Browsing by Author "Zhuang, Yuan"

The microphase segregation behavior, which exhibits periodic patterns on a mesoscale, has been found in many systems where it has demonstrated its extreme industrial usefulness in the diblock copolymer system. When studying more general isotropic colloidal systems, periodic microphases should ubiquitously emerge in systems for which short-range inter-particle attraction is frustrated by long-range repulsion~(SALR). The morphological richness of these phases makes them desirable material targets, but our relatively coarse understanding of even simple models hinders controlling their assembly both from thermodynamic and dynamic points of view. The thermodynamic question is what should be the appropriate potential to stabilize microphases, such as cluster crystal, cylindrical, double gyroid and lamellar, while the dynamic question is whether the current experiments are long enough for these phases to appear. This dissertation will focus on solving these two parts of problems and hopefully guide the experiments to discover a simple material that can have microphase segregation behavior. In order to answer the thermodynamic problem of the stability of the microphases, we use a novel thermodynamic integration method as well as density functional methods in comparing the free energy of the microphases with uniform liquids. With the thermodynamic integration, we locate FCC-cluster, cylindrical, double gyroid and lamellar phases as well as nontrivial interplay between cluster, gel and microphase formation for a model microphase former. We also extended the methods to the model with a shorter and longer repulsion region where we found that the shorter region of the system may be in the Wigner glass of clusters of different sizes rather than the microphases. We also compare our simulation results with that from the density functional theory where we demonstrate that the classical density functional theory is qualitatively right but the simple improvement of the radial distribution functional by assuming the system is the same as Percus-Yevick hard sphere does not make a quantitatively difference. Our finding confirms that if the colloidal system has proper SALR potential as well as the right regime of area fraction and temperature, the microphase will be found in these systems. We then answer the second question which is whether the slow dynamics hinders the formation of microphases. We study the modeled microphase former and track the change of the first peak in the structure factor as well as the structural correlation time. We found that the system has a very complex dynamical regimes, including homogeneous fluid, void micelle, liquid gel and solid gel. The system becomes extreme slow in the solid gel regime but if in the regime that density and temperature are near the order-disorder transition, the lamellar self-assembly is much faster than the relaxation time of the solid gel which may explain why in the experimental system, the colloids seem stuck forever. We have collaborated with an experimental group to realize the SALR self-assemble behavior in a well controlled system. We have calibrated the system using a high precision thermodynamic integration by determining and matching the critical point and triple point of the experiments when the system is set up in the purely attractive regime. The system, however, becomes unpredictable when it goes into the SALR system where both higher body and other interactions become dominant. Finally, we try to extend our system to a spherocylinder model, which is an anisotropic particles with SALR. We have developed a novel cell list method here to accelerate the simulation. By determining the percolation transition and the order parameter, we find that the simple anisotropic interaction will introduce a much complex phase behavior of the system even in the disordered regime. We have identified several disordered phases, including homogeneous liquid, micellar liquid, free rotator gel, nematic gel and smectic gel.

Proper regulation of the immune system with regards to development, reaction specificity, and duration is necessary to prevent immune reactions directed against the host that can have a number of potentially lethal consequences. This regulation is controlled in part through a wide variety of transcription factors that function to ensure the proper development, function, and regulation of the immune system.

E-proteins are widely expressed and have a multitude of functions in the immune system including proper lymphocyte development and function. E-proteins can be regulated by class V HLH factors, including the inhibitor of differentiation (Id). genes 1-4. Id proteins are expressed throughout the hematopoietic system and have crucial roles in cell fate decisions, differentiation and proliferation in a multitude of tissues and cell types.

Id3 plays a variety of important roles in the immune system including T cell homeostasis, activation, and effector function. The importance of Id3 has been demonstrated using a number of mouse models, and mice that lack Id3 develop an autoimmune condition similar to that of Primary Sjögren's syndrome (PSS). The goal of this dissertation is to further characterize disease initiation and progression in Id3-/- mice, with a focus on the specific targeting of exocrine glands and associated lymphoid tissues by the immune system.

T cells play a crucial role in the development of disease symptoms in Id3-/- mice, though much remains unknown about the relative contribution of the two major subsets of T cells, αβ and γδ T cells to disease severity. The importance of both αβ and γδ T cells is demonstrated in part by the use of two newly generated models, Id3/β-/- and Id3/δ-/- mice, which lack αβ or γδ T cells, respectively. These mice have allowed for a better understanding of the relative contribution of T cells in disease initiation and progression in Id3-/- mice.

Analysis of serum cytokine levels show that Id3-/- mice develop elevated levels of IL-13 at an early age, and this cytokine is associated with impaired saliva function. Significant populations of IL-13+ T cells have been identified in the peri-glandular lymph nodes of Id3-/- mice, though it appears that there are significant IL-13 producing cells not of the T cell lineage. Reduction of serum IL-13 levels via anti-IL-13 antibody treatment resulted in improved saliva production in response to cholinergic stimulation and reduced the number of mast cells detected in the mandibular and lachrymal glands. The importance of IL-13 in the initiation and progression of exocrinopathy in Id3-/- is being further investigated using a recently acquired IL-13 reporter mouse model. Additionally, IL-13Rα1+ cells have been identified in and around the gland tissues of Id3-/- mice, and while the origin and function of these cells remains unknown, these cells have a potential to serve as a biomarker for disease progression and severity.

Lastly, analysis of Id3-/- mice reveals an increased presence of mast cells in the peri-glandular lymph nodes and gland tissues as compared to wild type controls. These mast cells are localized in areas of significant tissue remodeling, serve as a potential source of IL-13 and are associated with impaired saliva production. These findings suggest an important role of mast cells in disease development in Id3-/- mice. Together, these studies have revealed a number of findings that will likely contribute to our understanding of the initiation and progression of exocrinopathy in Id3-/- mice.

Invariant Natural Killer T (iNKT) and γδNKT cells are well-characterized innate-like counterparts of αβ and γδ T cells respectively that express semi-invariant T cell receptors (TCRs) and are capable of mounting rapid immune responses. Although many key regulatory molecules have been shown to play important roles in the development of these cells, the mechanism of their lineage specification and acquisition of effector functions remain to be fully addressed.

Id proteins, or inhibitor of DNA binding and differentiation, act as antagonists of transcription factors known as E proteins. Id proteins are known promote the differentiation of conventional T cells, and suppress the expansion of innate-like T cells. We have previously found that expansion of iNKT and another subset of innate-like T cells leads to rapid lymphoma development in Id2/Id3-deficient mice. The goal of this dissertation is to elucidate the mechanisms by which Id proteins differentially regulate the lineage choice between the concurrently developing innate and conventional lineages in early stages of T cell development, as well as the mechanisms driving the malignant transformation of these expanding innate-like T cells.

Firstly, I tested the hypothesis whether uninhibited E2A activity in the absence of Id proteins transcriptionally promotes the development of iNKT cells. Indeed, I found E2A-mediated upregulation of critical genes, and biased rearrangement at the DP stage promotes iNKT cell lineage development in Id-deficient mice. The observed expansion of the iNKT cells in these mice is not abrogated by blocking pre-TCR signaling, which is required for conventional αβ T cell development. Finally, E2A is found to be a key transcriptional regulator of both iNKT and γδNKT lineages, which appear to have shared lineage history. Therefore, my study revealed a previously unappreciated role of E2A in the regulation of lineage choice between conventional and innate-like T cell fate as early as the pre-TCR checkpoint.

Second, I explored the origins and pathways that drive innate-like lymphomas in Id2/Id3-deficient mice. I found that CD1dTet- innate-like T cells develop independent of CD1d-mediated selection, and start expansion in neonatal mice. The transcriptional program in expanding neonatal iNKT cells is significantly modified, including upregulation of the cytokine-cytokine receptor interaction pathway which can promote their expansion and migration, ultimately leading to their malignant transformation. I also discovered shared dysregulation of the NF-kB pathway and genes with reported driver mutations between our iNKT-derived lymphomas and human iNKT tumors. My study demonstrates that Id2 plays a tumor suppressive role in collaboration with Id3 in developing T cells in mice. Contrary to the perception of Id proteins as potential therapeutic targets in some cancer models, these results also highlight the possibility of aggravated tumorigenesis upon non-targeted suppression of Id2 and Id3.

Macrophages play vital roles in pathogen clearance, initiation of immune responses, and maintenance of immune homeostasis; however, current understanding of the contributions of macrophages to these processes in vivo has been limited by the reagents and animal models available. Of the many macrophage-deficient mouse models that have been reported, none have specific, long-term loss of distinct macrophage populations, and most develop infections that complicate the study of immune homeostasis. By conditionally deleting the anti-apoptotic protein cellular FLICE-like inhibitory protein (c-FLIP) in myeloid cells, we have generated a novel mouse model that has proven useful in studying both the in vivo functions of macrophages under steady-state conditions and the requirements for macrophage survival at steady-state and during inflammation.

c-FLIPf/f Lysm-Cre mice specifically lack bone marrow macrophages and splenic marginal zone macrophages and develop severe neutrophilia, splenomegaly, extramedullary hematopoiesis, decreased body weight, and increased production of G-CSF and IL-1β, but not IL-17 secondary to the loss of these macrophage populations. c-FLIPf/f Lysm-Cre mice exhibit delayed clearance of circulating neutrophils, suggesting that failure of macrophages to efficiently clear apoptotic neutrophils causes production of cytokines that drive excess granulopoiesis. Further, blocking G-CSF, but not IL-1R signaling in vivo rescues this neutrophilia, suggesting that a G-CSF-dependent, IL-1β-independent pathway plays a role in promoting neutrophil production in mice with defective clearance of apoptotic cells.

Furthermore, using mice expressing only one c-FLIP isoform in myeloid cells (c-FLIP_S or c-FLIP_L), we have shown that although either isoform is sufficient to promote survival of macrophages under steady-state conditions, both c-FLIP_S and c-FLIP_L required for macrophage survival during inflammation. In contrast, c-FLIP_L is sufficient to promote survival of eosinophils in inflammatory conditions in the absence of c-FLIP_S. These data demonstrate distinct requirements for myeloid cell survival in the presence and absence of inflammation and point to a mechanism by which pathogenic organisms may target macrophages to evade the immune response.

Together, these findings demonstrate a critical role for c-FLIP in promoting macrophage survival, which is in turn required for neutrophil homeostasis, and provide an in vivo model system for continuing studies of the non-redundant functions of c-FLIP_S and c-FLIP_L in myeloid cells during infection and inflammation.

The E-protein transcription factors E2A and HEB regulate various cell processes during the development of B and T lymphocytes, including cell differentiation, lineage commitment, recombination of immune receptor genes, proliferation, and survival. B cell development is dependent on E2A from the earliest stages whereas T cell development relies on the cooperative efforts of both E2A and HEB. Established work demonstrates that the timing and dosage of E-protein expression is critical for mediating these diverse functions. The goal of this dissertation is to develop and utilize new genetic tools to manipulate the timing and dosage of E2A and HEB expression in order to enhance our understanding of E-protein function. Here we develop two new mouse models to identify novel lineage and stage specific roles of E-proteins during B lineage commitment and thymocyte selection.

First, we have generated an E2A inducible mouse model to allow reversible regulation of E2A function and precise timing of induction at the protein level. This system was created by inserting a tamoxifen responsive region of the estrogen receptor ligand binding domain (ER) at the carboxyl end of the tcfe2a gene, encoding E2A, to generate E2AER fusion proteins. To our knowledge, the ER fusion system has not yet been tested from an endogenous locus in live animals. Using the E2AER system, we have demonstrated rapidly induced E2AER activity upon tamoxifen treatment that is capable of supporting B cell development in an ex vivo culture system. In addition to characterizing the kinetics and reversibility of this inducible system, we have utilized tamoxifen treatment of E2AER B cell progenitors to identify potential novel E2A target genes driving B lineage commitment.

Second, we have analyzed E-protein function during the double positive (DP) stage of alpha beta T cell development by using a Cre-loxp conditional deletion system. Here, E-protein dosage was manipulated by removal of both E2A and HEB, and the timing of deletion was controlled by using a CD4Cre transgene. During development, survival through the DP stage and initiation of differentiation to the subsequent single positive (SP) stage for generation mature alpha beta T cells is dependent on the production of a functional alpha beta T cell receptor (TCR). The mechanism that maintains cells at the DP stage prior to expression of a mature TCR remains unclear. In this study, we have shown that E2A and HEB together are required to maintain DP fate and regulate the transition to the SP stage. Loss of E2A and HEB in DP thymocytes was sufficient to trigger DP to SP differentiation, even in the absence of a TCR. Deletion of E2A and HEB allowed cells to bypass the requirement for a TCR-mediated positive selection signal. These findings identify E2A and HEB as key regulators enforcing thymocyte positive selection to ensure maturing T cells express a functional receptor.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is crucial in linking TCR engagement to downstream signaling events, such as calcium flux and Ras-MAPK pathway. Following TCR engagement, LAT is phosphorylated at its membrane-distal tyrosine residues, which mediates the binding of Grb2/Sos, PLC-1, and GADS/SLP-76 complexes. This multi-protein signaling complex initiates signaling cascades eventually leading to the activation of transcription factors that regulate the genes required for T cell proliferation and effector functions. The indispensable role of LAT in thymocyte development has been evidenced as LAT-deficient mice completely lack peripheral T cells. To study the function of LAT in mature T cells, our lab previously generated a conditional knock-in mouse line in which the lat gene can be deleted by Cre recombinase. Deletion of LAT in mature T cells revealed the critical role of LAT in T cell activation. Here, we used this inducible LAT deletion mouse line crossed with the OT-I transgenic mice to study the role of LAT in mature CD8 T cells.

To analyze the contribution of LAT in CD8 T cells during the course of pathogen infection, we infected mice with Listeria monocytogenes-expressing Ova to elicit activation of antigen-specific CD8 T cells, and then inducibly deleted LAT in these cells at different stages of infection under the control of tamoxifen treatment. We show that LAT is important for maintaining CD8 T cell expansion during the priming phase; however, it is not required for CD8 T cell contraction. In addition, memory CD8 T cell can persist in the absence of LAT, suggesting that LAT-signaling is not necessary for memory maintenance. Nonetheless, these LAT-deficient memory T cells were unable to proliferate or produce cytokines upon secondary infection. Moreover, LAT deficiency accelerates memory differentiation during the effector-to-memory transition, leading to a higher frequency of KLRG1lowIL-7RhighCD62Lhigh memory T cells. Together, these data demonstrate that, while it is dispensable for contraction and memory maintenance, LAT-signaling regulates CD8 T cell memory differentiation and is essential for the memory response against pathogens.

The fundamental activity of CD8 T cells is to elicit cytotoxicity toward target cells that express foreign antigens, and this is mediated through granule-dependent and Fas ligand-dependent mechanisms. The signaling events that regulate these processes remain unclear. We showed that LAT-deficient cytotoxic T cells (CTLs) failed to upregulate FasL and produce IFN- after engagement with target cells. Moreover, they displayed reduced granule-mediated killing. We further dissected the effect of the LAT deletion on each step of granule exocytosis. LAT-deficiency led to altered synapse formation, subsequently causing unstable T cell:APC conjugates. MTOC polarization and granule reorientation were also impaired by LAT-deficiency, leading to reduced granule delivery. Despite these defects, granule release was still observed in LAT-deficient CTLs due to residual calcium flux and PLC activity. This revealed an unexpected finding that CTL function is not entirely dependent on LAT. Collectively, these data indicate that the signaling circuits governing CTLs are programmed to adopt multiple pathways, allowing CTLs to effectively eliminate various pathogens during adaptive immune responses.

Within the field of immunology, the existence of autoimmune diseases presents a unique set of challenges. The immune system typically protects the host by identifying foreign pathogens and mounting an appropriate response to eliminate them. Great strides have been made in understanding how foreign pathogens are identified and responded to, leading to the development of powerful immunological tools, such as vaccines and a myriad of models used to study infectious diseases and processes. However, it is occasionally possible for host tissues themselves to be inappropriately identified as foreign, prompting an immune response that attempts to eliminate the host tissue. The immune system has processes in place, referred to as selection, designed to prevent the development of cells capable of recognizing the self as foreign. While a great deal of work has been invested in understanding these processes, many concrete answers remain elusive.

Our laboratory, which focuses on understanding the roles of E and Id proteins in lymphocyte development, has established the Id3 knockout mouse as a model of autoimmune disease. Id3 knockout mice develop a disease reminiscent of human Sjӧgren's Syndrome, an autoimmune disease that progressively damages the salivary and lachrymal glands. Continued study of this model has yielded interesting results. These include the identification of CD4+ T cells as initiators of disease as well as the identification of the cytokine Interleukin 13 (IL-13) as a potential causative agent. However, the source of IL-13, its true role as a causative agent of disease, as well as the developmental basis for its elevated expression remained elusive.

To this end, I utilized a reporter gene that enabled me to detect cells producing IL-13 as well as test the effects of IL-13 deletion on disease progression. Using this system, I was able to identify both CD4+ T cells and γδ T cells as major sources of IL-13. I was also able to determine that elimination of IL-13 in Id3 knockout mice was sufficient to block the development of disease symptoms, reinforcing the hypothesis that IL-13 is a causative agent in disease initiation. Finally, I attempted to better characterize the phenotype of cells producing IL-13. These experiments indicated that the T cell receptor (TCR) repertoire of Id3 knockout mice is markedly different than that of wild-type (WT) mice. Furthermore, cells bearing certain TCRs appeared to express IL-13 at dramatically different rates, indicating that certain TCRs may be predisposed to IL-13 particular effector fates.

Several HIV-1 neutralizing antibodies (e.g. 2F5, 4E10) have been shown to react with self-antigens, suggesting that effective humoral responses to HIV-1 may be constrained by the tolerization of HIV-reactive B cells that also recognize self-antigens. I have tracked the development of 2F5-like HIV-1 gp41 membrane proximal external region (MPER)-reactive B cells throughout ontogeny using B-cell tetramer reagents. In BL/6 mice, MPER-binding populations are lost during normal B-cell development and immunization with HIV-1 MPER antigen does not elicit robust humoral responses. I have identified Kynureninase as a self-antigen that is recognized by 2F5 antibody and, therefore, is a molecule that could mediate the developmental loss of B cells reactive to an epitope shared by HIV gp41 and Kynureninase. To recover these MPER-reactive cells, I describe and characterize a stromal-cell independent culture system that efficiently supports pro-B cell to IgM+ B-cell development with near normal levels of IgH and Igkappa diversity. B-cell development in vitro closely follows the patterns of development in vivo with culture derived (CD) B cells demonstrating characteristic patterns of surface antigen expression and gene activation. Immature and transitional B-cell compartments are reduced, due to the induction of tolerance, in the bone marrow of 3H9 IgH knockin mice ; however, cultures of 3H9 IgH knockin pro-B cells yields high frequencies of "forbidden", autoreactive IgM+ B cells. Furthermore, serum IgG autoantibody exceeded that present in autoimmune, C4-/- animals following the reconstitution of RAG-1-/- mice with IgM+ CD cells derived from BL/6 mice. I show that HIV-1 MPER-reactive B cells are recovered from both BL/6 and 2F5 IgH knockin bone marrow using this in vitro culture system. RAG-1-/- mice reconstituted with these culture-derived B and T cells generate strong germinal center and antibody responses to HIV-1 MPER antigens. These data demonstrate that the humoral immune response to this HIV-1 gp41 MPER antigen can be restored in mice when the constraints of B-cell tolerance have been relaxed.

Invariant NKT (iNKT) cells represent a unique group of αβ T cells that have been classified based on their exclusive usage of the invariant Vα14Jα 18 TCRα –chain and their innate–like effector function. Thus far, the transcriptional programs that control Vα14Jα18 TCRα rearrangements and the population size of iNKT cells remain incompletely defined.

E protein transcription factors have been shown to play multiple roles in T cell development including lineage commitment, receptor gene recombination, proliferation and lineage choice. Inhibitor of DNA–binding (Id) proteins are the natural inhibitors of E protein transcription factors. The goal of this dissertation is to examine E protein functions in the development of iNKT cells in the mouse after combined deletion of genes encoding E protein inhibitors Id2 and Id3.

We revealed important roles of Id proteins and E proteins in regulating iNKT cell development. Deletion of Id2 and Id3 in T cell progenitors resulted in a partial block at the pre–TCR selection checkpoint and a dramatic increase in numbers of iNKT cells. This increase in iNKT cells is accompanied with a biased rearrangement involving Vα14 to Jα18 recombination at the double–positive stage and enhanced proliferation of iNKT cells. We further demonstrate that a 50 percent reduction of E proteins can cause a dramatic lineage shift from iNKT cells to innate–like gd T cells in Id2 and Id3 double–deficient mice. Collectively, these findings suggest that Id2– and Id3–mediated inhibition of E proteins controls iNKT development by restricting lineage choice and population expansion.

Our study also uncovered a novel role of Id proteins in development of NKT lymphoma. Id deficient NKT cells gradually progresses into NKT lymphoma, a rare form of tumor with no clearly defined etiology. Id and E proteins have been demonstrated to be involved in multiple lymphoma and cancer subtypes, but their role in the development of NKT lymphomas is unexplored. Adoptive transfer experiments confirmed that the malignant cells are able to invade healthy tissues. cDNA Microarray analysis of NKT lymphoma and pre–malignant NKT cells revealed alterations in several cytokine signaling pathways during tumor progression. These findings indicate that regulation of E proteins by Id2 and Id3 may play important roles in the development of NKT lymphoma. To our knowledge, this study represents the first mouse model in which NKT lymphoma develops at such high frequency and fast kinetics. Our double knockout mice provide a unique model to study mechanisms of human NKT lymphoma progression.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that lacks any intrinsic enzymatic or transcriptional activity. Upon TCR engagement, LAT is phosphorylated at its membrane-distal tyrosine residues, which mediate the binding of Grb2/Sos, PLCγ1, and GADS/SLP-76 complexes. SLP-76 (SH2 domain-containing leukocyte protein of 76kD) is a cytosolic adaptor protein that can interact with a variety of other adaptor proteins and signaling effectors. Through its constitutive binding of GADS, SLP-76 is recruited to the plasma membrane via LAT following TCR stimulation. Together, LAT and SLP-76 nucleate a large multi-molecular signaling complex, which couples TCR proximal signaling to downstream biochemical events, including calcium mobilization and Ras-MAPK pathway activation.

LAT is important in early thymocyte development as LAT-deficient mice have a complete block at the DN3 stage. To study the role of LAT beyond the DN3 stage, we generated mice in which the lat gene could be deleted by Cre recombinase. Deletion of LAT after the DN3 stage allowed largely normal development of DP thymocytes. However, LAT-deficient DP thymocytes were severely defective in responding to stimulation via the TCR and failed to efficiently differentiate into SP thymocytes. Moreover, deletion of LAT in peripheral mature T cells rendered these T cells completely unresponsive to CD3 crosslinking due to abolished calcium mobilization and Ras-ERK activation. Long-term survival and lymphopenia-driven homeostatic proliferation of the LAT-deficient naïve T cells were also severely impaired. Together, these data indicate that, in addition to its role in pre-TCR signaling, LAT also plays an essential role in thymocyte development during the transition from the DP to SP stage, as well as in mature T cell activation and homeostasis.

Similar to LAT, SLP-76 is also critical for T cell function and thymocyte development. While the functions of various SLP-76 domains have been extensively studied, the role of the sterile alpha motif (SAM) domain in SLP 76 function remains unknown. By generating SLP 76 knock in mice with the SAM domain deleted, we showed that the absence of the SAM domain resulted in impaired positive and negative thymic selections, leading to a partial block of thymocyte development at the DP to SP transition. TCR-mediated IP3 production, calcium flux, and ERK activation were all decreased in these ΔSAM-SLP-76 knockin T cells, leading to defective IL 2 production and proliferation. Moreover, despite normal association between GADS and SLP-76, TCR-mediated SLP 76 clustering was inhibited by the deletion of the SAM domain, likely causing the aforementioned TCR signaling defects. These data demonstrated for the first time that the SAM domain is indispensable for optimal SLP-76 signaling.

The role of CD4 T cell help in primary and secondary CD8 T cell responses to infectious pathogens remains incompletely defined. The primary CD8 T response to infections was initially thought to be largely independent of CD4 T cells, but it is not clear why some primary, pathogen-specific CD8 T cell responses are CD4 T cell-dependent. Furthermore, although the generation of functional memory CD8 T cells is CD4 T cell help-dependent, it remains controversial when the "help" is needed. The goal of this thesis project is to determine requirement and mechanisms of CD4 help during the CD8 response to vaccinia viral (VV) infection.

The first aim of this project was to determine when CD4 T cell help is required during the CD8 response to VV infection. Using both CD4-deficient mice and mice with transient depletion of CD4 T cells, we demonstrated that CD4 T cell help was not needed for the activation and effector differentiation of CD8 T cells during the primary response to VV infection. However, the activated CD8 T cells showed poor survival without CD4 T cell help, leading to a reduction in clonal expansion and a diminished, but stable CD8 memory pool. In addition, we observed that CD4 T cell help provided during both the primary and secondary responses was required for the survival of memory CD8 T cells during recall expansion. Our study indicates that CD4 T cells play a crucial role in multiple stages of CD8 T cell response to VV infection and may help to design effective vaccine strategies.

Given that CD4 T cell help is critical for the survival of activated CD8 T cells during both the primary and memory recall responses, it is still unclear how CD4 T cell help promotes CD8 T cell survival. The second aim of this project was to determine the mechanism of CD4 help for the survival of activated CD8 cells. We first showed that CD4 help in vitro was mediated by IL-21, a cytokine produced predominantly by activated CD4 T cells. We then demonstrated direct action of IL-21 on CD8 T cells was critical for the VV-specific CD8 T cell response in vivo. This intrinsic IL-21 signaling was essential for the survival of activated CD8 T cells and the generation of long-lived memory cells. We further revealed that IL-21 promoted CD8 T cell survival in a mechanism dependent on activation of the STAT1 and STAT3 pathways and subsequent upregulation of the pro-survival molecules Bcl-2 and Bcl-xL. Collectively, these results identify a critical role for CD4-derived IL-21 signaling in CD8 T cell responses to acute VV infection in vivo and may help design effective vaccine strategies in situations where CD4 cells are not fully functional.

E and Id proteins are members of the basic helix-loop-helix (bHLH) transcription regulator family. These proteins control a broad range of lymphocyte biology, from the development of multiple lineages to execution of their effector functions. With the development of new experiment models, novel functions of E and Id proteins continued to be discovered. In this thesis, I focused my study on the role of Id2 in gamma delta T cells and CD4+ alpha beta T cells, as well as the role of Id3 in B cells.

Id proteins have been shown to control gamma delta T cell development. Id3 knockout mice demonstrate a dramatic expansion of innate-like Vgamma1.1+ Vdelta6.3+ T cells in the neonatal stage, suggesting that Id3 is an inhibitor of their development. Interestingly, Id3 knockout mice with a B6/129 mix background have much less expansion of the Vgamma1.1+ Vdelta6.3+ T cells compared to mice with pure B6 background. Genetic studies showed that this difference is strongly influences by a chromosome region very close to the Id2 locus. Using the Id2f/f CD4Cre+ mice, I found that Id2 is also an inhibitor of gamma delta T cell development. Deletion of Id2 alone is sufficient to enhance the maturation of these cells in the thymus and induce a moderate expansion of gamma delta T cells in the periphery. This study demonstrated the delicate balance of transcription control in cells of the immune system.

The Id2f/f CD4Cre+ mice also enabled me to study the role of Id2 in peripheral CD4+ alpha beta T cell functions, which was difficult in the past because Id2 knockout mice lack lymph node development. I found that CD4 T cells in these mice have a profound defect in mounting immune responses, demonstrated by a complete resistance to induction of experimental autoimmune encephalomyelitis (EAE). I found that Id2-deficient CD4 T cells fail to infiltrate the central nervous system, and the effector CD4 T cell population is smaller compared to that in control mice. Id2 is important for the survival and proliferation of effector CD4 T cells, and this phenotype was correlated with an increased expression of Bim and SOCS3. This study revealed a novel role of Id2 in the functioning of CD4+ alpha beta T cells.

Switching my focus to B cells, recent next generation sequencing of human Burkitt lymphoma samples revealed that a significant proportion of them have mutations of Id3. This finding suggests that Id3 may be a tumor suppressor gene in the lymphoid system. Utilizing various Id3 knockout and conditional knockout mouse models, I showed that Id3 deficiency can accelerate lymphoid tumor genesis driven by the over-expression of oncogene c-Myc. This work may lead to development of a more realistic mouse model of human Burkitt lymphoma, allowing more mechanistic studies and perhaps preclinical tests of new therapies.

The homeostasis of naïve T lymphocytes is maintained by several mechanisms involving basal TCR and cytokine signaling, and nutrient factors. One of the common net results of these input signals is the production and stabilization of anti-apoptotic Bcl-2 family members. A second result of these processes is the induction of autophagy, an intracellular, catabolic, lysosomal targeting pathway. Autophagy induction in most systems involves the class III phosphatidylinositol-3 kinase (PI3K), Vps34, to produce phosphatidylinositol-3-phosphate (PI(3)P). To test this in T lymphocytes, I generated mice specifically lacking Vps34 in T cells (Vps34f/fLck-cre mice). However, Vps34-deficient T lymphocytes have normal levels of basal autophagy, and upregulate autophagy normally in response to cytokine or nutrient withdrawal, or TCR stimulation. Therefore I conclude that Vps34 activity is not required for autophagy induction in T lymphocytes. T lymphocytes lacking Vps34 do have enhanced rates of apoptosis, but this is due to defects in intracellular trafficking, specifically of the Interleukin-7 receptor alpha subunit (IL-7Rα). Additionally, multivesicular body (MVB) maturation is impaired in T cells lacking Vps34 such that extracellular ligands are not efficiently targeted to the lysosome.

Autophagy induction in Vps34-deficient T lymphocytes is still sensitive to pan-PI3K inhibitors, such as wortmannin and 3-methyladenine (3MA). Therefore, I hypothesized that other classes of PI3K are necessary to induce autophagy in T lymphocytes through the production of PI(3)P. Autophagy induction is sensitive to specific class I PI3K (PI3KI) inhibitors, such as PIK75. Additionally, T cells lacking the p85 regulatory subunit of PI3KI also have severe defects in T cell receptor (TCR) mediated autophagy induction. PI3KI activity results in the production of PI(3,4,5)P3, though, and not PI(3)P. Because of this specificity, I hypothesize that additional inositol polyphosphatases (Inpp) are required for autophagy induction downstream of PI3KI activity. Indeed, utilizing both inhibitors of pharmacological inhibition and siRNA-mediated knockdown of two classes of phosphatidylinositol phosphatases, inositol polyphosphate-4-phosphatase (Inpp4) and SH2 containing inositol phosphatase (SHIP), had dramatic impacts on autophagy induction. Furthermore, exogenous addition of PI(3,4)P2, a hypothesized intermediate in this pathway, positively regulates autophagy induction and leads to enhanced progression of autophagy. These observations indicate that PI3KI activity, linked to Inpp activity, are necessary and positive regulators of autophagy through the production of PI(3)P.