Ag-loaded monocytes appeared to be dose-dependent and required live monocytes with no need of ex vivo stimulation. I found that Ag-specific CD8+ T cells induced by Ag-loaded monocytes were functionally more robust than those induced by protein Ag emulsified in a traditional adjuvant CFA.

In the second part of this study, I investigated how IV injected Ag-loaded monocytes stimulate T cell responses. I identified that the spleen is the primary immune niche for Ag-loaded monocytes to induce T cell responses. I found that Ag-loaded monocytes mainly retain in the spleen where they begin to differentiate into phenotypic DC. Surprisingly, major histocompatibility complex (MHC)-deficient monocytes maintain full capacity to stimulate T cell responses, suggesting that Ag-loaded monocytes do not present Ag by themselves. I determined that endogenous splenic DC is absolutely required for monocyte-induced T cell responses. Therefore, Ag-loaded monocytes induce T cell responses indirectly via transferring Ag to splenic DC even they do differentiate into phenotypic DC in the spleen. I elucidated that this monocyte-to-DC Ag transfer occurs via gap junctions for CD8+ T cell responses and via macrophages for CD4+ T cell responses.

In the final part of this study, I demonstrated that IV injected Ag-loaded monocytes have robust anti-tumor efficacy targeting both model and validated tumor Ag in prophylactic, memory and therapeutic murine SQ melanoma models. The anti-tumor efficacy is superior to that seen with traditional adjuvants or RNA-pulsed DC vaccines, and can be combined with checkpoint blockade to increase their efficacy. Furthermore, I demonstrated that Ag-loaded monocytes have a clear anti-tumor efficacy in an intracranial glioblastoma (GBM) model targeting against mutant isocitrate dehydrogenase 1-R132H (mIDH1-R132H), a validated tumor Ag of GBM.

In conclusion, IV injection of unactivated Ag-loaded monocytes without adjuvants induces highly efficacious anti-tumor T cell responses via dual independent and efficient Ag transfer pathways to splenic DC. These findings revise the paradigm that monocytes have to be activated ex vivo to achieve optimal vaccine efficacy and reveal unappreciated cell-associated Ag acquiring pathways of splenic DCs that can be specifically manipulated for future vaccine design in the treatment of human cancers.

Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. Although pyroptosis and apoptosis have distinct signaling pathways, when a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we also now engineer S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. In both engineered strains, apoptosis occurred somewhat slower than pyroptosis. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche, but failed to clear the bacteria from the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. In order to clear an infection, distinct cell types may have specific tasks that they must complete before they die. In some cells, either apoptotic or pyroptotic signaling may initiate the same tasks, whereas in other cell types these modes of cell death may lead to different tasks that may not be identical in defense against infection. We recently suggested that such diverse tasks can be considered as different cellular “bucket lists” to be accomplished before a cell dies. As demonstrated here, engineering pathogens is a useful method for discovering new details of microbial pathogenesis and host defense. However, engineering can result in off-target effects. We engineer S. Typhimurium to overexpress the secretion signal of the type 3 secretion system effector SspH1 fused with domains of other proteins as cargo. Such engineering had no virulence cost to the bacteria for the first 48 hours post infection in mice. However, after 48 hours the engineered bacteria manifest an attenuation that correlates with the quantity of the SspH1 translocation signal expressed. In IFNg-deficient mice this attenuation was weakened. Conversely, the attenuation was accelerated in the context of a pre-existing infection. We speculate that inflammatory signals change aspects of the target cell’s physiology that make host cells less permissive to S. Typhimurium infection. This increased degree of difficulty requires the bacteria to utilize its T3SS at peak efficiency, which can be disrupted by engineered effectors.

B cells and humoral immunity are critical components of an effective immune response. However, B cells are also a significant driver of a variety of autoimmune diseases and can also become malignant. Antibody-mediated B cell depletion is now regularly used in the clinic to treat both B cell-derived cancers and B-cell driven autoimmunity, and while depletion itself is effective in some patients, removal of B cells is not often curative for patients and may present additional, unforeseen risks. The overall goal of this dissertation was therefore to determine the impact of B cell depletion on T cell homeostasis and function during infection and to elucidate the genetic factors that determine the effectiveness of antibody-mediated therapy.

In Chapter 3 of this dissertation, the role of B cells in promoting T cell homeostasis was investigated by depleting mature B cells using CD20 monoclonal antibody (mAb). Acute B cell depletion in adult mice significantly reduced spleen and lymph node T cell numbers, including naïve, activated, and cytokine-producing cells, as well as Foxp3+ regulatory T cells, whereas chronic B cell depletion in aged mice resulted in a profound decrease in activated and cytokineproducing T cell numbers. To determine the significance of this finding, B cell-depleted adult mice were infected with acute lymphocytic choriomeningitis virus (LCMV). Despite their expansion, activated and cytokine-producing T cell numbers were still significantly reduced one week later. Moreover, viral peptide-specific T cell numbers and effector cell development were significantly reduced in mice lacking B cells, while LCMV titers were dramatically increased. Thus, B cells are required for optimal T cell homeostasis, activation, and effector development in vivo, particularly during acute viral infection.

In Chapter 4 of this dissertation, lymphoma genetic changes that conferred either sensitivity or resistance to CD20 mAb therapy were examined in a preclinical mouse lymphoma model. An examination of primary lymphomas and extensive lymphoma families demonstrated that sensitivity to CD20 mAb was not regulated by differences in CD20 expression, prior exposure to CD20 mAb, nor serial in vivo passage. An unbiased forward genetic screen of CD20 mAb-resistant and -sensitive lymphomas identified galectin-1 as a significant factor driving CD20 mAb therapy resistance. As lymphomas acquired therapy resistance following serial in vivo passage, galectin-1 expression also increased. Furthermore, inducing lymphoma galectin-1 expression within the tumor microenvironment ablated lymphoma sensitivity to CD20 mAb. Therefore, lymphoma acquisition of galectin-1 expression confers CD20 mAb therapy resistance.

In Chapter 5 of this dissertation, the distinct germline components that control the efficacy of host CD20 mAb-dependent B cell and lymphoma depletion were evaluated using genetically distinct lab mouse strains. Variations in B cell depletion by CD20 mAb among several lab mouse strains were observed, where 129 mice had significantly impaired mAb-dependent depletion of endogenous B cells and primary lymphomas relative to B6 mice. An unbiased forward genetic screen of mice revealed that a 1.5 Mbp region of Chromosome 12 that contains mycn significantly altered CD20 mAb-dependent lymphoma depletion. Elevated mycn expression enhanced mAb-dependent B cell depletion and lymphoma phagocytosis and correlated with higher macrophage numbers. Thus, host genetic variations in mycn expression in macrophages alter the outcome of Ab-dependent depletion of endogenous and malignant cells.

These studies collectively demonstrate that B cells are required for effective cellular immune responses during infection and identified factors that alter the effectiveness of mAb-dependent B cell depletion. This research also established and validated an unbiased forward genetics approach to identify the totality of host and tumor-intrinsic factors that influence mAb therapy in vivo. The findings of these studies ultimately urge careful consideration in the clinical application of B cell depletion therapies.

Helminth exposure, allergy and asthma each induce cellular responses in lymphoid and peripheral tissues that give rise to type-2 inflammation. Essential molecular mediators of this response are type-2 cytokines interleukin(IL)-4 and IL-13 derived from various subsets of immune cells. In lymphoid tissues, CD4+ Tfh cells make IL-4 to elicit IgE and high-affinity IgG1 production. In peripheral sites of infection, group 2 innate lymphoid (ILC2) cells make IL-13 and Th2 cells make both IL-13 and IL-4. Together, these cells mediate smooth muscle contraction, mucus production and recruitment of other innate effector cells, all of which are hallmarks of type-2 inflammation. As central mediators of type-2 inflammation, understanding the cell-specific expression and molecular regulation of type-2 cytokines in CD4+ T cells and ILC2 cells may lead to new therapies that ameliorate allergic disease and helminth infections.

The AP-1 factor basic leucine zipper transcription factor ATF-like (BATF) has been identified as a pioneer factor in in vitro-generated Th17 cells. BATF facilitates chromatin remodeling at the IL-17 locus as well as loci of key Th17-associated lineage specifying factors. It has also been deemed essential to the generation of functional humoral immunity through the development of follicular helper T (Tfh) cells and germinal center B cells. However, the role of BATF in the development and function of other CD4+ T helper subsets and innate immune cells in vivo has remained unclear. I show here that mice deficient in BATF do not develop type-2 inflammation after exposure to the parasitic helminth Nippostongylus brasiliensis. Since type-2 cytokine expression by Th2 and ILC2 cells is essential for expedient helminth expulsion, I hypothesized that BATF likely has a role in the development and/or induction of cytokine expression in CD4+ Th2 cells and ILC2 cells. Consistent with this hypothesis, I found that BATF utilizes a novel mechanism to control Th2 cytokine expression in Th2 cells. Specifically, BATF promotes permissive epigenetic modifications to alter the chromatin landscape early during Th2 cell differentiation. In addition, my data show that BATF deficiency inhibits the activation of ILC2 cells, preventing ILC2-mediated helminth clearance.

In addition to uncovering BATF-mediated regulations of type-2 inflammation, my work has revealed new insight into the role of a second bZIP transcription factor, cMaf, during type-2 immunity. As mentioned above, helminth exposure elicits IL-4 production by both CD4+ Tfh and Th2 cells. Although type-2 cytokine transcription has been well characterized in Th2 cells, Tfh cell-mediated IL-4 production has yet to be fully defined. Importantly, I show that IL-4 production by Tfh cells is sustained upon deletion of classical IL-4 regulatory factors signal transducer and activator of transcription 6 (STAT6) and STAT5 and is not dependent on high GATA-3 expression. In sum, Tfh-driven IL-4 production is induced independent of classical pathways in Th2 cells.

Presently, the non-canonical transcription factors involved in IL-4 production by Tfh cells remain unclear. C-Maf works with BCL6, the master regulator of Tfh cells, to elicit Tfh formation. However, the precise role of c-Maf in Tfh cell fate and function remains unclear. So far, it has been shown that in Th2 cells, c-Maf binds to the IL-4 promoter and in Tfh cells, c-Maf binds to the CNS2 enhancer of the IL-4 locus to regulate IL-4 expression. Therefore, I hypothesized that c-Maf is important in non-canonical, GATA-3-independent IL-4 production by Tfh cells.

Here, I show that Tfh cells lacking canonical Th2 pathways for IL-4 expression express high levels of c-Maf and IL-4 transcript. Deletion of c-Maf in CD4+ T cells resulted in normal induction of BCL6 expression. Thus the initial stages of Tfh cell generation were induced. However, cMaf-deficient CD4+ T cells did not express important molecules associated with Tfh cell migration. Immunohistochemistry also confirmed that c-Maf deficiency inhibited CD4+ T cell migration from the paracortex into the B cell follicle.

These defects did not inhibit cMaf-deficient CD4+ T cells from making IL-4 transcript; however, IL-4 protein production was significantly impaired. Together, these results demonstrate that c-Maf is essential for Tfh cell-mediated immunity by promoting CD4+ T cell migration to the B cell follicles and the production of IL-4 protein in the germinal centers.

Collectively, the objective of my thesis research is to define the roles of the bZIP transcription factors BATF and c-Maf in type-2 inflammation. My data demonstrate that BATF is essential for the differentiation and function of Tfh, Th2, and ILC2 cells during helminth infection. Additionally, I have shown that c-Maf is required for Tfh function and CD4+ T cell migration to the B cell follicle. Thus, BATF and c-Maf are central to the development of humoral and peripheral type-2 inflammatory responses against helminth infection. Given the wide spectrum of disorders associated with type-2 inflammation, the identification of factors relevant to the development and function of Th2-, ILC2- and Tfh-driven allergic pathologies is broadly relevant. A comprehensive characterization of core factors like BATF and c-Maf provide new avenues in which to explore novel therapies to modulate type-2 inflammatory responses.

Bronchiolitis Obliterans (BO) is a major cause of chronic airway dysfunction after toxic chemical inhalation. The pathophysiology of BO is not well understood, but epithelial cell injury has been closely associated with the development of fibrotic lesions in human studies and in animal models of both toxin- and transplant-induced BO. However, while almost all cases and models of BO include epithelial injury, not all instances of epithelial injury result in BO, suggesting that epithelial damage per se is not the critical event leading to the development of BO. In this dissertation, we describe a model of chlorine (Cl2)-induced BO in which mice develop tracheal and large airway obliterative lesions within 10 days of exposure to high (350 ppm), but not low (200 ppm), concentrations of Cl2 gas. Lesions develop in a series of well-demarcated pathological changes that include epithelial denudation, inflammatory cell infiltration by day 2 after exposure, fibroblast infiltration and collagen deposition by day 5, and in-growth of blood vessels by day 7, ultimately leading to lethal airway obstruction by days 9-12. Using this model, we were able to test our hypothesis that loss of epithelial progenitor cells is a critical factor leading to the development of obliterative airway lesions after chemical inhalation. Indeed, these lesions arise only under conditions and in areas in which basal cells, the resident progenitor cells for large airway epithelium, are eliminated by Cl2 exposure.

The molecular pathways contributing to BO development are not well understood. Mechanisms of epithelial injury differ across BO models, but we hypothesized that after the inciting injury, BO models share common pathways. We compared microarray analysis from day 5 non-BO- and BO-inducing chemical injuries and subsequently identified biological pathways that may contribute to BO pathogenesis. Our findings add support to pathways previously implicated in BO development and more importantly, suggest potential new pathways and molecular mediators of BO. Furthermore, we evaluated the efficacy of therapeutic inhibition of neovascularization or inflammation to prevent Cl2-induced BO. To date, our therapeutic interventions were ineffective. Nonetheless, our findings suggest that in the context of Cl2-induced BO, vascular endothelial growth factor receptor 2 (a mediator of neovascularization) and inducible nitric oxide synthase (a mediator of inflammation) are not critical in BO pathogenesis.

In sum, our work introduces and characterizes a novel Cl2-induced murine model of BO. Using this model we demonstrated that in the absence of basal cells, epithelial regeneration does not occur and regions of epithelial denudation persist from which an aberrant repair process is initiated, leading to obliterative airway lesions. Our findings suggest that, irrespective of the cause, loss of epithelial progenitor cells may be a critical factor leading to the development of BO. Furthermore, our gene expression analysis implicates novel mediators and signaling pathways in the development of BO. Our analysis lays the foundation for more rigorous exploration of these targets in the pathogenesis of BO.

Lymph nodes are organs of efficiency. Once activated, they essentially function to optimize and accelerate the production of the adaptive immune response, which has the potential to determine survival of the host during an initial infection and protect against repeated infections, should specific and appropriate immunological memory be sufficiently induced. We now have an understanding of the fundamental structure of lymph nodes and many of the interactions that occur within them throughout this process. Yet, lymph nodes are dynamic and malleable organs and much remains to be investigated with regards to their responses to various types of challenges. In this work, we examined multiple inflammatory scenarios and sought to understand the complex ways that lymph nodes can be externally targeted to impact immunity. First, we outline a novel mechanism of cellular communication, where cytokine messages from the periphery are delivered to draining lymph nodes during inflammation. These signals are sent as particles, released by mast cells, and demonstrate the ability of the infected tissue to communicate to lymph nodes and shape their responses. Based on these interactions, we also explored the ability to therapeutically or prophylactically modulate lymph node function, using bioengineered particles based on mast cell granules, containing encapsulated cytokines. When we used these particles as a vaccine adjuvant, we were able to polarize adaptive immune responses, such as to promote a Th1 phenotype, or enhance a specific attribute of the immune response, such as the production of high avidity antibodies. We then explore three examples of lymph node-targeting pathogens: Salmonella typhimurium, Yersinia pestis and Dengue virus. Each of these pathogens has a well-characterized lifecycle including colonization of draining lymph node tissue. In the case of S. typhimurim, we report that the virulence this pathogen depends on a specific shut down of the chemotactic signals in the lymph node that are required to maintain appropriate cellular localization within it. Our results demonstrate that these architecture changes allow S. typhimurim to target the adaptive immune process in lymph nodes and contribute to its spread in vivo and lethality to the host. With Y. pestis, similar targeting of cellular trafficking pathways occurs through the modulation of chemokine expression. Y. pestis appears to use the host's cellular trafficking pathways to spread to lymph nodes in two distinct waves, first exploiting dendritic cell movement to lymph nodes and then enhancing monocyte chemoattractants to replicate within monocytes in draining lymph nodes. These processes also promote bacterial spread in vivo and we further demonstrate that blocking monocyte chemotaxis can prolong the host's survival. In the third example of pathogen challenge, we report for the first time that mast cells can contribute functionally to immunosurveillance for viral pathogen, here, promoting cellular trafficking of innate immune cells, including NK cells, and limiting the spread of virus to draining lymph nodes. For each of these three examples of lymph node targeting by microbial pathogens, we provide data that modulation of cellular trafficking to and within lymph nodes can drastically influence the nature of the adaptive immune response and, therefore, the appropriateness of that response for meeting a unique infectious challenge. Cumulatively this work highlights that a balance exists between host and pathogen-driven modulation of lymph nodes, a key aspect of which is movement of cells within and into this organ. Cytokine and chemokine pathways are an area of vulnerability for the host when faced with host-adapted pathogens, yet the lymph node's underlying plasticity and the observation that slight modulations can be beneficial or detrimental to immunity also suggests the targeting of these pathways with therapeutic intentions and during vaccine design.

B lymphocytes are multifunctional and play important roles in both innate and adaptive immunity. The diverse roles of B cells can be attributed to the various and distinct types of B cells as determined by their origin, developmental stage, antigen specificity, and function.

Evidence suggests that human innate-like B cells (i.e., marginal zone and/or B1-like B cells) develop during fetal life. However, the characteristics of human fetal B-lineage cells are less understood. Recent studies of fetal and human umbilical cord B cells indicated that CD27, a well-established marker of human memory B cells, may also be expressed on human B1-like B cells. Indeed, CD27+ B cells are present in patients with hyper-IgM 1 (HIGM1) syndrome who are unable to generate GCs or memory B cells. In order to define the origin of naïve CD27+IgD+ human B cells, I studied B-cell development in both fetal and adult tissues.

In human fetal liver, most CD19+ cells co-express CD10, a marker of human developing B cells. Some CD19+CD10+ B cells express CD27, and these fetal CD27+ cells are present in the pro-B, pre-B, and immature/transitional B-cell compartments. Lower frequencies of phenotypically identical cells are also identified in adult bone marrow. CD27+ pro-B, pre-B, and immature/transitional B cells express recombination activating gene-1, terminal deoxynucleotidyl transferase, and Vpre-B mRNA comparable to their CD27− counterparts. CD27+ and CD27− developing B cells show similar immunoglobulin heavy chain gene usage with low levels of mutations, suggesting that CD27+ developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27+ developing B cells differ from CD27− developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27+ pro-B cells efficiently generate IgM+IgD+ immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B-cell development identifies a physiologic state or lineage for human B-cell development distinct from the memory B-cell compartment.

Regarding B-cell repertoire, due to the random recombination of immunoglobulin V, D, and J gene segments during B-cell development, B cells are highly diversified in their antigen specificity. Through their specific B-cell antigen receptors (BCRs), B cells recognize foreign (and self-) antigens, and present these antigens to cognate T cells to elicit/establish humoral responses, such as germinal centers, immunological memory, and long-lasting circulating antibodies. Some bacteria and viruses escape the host’s immune system by mimicking host antigens, as B cells that recognize shared epitopes on self- and foreign antigens may provide protection against such pathogens; however, these B cells are normally eliminated by tolerance mechanisms during development. The extent of tolerization manifest among human B cells that recognize both self- and foreign antigens is unknown. Here, I and my colleagues use an efficient single B-cell culture method and multiplexed antigen-binding assays to determine the specificity of about 2,300 clonal IgG antibodies produced by the progeny of single transitional and mature B cells. We show that in healthy individuals, half of the self-reactive B cells crossreact with foreign antigen, and that the frequencies of crossreactive B cells decrease by half between the transitional and mature B-cell stages, indicating that a substantial fraction of foreign specificities is lost by the second tolerance mechanisms. In SLE patients, who show defective peripheral tolerance, frequencies of crossreactive B cells are unchanged between the B-cell stages. The crossreactive, mature B cells in SLE patients show distinct reactivity to foreign antigens. We propose that activating forbidden B cells may be a good strategy for protection against host-mimicking pathogens if we can control tolerance.

Activated B cells can present antigen to T cells, as well as differentiate into memory B cells and plasma cells. Indeed, activated B cells express high levels of MHCII and are considered to be professional antigen presenting cells (APC), along with dendritic cells and macrophages. APC can be used to discover the epitopes targeted in T-cell responses; T cells are co-cultured with autologous APC in the presence of antigens and T-cell responses are evaluated. With numerous epitope candidates, mapping T-cell epitopes requires large numbers of APC; the availability of APC in blood is a limiting component and leukapheresis is often required. Since B cells can be expanded in vitro more easily than other APC, they represent a solution for the challenge of isolating adequate numbers of APC from blood in order to determine T-cell antigen specificity. I modified our single B-cell culture to support efficient activation and proliferation of both naïve and memory human B cells for the purpose of generating large numbers of autologous APC. Briefly, naïve or memory B cells recovered from blood are cultured with recombinant human IL-2, IL-4, IL-21, and BAFF on CD154+ feeder cells; this culture supports extensive B-cell proliferation, with approximately 103 fold increases following 8 days in culture, and 106 fold increases when cultures are split and cultured for 8 more days. The capacity for continued proliferation is stable for at least another week. In culture, a significant fraction of naïve B cells undergo isotype switching and terminally differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved, and when recovered, retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHCII, CD80, and CD86. I have examined the APC function of CD B cells and found that they present both allo- and microbial antigens to autologous T cells with comparable efficiency to PBMC. Moreover, I am able to activate and expand antigen-specific memory B cells; these cultured cells are highly effective in presenting antigen to T cells. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual.

Foxp3-expressing regulatory T cells (Tregs) are essential mediators of immunological tolerance in both mice and humans. Although Tregs have traditionally been considered a relatively homogeneous population, they are now thought to comprise numerous cellular subtypes each with distinct roles in maintaining organismal homeostasis. In this regard, Tregs undergo functional specialization leading to diverse phenotypes and effector functions depending on (1) the inflammatory milieu encountered during an immune response, and (2) the specific anatomical location in which they reside. This process of context-dependent adaptation of effector function is facilitated by changes in transcriptional programming resulting from the coordinated action of numerous transcription factors (TFs). However, aside from the demonstrated roles for classical lineage-defining transcription factors – such as RORt, GATA-3, T-bet and Bcl-6 – the transcriptional regulatory circuity underlying Treg specialization has remained largely unexplored.

The activator protein-1 (AP-1) superfamily consists of a large number of TFs which bind similar DNA sequences as either homo- or hetero-dimers to influence transcription. Transcriptomic profiling studies have demonstrated that numerous AP-1 TFs exhibit subset- or tissue-specific expression patterns in Tregs, suggesting that these factors might regulate Treg specialization. Specifically, AP-1 TFs c-Maf and JunB were shown to exhibit preferential expression in specialized Tregs and have established regulatory roles in the differentiation of helper T cells. Therefore, I hypothesized that c-Maf and JunB might play undiscovered roles controlling the process of Treg specialization.

Using mice with Treg-specific ablation of Maf (encoding c-Maf), I evaluated the role of c-Maf in Treg specialization within the intestinal immune compartment that hosts a variety of specialized Treg subsets. Mice with Tregs lacking c-Maf appeared largely normal, with no apparent health problems, indicating that c-Maf was dispensable for general Treg function. However, using multicolor flow cytometry to perform comprehensive immunophenotyping of these mice, I found that c-Maf played a critical role in the generation of RORt+ Tregs, which constitute a major subset of specialized Tregs in the intestine but are rarely found in other organs. Additionally, these analyses showed that c-Maf was important for the generation of a second specialized Treg subset, T follicular regulatory (Tfr) cells, which are found in secondary lymphoid organs and are important for controlling antibody responses. However, in contrast to RORt+ Tregs and Tfr cells, c-Maf was not required for differentiation of other types of specialized Tregs, indicating a novel and specific role for c-Maf in Treg specialization. Mechanistically, in vitro culture experiments showed that upregulation of c-Maf and RORt in Tregs could be driven by exposure to IL-6 signaling in the presence of TGF, suggesting that c-Maf functions to link environmental signals to Treg specialization in the intestine. This work demonstrated that c-Maf is a novel and critical regulator of subset-specific Treg specialization for RORt+ Tregs and Tfr cells.

Separately, I investigated the role of JunB in Treg specialization by employing a similar approach as for c-Maf. Surprisingly, I found that mice with Treg-specific ablation of JunB developed a spontaneous phenotype of immune dysregulation which lead to weight loss and colonic inflammation, indicating that JunB was critical for normal Treg functioning. JunB expression was greatly elevated in intestinal Tregs relative to other populations and inflammation was most pronounced within the colons of animals with JunB-deficient Tregs, suggesting that JunB played a specific role in the function of colon-resident Tregs. Like c-Maf, JunB was absolutely required for the development of Tfr cells; however, JunB was otherwise dispensable for the development of all other specialized Tregs examined. Unexpectedly, I found that the loss of Tfr cells in mice with Tregs lacking JunB stemmed from impaired maintenance of the CD25- Treg population, which may be due to novel JunB-dependent metabolic requirements in these cells. Examining the colon-specific effects of JunB ablation using RNA-sequencing of intestinal Tregs, I found that JunB was required for expression of a tissue-specific set of Treg effector genes, such as granzyme B, which may represent a novel suppressive mechanism employed by multiple subtypes of colonic Tregs. Therefore, JunB plays a novel, organ-specific role in Treg specialization of both Tfr cells and all colonic Tregs.

Taken together, my work has revealed novel functions for two AP-1 family TFs – c-Maf and JunB – in the control of Treg specialization. Although c-Maf and JunB are both important for Treg specialization, their contributions to this process are distinct. c-Maf predominantly plays a subset-specific role in RORt+ Tregs and Tfr cells, whereas JunB plays both subset-specific and organ-specific roles depending on anatomical location. Of note, both c-Maf and JunB are essential for differentiation of Tfr cells, although the underlying molecular mechanisms appear to be different. These findings highlight the importance of AP-1 TFs in the control of Treg diversification and functional specialization and suggest that AP-1-mediated transcriptional regulatory circuits are an important mechanism controlling subset- and tissue-specific gene expression in Tregs.

Dectin-1 is a C-type lectin receptor with diverse functions in orchestrating the innate immune response. Previous studies have primarily focused on the function of Dectin-1 in the setting of fungal infection. However, emerging evidence supports an important role for Dectin-1 in the context of autoimmunity and sterile inflammation. In this thesis, I investigated the function of Dectin-1 in central nervous system (CNS) autoimmunity. Specifically, I studied the role of Dectin-1 signaling in experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis (MS). Genetically modified mouse lines and immunophenotyping approaches were used to study Dectin-1 function in EAE. In addition, ex vivo studies using small molecule inhibitors and next-generation sequencing were used to perform in-depth characterization of Dectin-1 signaling mechanisms. Here I report that Dectin-1 limited EAE, while its downstream signaling molecule, Card9, promoted the disease. Myeloid cells mediated the pro-resolution function of Dectin-1 in EAE with enhanced gene expression of the neuroprotective molecule, Oncostatin M (Osm), through a Card9-independent pathway, mediated by the transcription factor NFAT. Furthermore, I found that the Osm receptor (OsmR) functioned specifically in astrocytes to reduce EAE severity. Notably, Dectin-1 did not respond to heat-killed Mycobacteria, an adjuvant to induce EAE. Instead, endogenous Dectin-1 ligands, including galectin-9, in the central nervous system (CNS) were involved to limit EAE. This thesis research reveals a mechanism of beneficial myeloid cell-astrocyte crosstalk regulated by a Dectin-1 pathway and identifies potential therapeutic targets for autoimmune neuroinflammation.

CD4+ and CD8+ T cells are dichotomous lineages in adaptive immunity. While conventionally viewed as distinct fates that are fixed post-thymic development, accumulating evidence indicates that these two populations can exhibit significant lineage plasticity, particularly upon TCR-mediated activation. In this work, I define a novel murine CD4-CD8ab+ MHCII-recognizing population generated from effector CD4+ T cells. Effector CD4-CD8ab+ MHCII-recognizing T cells downregulate expression of T helper cell-associated costimulatory molecules and increase expression of cytotoxic T lymphocyte-associated cytotoxic molecules. TCRb repertoire sequencing and in vivo genetic lineage tracing in acutely-infected wild-type mice demonstrate fundamental lineage reprogramming resulting in an “ex-CD4” T cell phenotype, rather than expansion of an aberrantly-developed CD8+ MHCII-restricted population. Impairing autophagy by functional deletion of the initiating kinase Vps34 or a downstream enzyme Atg7 dramatically enhances the generation of effector CD4-CD8ab+ MHCII-recognizing T cells. These findings suggest that effector CD4+ T cells can exhibit a previously unreported degree of skewing towards the CD8+ T cell lineage, which may point towards a novel direction for vaccine design, particularly against HIV.

Dendritic cells (DCs) are potent antigen presenting cells (APC) that sense microbes and induce T cell activation and functional differentiation. The APC function of DCs is upregulated by the signaling pathway downstream of the microbial sensing receptor, a process well studied during pathogen infection and immunization. Multiple lines of evidence suggested that DCs in the intestine lamina propria (LP-DCs) frequently interact with the innocuous microbiota, and through these interactions LP-DCs support intestinal immune homeostasis. However, DC responses to microbiota, if not regulated, can give rise to inflammatory T cells and trigger inflammatory bowel disease (IBD). The DC subsets, DC functions and signaling pathways that induce inflammatory T cells remain incompletely characterized. Here, we demonstrated that mice lacking signaling attenuator A20 (A20cko mice) in DCs develop spontaneous small intestine inflammation that is dependent of microbiota, DCs and T cells. LP-DCs induce inflammatory T cells and that the signals perceived and APC functions are unique for three distinct LP-DC subsets. Thus, while CD103+CD11b- DCs exclusively upregulate their ability to instruct IFNγ+ T cells, CD103+CD11b+ DCs exclusively upregulate their ability to instruct IL-17+ T cells. Of note, APC functions of both DC subsets are upregulated in a MyD88-independent fashion. In contrast, CD103-CD11b+ DCs instruct both IFNγ+ and IL-17+ T cells, and only the IL-17-inducing APC functions require MyD88. In disease pathogenesis, both CD103-CD11b+ and CD103+CD11b+ DCs expand pathologic Th17 cells.

Although MyD88 pathways are potent inducer of intestinal inflammation in the colitis of IL-10 knockout mice and upon transferring of naïve T cells into Rag-deficient hosts, MyD88 pathways are not required for the inflammation of small intestine in A20cko mice. Among the MyD88-independent signaling pathways that could mediate host interaction with microbiota, Dectin-1 pathway is of particular interest because both the receptor Dectin-1 and the downstream signaling molecule CARD9 are IBD-associated genes. Additionally, the defect in either molecule influences the severity of the intestinal inflammation in mouse. We established that the production of inflammatory cytokines downstream of the Dectin-1 pathway is restricted by A20. Mechanistically, A20 inhibits TRAF6 ubiquitination downstream of the Dectin-1 pathway, thereby controlling NFκB and Jnk activation. Although we showed that CD103-CD11b+ and CD103+CD11b+ DCs express Dectin-1 and CARD9, the Dectin-1 pathway is not required for the upregulation of DC function and expansion of inflammatory T cells in the intestine of A20cko mice. Thus, our studies have unveiled a critical role of MyD88-independent pathways in mediating the interaction of the microbiota and LP-DCs. MyD88-independent pathway is capable of driving functional maturation of LP-DCs, pathological expansion of CD4 T cells, and the inflammatory disease in the small intestine.

Despite decades of research, a safe and effective vaccine for Human Immunodeficiency Virus-1 (HIV-1) remains elusive. The HIV-1 envelope (Env) glycoprotein expressed on virion surface is the sole target for neutralizing antibodies. HIV-1 sequences are diverse and most neutralizing antibodies are strain-specific. However, broadly neutralizing antibodies (bnAbs) have been isolated from people living with HIV (PLWH). These bnAbs target conserved regions on the Env protein and neutralize heterologous HIV-1 isolates. Thus, inducing bnAbs is a current major goal of HIV-1 vaccine development.

Nucleoside modified, purified mRNA in lipid nanoparticles (mRNA–LNP) has emerged as a novel vaccine platform. The success of using mRNA–LNP forvaccines was demonstrated first by an experimental Zika vaccine and then by two clinically approved COVID-19 vaccines. mRNA vaccine is safe and effective, and is easier to manufacture. The use of mRNA–LNP for HIV-1 vaccine has not been explored.

This thesis focuses on designs of HIV-1 vaccine immunogens encoded by modified mRNA and the investigation of their antigenicity and immunogenicityin animal models. In the first study (Chapter 3), I demonstrated that mRNA can encode immunogenic Env trimers in the forms of full-length membrane-bound gp160 (MBEnv) or Env–ferritin nanoparticles (Env–NP). I tested mRNAs encoding HIV-1 Env forms with various stabilizing mutations and demonstrated in vitro that mRNA-encoded Env forms were well-folded in native-like conformation when optimal stabilizing mutations were included. Following this, I studied the immunogenicity of optimally stablilized MBEnv and Env–NP encoded by mRNA in a bnAb precursor knock-in mouse model. I demonstrated that mRNA immunization initiated bnAb precursor B cell expansion and induced serum autologous tier-2 neutralizing activities. Next-generation sequencing demonstrated the acquisition of critical bnAb mutations and monoclonal antibodies that neutralized heterologous HIV-1 isolates were isolated. In summary, mRNA–LNP can encode complex HIV-1 immunogens and may be of use in design of HIV-1 vaccines.

In the second study (Chapter 4), I studied the top Env–NP mRNA candidate from the first study in rhesus macaques in comparison with the same immunogen in the form of purified recombinant protein. I demonstrated that purified protein Env–NP induced higher binding and neutralizing antibody responses than mRNA did in rhesus macaques. By immunizing bnAb precursor KI mice at a wide dose range, I demonstrated that this mRNA was immunogenic at 20 µg only and was minimally immunogenic at 1 µg dose. In an effort to improve this mRNA vaccine candidate, I demonstrated that mRNA encoding MBEnv was more immunogenic than mRNA encoding Env–NP. Immunohistochemistry staining of mouse inguinal lymph node (vaccine draining) sections showed that mRNA encoding MBEnv expressed higher levels of Env antigens compared to mRNA encoding Env–NP.

Taken together, these two studies demonstrated the importance of stabilizing mutations in mRNA-encoded Env designs and the significance of evaluationof the immunogenicity of mRNA vaccine candidates at proper doses in animals before moving to next steps.

The B and T lymphocytes utilize B and T cell receptors, respectively, for the specific recognition of pathogen. Somatic recombination of the antigen receptor loci imparts the immense diversity and specificity of the adaptive immune system’s antigen recognition. The recombination activating gene (RAG) 1 and 2 mediated V(D)J recombination process is a highly developmentally regulated process, occurring at strict stages of B and T lymphocyte development. Both the expression of RAG1/2 and the accessibility of the recombination substrates come under strict developmental stage-specific regulation. E protein transcription factors and the inhibitory Id proteins mediate many of the signaling and transcriptional changes that occur during lymphocyte development. While E protein binding has been identified at antigen receptor loci, the understanding of their cis-regulatory function is lacking.

Recombination of the Tcra-Tcrd locus occurs during two temporally separate stages of T lymphocyte development. Tcrd, nested with Tcra, undergoes recombination first, during the double negative (DN) stage. Cells that commit to the αβ T lymphocyte lineage will then undergo Tcra recombination during the double positive (DP) stage. These processes are highly dependent on cis-acting enhancers termed Eδ and Eα. E proteins have long been known to bind at Eα, while recent DN cell E protein ChIP-seq allowed identification of a vast number of additional targets within the Tcra-Tcrd locus. A series of mutagenesis studies were pursued to better understand E protein cis-regulatory function during Tcra-Tcrd locus recombination.

Eα mutants lacking one or two of the three E protein binding sites were generated. The mutant lacking two binding sites displayed a partial block in αβ T lymphocyte development at the positive selection stage. Loss of transcription at the Jα array reflected a loss in accessibility, which was mirrored by dysregulated primary and impaired secondary Vα-Jα rearrangement. Therefore, Eα E protein binding increases Tcra locus accessibility, which is required for efficient Tcra recombination and the resulting Tcra repertoire diversity.

Analysis of available E protein ChIP-seq revealed strong E protein binding downstream of Trav15-dv6 family V segments. Mutagenesis of the E protein binding region directly downstream of Trav15d-1-dv6d-1 resulted in a loss of Vγ1.1Vδ6.3 T lymphocytes, whose development relies on Tcrd recombination of Trav15d-1-dv6d-1. This has established a new method of E-Id protein axis regulation of Vγ1.1Vδ6.3 T lymphocyte development. Furthermore, analysis suggested that the Vγ1.1Vδ6.1 and Vγ1.1Vδ6.3 T lymphocyte subsets compete during development, with expansion of Vγ1.1Vδ6.1 T lymphocytes only possible in the absence of Vγ1.1Vδ6.3 T lymphocytes. Competition and affinity for ligand likely play a tremendous role in this process. Analysis of two Vγ1.1Vδ6.3 TCR transgenic lines revealed that small differences in CDR3γ and CDR3δ are sufficient to significantly modulate Vγ1.1Vδ6.3 T lymphocyte development and immune profile. These findings and the generated genetic models can form the basis of elucidating the ligand of the Vγ1.1Vδ6.3 TCR.

The findings presented here have advanced the understanding of E protein regulation of the Tcra-Tcrd locus, and by extension the lineage-defining potential of the Vγ1.1Vδ6.3 TCR. This is the first report of E protein cis-regulation of the Tcra locus and of Tcrd recombination of Trav15-dv6 family V segments (specifically Trav15d-1-dv6d-1). Extensive E protein binding of the Vα/Vδ array suggests further regulatory mechanisms and action.

Diacylglycerol (DAG), a lipid messenger generated upon T cell receptor (TCR) engagement, mediates signaling through the IKK/NF-κB and Ras/ERK pathways. Further downstream of the Ras/ERK pathway are mammalian target of rapamycin (mTOR) and MAP kinase signal integrating kinases Mnk1 and Mnk2. While mTOR acts as a critical regulator of T cell metabolism, homeostasis and function, Mnk1 and Mnk2 phosphorylate the initiation factor eIF4E that plays an important role in cap-dependent mRNA translation. Diacylglycerol kinases (DGKs) terminate DAG-mediated signals by phosphorylating DAG into phosphatidic acid. T cells that lack both α and ζ isoforms of DGK accumulate excess DAG upon activation, resulting in hyper-activation of the IKK/NF-κB, Ras/ERK and mTOR pathways, hypersensitivity to TCR stimulation, and loss of self-tolerance. Here, we have examined the mechanisms by which dysregulated DAG-mediated signaling affects T cell function. To this end, we studied the effects of hyper-activating individual DAG-mediated pathways (IKK/NF-κB and TSC/mTOR) on T cell function. We also examined the role of ERK-activated kinases Mnk1 and Mnk2 in T cell function.

Using mice with T cell-specific expression of a constitutively active form of IKKβ (`IKK' mice), we found that uncontrolled IKKβ/NF-κB signaling promotes T cell apoptosis and attenuates responsiveness to TCR stimulation. Defective IL-2 production and increased FasL expression contributed to enhanced IKK T cell apoptosis. Impaired IKK T cell activation and proliferation were associated with defects in TCR signaling, and upregulation of the cell surface inhibitory receptor PD1. In vivo, IKK T cells mounted a compromised antigen-specific CD8 T cell response with curtailed expansion and exaggerated contraction phases. Notably, expression of transcriptional repressor Blimp1 (a regulator of T cell exhaustion) was increased in IKK T cells, and conditionally deleting Blimp1 was able to largely restore responsiveness to TCR stimulation.

Investigating Mnk1/2 double knockout (DKO) mice, we found that Mnk1 and Mnk2 are dispensable for T cell development and function, but important for the pathogenesis of experimental autoimmune encephalomyelitis (EAE). TCR engagement activated Mnk1/2 in a Ras/ERK-dependent manner in primary T cells, and was inhibited by DGK α and ζ. Mnk1/2 deficiency did not affect the development of conventional αβ T cells, regulatory T cells, or invariant NKT cells. Mature T cells from DKO mice showed normal activation and CD4 TH differentiation ex vivo, but DKO mice developed lower clinical scores than WT counterparts in an EAE model, correlating with a smaller pool of MOG-reactive IL-17-producing and IFNγ-producing CD4 cells. These results suggest that Mnk1/2 may play a minimal role in T cell development and function but may control non-T cell lineages to regulate TH1 and TH17 differentiation in vivo.

To determine the effect of constitutive mTOR complex 1 activity on anti-bacterial CD8 responses, we investigated mice with T cell-specific deletion of TSC1, a suppressor of mTOR complex 1 activity. Using an established model system of transgenic (OT1) CD8 cell adoptive transfer and challenge with Listeria monocytogenes expressing a cognate antigen, we found that TSC1 deficiency impairs antigen-specific CD8 responses. Fewer TSC1-deficient OT1 cells were present in the peripheral blood and spleen at the peak of the response and fewer memory cells were found at later time points, in individual and competitive adoptive transfer experiments with WT counterparts. Weak expansion of TSC1-deficient cells was correlated with defects in survival and proliferation in vivo, while exaggerated contraction was associated with an increased ratio of SLECs to MPECs in the effector cell population. This perturbation in effector-memory differentiation was concomitant with enhanced T-bet expression and decreased Eomes expression among activated TSC1 KO cells. Upon competitive adoptive transfer with WT counterparts and antigen re-challenge, TSC1-deficient memory cells showed moderate defects in expansion but not cytokine production. Taken together, these findings provide direct evidence of a CD8 cell-intrinsic role for TSC1 in regulating antigen-specific primary and memory responses.

In sum, findings from these studies provide deeper insight into the regulation of T cell function by DAG-mediated pathways, and may have implications for the design of immune-modulation strategies during vaccination, autoimmunity and cancer immunotherapy.

Human CMV (HCMV), a ubiquitous β-herpesvirus, remains the most common congenital infection and infectious complication in immunocompromised patients. Despite the severe clinical impacts, there is no current approved HCMV vaccine or immunotherapy. Two major hurdles have been identified for the HCMV vaccine development. First, the HCMV genome is highly diverse and variable, i.e., a HCMV vaccine design that only includes immunogens from a single strain might not provide effective protection against other HCMV strains. Second, a large gap remains to understand what virologic determinants are essential for congenital CMV transmission.

HCMV glycoprotein B (gB) and pentameric glycoprotein complex (PC) are currently the most promising vaccine targets. gB is essential for viral entry into all host cells and was shown to elicit both neutralizing and non-nAb responses. The most successful HCMV vaccine to-date, a gB subunit vaccine adjuvanted with MF59, achieved 50% efficacy against primary HCMV infection. The gB/MF59 vaccinees were less frequently infected with HCMV gB genotype strains most similar to the vaccine strain than strains encoding genetically distinct gB genotypes, suggests that the strain-specific immunity might account for the limited efficacy. Applying the lipid nanoparticles-encapsulated nucleoside-modified mRNA (mRNA-LNP) vaccine platform, we hypothesized that vaccination with multiple HCMV gB genotypes could increase the breadth of gB-specific humoral and cellular responses, leading to broader protection.

To test our hypothesis, we intradermally immunized female rabbits with three doses of the monovalent or multivalent gB mRNA-LNP vaccines and measured the vaccine-elicited humoral and cellular responses. Compared to the monovalent vaccine, the multivalent vaccines did not demonstrate a higher magnitude or breadth of gB-specific IgG binding or functional antibody responses against multiple gB genotype. In addition, the multivalent vaccines did not elicit a stronger T cell response against variable regions among gB genotypes. Our data suggests that inclusion of multivalent gB antigens is not an effective strategy to increase the breadth of anti-HCMV gB antibody and T cell responses.

PC, composed of the subunits gH/gL/UL128/UL130/UL131A, has been demonstrated to be essential for CMV entry into non-fibroblast cells in vitro. The PC was also identified as the major target of neutralizing antibodies. As HCMV infection of most cell types found near the maternal-fetal interface depends on the PC-mediated entry in vitro, it is conceivable that this complex could be required for cross-placental CMV transmission in vivo. These findings link the PC to broad cell tropism and virus dissemination in vivo, denoting all subunits as potential targets for intervention strategies and vaccine development.

To determine the importance of the PC for congenital transmission in a translational non-human primate (NHP) model, we engineered a rhesus CMV (RhCMV) mutant lacking the homologues of UL128 and UL130 which demonstrated diminished infection of epithelial cell in vitro. Nevertheless, intravenous inoculation of immunocompetent and CD4+ T cell-depleted, RhCMV-seronegative, pregnant rhesus macaques with the PC-deficient mutant resulted in similar maternal RhCMV peak plasma viremia levels to that of PC-intact RhCMV, while virus shedding in saliva and urine was limited. Infections with the PC-intact virus induced strong IgG responses that were able to neutralize RhCMV entry into epithelial cells. These responses were reduced, but not absent, from animals infected with the PC-deficient mutant, which also induced IgG responses against gH. Most importantly, congenital infection rates determined by the viral DNA detection in amniotic fluid was identical between the PC-deficient and PC-intact RhCMV. Our data indicates that the PC is dispensable for transplacental transmission in non-human primates.