Browsing by Author "Krangel, Michael S"

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.

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.

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.

B cell abnormalities contribute to the development and progress of autoimmune disease. Traditionally, the role of B cells in autoimmune disease was thought to be predominantly limited to the production of autoantibodies. Nevertheless, in addition to autoantibody production, B cells have other functions potentially relevant to autoimmunity. Such functions include antigen presentation to and activation of T cells, expression of costimulatory molecules and cytokine production. Recently, the ability of B cells to negatively regulate cellular immune responses and inflammation has been described and the concept of “regulatory B cells” has emerged. A variety of cytokines produced by regulatory B cell subsets have been reported with interleukin-10 (IL-10) being the most studied. IL-10-producing regulatory B cells predominantly localize within a rare CD1dhiCD5+ B cell subset in mice and the CD24hiCD27+ B cell subset in adult humans. This specific IL-10-producing subset of regulatory B cells have been named “B10 cells” to highlight that the regulatory function of these rare B cells is primarily mediated by IL-10, and to distinguish them from other regulatory B cell subsets that regulate immune responses through different mechanisms. B10 cells have been studies in a variety of animal models with autoimmune disease and clinical settings of human autoimmunity. There are many unsolved questions related to B10 cells including their surface phenotype, their origin and development in vivo, and their role in autoimmunity.

In Chapter 3 of this dissertation, the role of the B cell receptor (BCR) in B10 cell development is highlighted. First, the BCR repertoire of mouse peritoneal cavity B10 cells is examined by single cell sequencing; peritoneal cavity B10 cells have clonally diverse germline BCRs that are predominantly unmutated. Second, mouse B10 cells are shown to have higher frequencies of λ+ BCRs compared to non-B10 cells which may indicate the involvement of BCR light chain editing early in the process of B10 cell development in vivo. Third, human peripheral blood B10 cells are examined and are also found to express higher frequencies of λ chains compared to non-b10 cells. Therefore, B10 cell BCRs are clonally diverse and enriched for unmutated germline sequences and λ light chains.

In Chapter 4 of this dissertation, B10 cells are examined in the healthy developing human across the entire age range of infancy, childhood and adolescence, and in a large cohort of children with autoimmunity. The study of B10 cells in the developing human documents a massive transient expansion during middle childhood when up to 30% of blood B cells were competent to produce IL-10. The surface phenotype of pediatric B10 cells was variable and reflective of overall B cell development. B10 cells down-regulated CD4+ T cell interferon-gamma (IFN-γ) production through IL-10-dependent pathways and IFN-γ inhibited whereas interleukin-21 (IL-21) promoted B cell IL-10 competency in vitro. Children with autoimmunity had a contracted B10 cell compartment, along with increased IFN-γ and decreased IL-21 serum levels compared to age-matched healthy controls. The decreased B10 cell frequencies and numbers in children with autoimmunity may be partially explained by the differential regulation of B10 cell development by IFN-γ and IL-21 and alterations in serum cytokine levels. The age-related changes of the B10 cell compartment during normal human development provide new insights into immune tolerance mechanisms involved in inflammation and autoimmunity.

These studies collectively demonstrate that BCR signals are the most important early determinant of B10 cell development in vivo, that human B10 cells are not a surface phenotype defined developmental B cell subset but a functionally defined regulatory B cell subset that regulates CD4+ T IFN-γ production through IL-10-dependent pathways and that human B10 cell development can be regulated by soluble factors in vivo such as the cytokine milieu. The findings of these studies provide new insights into immune tolerance mechanisms involved in human autoimmunity and the potent effects of IL-21 on human B cell IL-10 competence in vitro open new horizons in the development of autologous B10 cell-based therapies as an approach to treat human autoimmune disease in the future.

Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance since mice expressing the VH and VL of 2F5 have an arrested B-cell development characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that mitigate effective humoral responses. I hypothesize that discreet human antigens are mimicked by the membrane-proximal external region (MPER) of HIV-1 gp41, and that such mimicry is a wide-spread strategy for HIV-1 to evade immune attacks to its vulnerable neutralizing epitopes.

In the first part of the study, I propose to identify autoantigens mimicked by the 2F5 and 4E10 epitopes. I used immunoprecipitation coupled with mass spectrometry as well as protein arrays to identify the self-antigens recognized by 2F5 and 4E10. The binding of antigens was confirmed using serological assays and targeted mutagenesis was used to map the binding epitope. We identified human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes a conformational epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but retain all SF3B3 4E10 epitopes. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant.

Our identification of structural motif shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1. In the second part of the project, I propose to study the mechanisms of immune tolerance to B cells expressing the 2F5 antibodies. To determine the B cell repertoire before and after tolerance checkpoints, I used the Nojima-Kitamura single B-cell culture that supports differentiation into IgG-secreting plasma cells, even autoreactive cells that are normally subject to tolerization in vivo. I found that the pre-tolerance compartment (small pre-B) from 2F5 KI mice are cells that express the 2F5 V(D)J rearrangements and bind HIV-1 gp41, KYNU, and cardiolipin. Mature, post-tolerance B cells from 2F5 KI mice, however, are purged of gp41- and KYNU-reactivity, but retain cardiolipin-binding, and sequence analysis revealed extensive light-chain editing. The anergic B cells in the post-tolerance compartment are enriched with self-reactivity to KYNU and maintain binding to HIV-1 gp41. Our results demonstrate that tolerance of the 2F5 epitope is driven by specific reactivity to KYNU, but not general cross-reactivity to cardiolipin. In addition, that the peripheral anergic B cells retain self-reactivity and binding to HIV-1 gp41 suggests a potential target for activation by immunizations.

Lastly, we sought to determine whether the host mimicry by 4E10 and 2F5 epitopes is also present in other HIV-1 epitopes, including additional conserved neutralizing epitopes and more importantly, non-neutralizing epitopes. We used protein microarrays to assess autoreactivity of HIV-1 broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (nnAbs) and found that as a class, bnAbs are more polyreactive and autoreactive than nnAbs. The poly- and autoreactive property is therefore not a result of chronic inflammation, but rather uniquely associated with neutralization, consistent with the role of heteroligation for HIV-1 neutralizing activity. In addition, mutation frequencies of bNAbs and nnAbs per se do not correlate with poly- and autoreactivity. Our results demonstrate that HIV-1 bnAbs are significantly more polyreactive and self-reactive than non-neutralizers, which may subject them to immunological tolerance control in vivo. Infrequent poly- or autoreactivity among nnAbs implies that their dominance in humoral responses is due to the absence of negative control by immune tolerance.

The results of this study indicate that mimicry of host antigens by HIV-1 is an effective mechanism to camouflage vulnerable neutralizing epitopes of HIV-1 and evade host immune responses. As a result, protective HIV-1 bnAbs are rare and often poly- or autoreactive, constituting a major hurdle that must be overcome to effectively elicit protective responses by an HIV-1 vaccine.

For effective adaptive immune responses, V(D)J recombination must produce diverse, clonally distributed B and T cell receptors (BCRs and TCRs) during lymphocyte development. Recombination of the genes encoding the TCR and TCR chains (Tcra and Tcrd) in developing T cells poses distinct regulatory challenges, as the two genes are encoded by a single locus (Tcra-Tcrd) and share an array of V segments. Tcrd undergoes a single round of V-D-J in CD4-CD8 double-negative (DN) thymocytes, while Tcra is assembled through multiple rounds of V-J rearrangement in the CD4-CD8 double-positive (DP) compartment. However, the mechanisms ensuring that diverse repertoires result from rearrangements at Tcra-Tcrd are poorly understood. Here, we demonstrate that the developmentally-dynamic chromatin architecture of Tcra-Tcrd ensures diverse gene segment usage during both Tcrd and Tcra rearrangement.

We began by seeking a better understanding of how the Tcra repertoire is assembled through several rounds of V-J rearrangement. To do this, we developed and applied a high-throughput sequencing (HTS)-based strategy to analyze the pre-selection Tcra repertoire in mice carrying wild-type and genetically modified Tcra-Tcrd alleles. We found that Tcra rearrangement in individual thymocytes is intrinsically highly processive and coordinated along the V and J arrays through multiple rounds of rearrangement, imposing severe constraints on combinatorial diversity. Repertoire diversity is nonetheless achieved by mechanisms that impart combinatorial diversity to primary V-J rearrangement, effectively scrambling the starting points for progressions of secondary rearrangements in individual DP thymocytes. Using 4C-seq and 3D DNA-FISH, we determined that the processive usage of V segments is supported by an extended V-V array architecture in DP thymocytes.

We next explored how assembly of the Tcrd repertoire is regulated. Using 4C-seq, we identified a stable chromatin loop mediated by CTCF binding that contains the D and J segments used by Tcrd. This loop was essential for diverse V usage, as disruption of one end by gene targeting biased Tcrd rearrangements toward D-J proximal V segments. We demonstrated that V usage during Tcrd rearrangement also has a substantial impact on the Tcra repertoire. V-D rearrangement in DN thymocytes variably truncates the V-V array on Tcra-Tcrd alleles that will later recombine Tcra in the DP compartment, scrambling the starting point for primary V-J rearrangement. We revealed that these variable truncations impart V diversity during primary V-J rearrangements and allow otherwise disfavored V-J rearrangements to occur. Such diversification is functionally significant, as Trav1-Traj33 mucosa-associated invariant T (MAIT) cells are depleted when Tcrd rearrangement is impaired or absent. Therefore, the regulation of Tcrd and Tcra recombination by Tcra-Tcrd chromatin architecture represents an integrated mechanism for boosting TCR diversity in both the  and  T cell lineages.

Natural IgM (nIgM) is constitutively present in the serum, where it aids in the early control of viral and bacterial expansions. nIgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. However, the cells that maintain high titers of nIgM in the circulation had not yet been identified. Several studies have linked serum nIgM with the presence of fetal-lineage B cells, and others have detected IgM secretion directly by B1a cells in various tissues. Nevertheless, a substantial contribution of undifferentiated B1 cells to nIgM titers is doubtful, as the ability to produce large quantities of antibody (Ab) is a function of the phenotype and morphology of differentiated plasma cells (PCs). No direct evidence exists to support the claim that a B1-cell population directly produces the bulk of circulating nIgM. The source of nIgM thus remained uncertain and unstudied.

In the first part of this study, I identified the primary source of nIgM. Using enzyme-linked immunosorbent spot (ELISPOT) assay, I determined that the majority of IgM Ab-secreting cells (ASCs) in naïve mice reside in the bone marrow (BM). Flow cytometric analysis of BM cells stained for intracellular IgM revealed that nIgM ASCs express IgM and the PC marker CD138 on their surface, but not the B1a cell marker CD5. By spinning these cells onto slides and staining them, following isolation by fluorescence-activated cell sorting (FACS), I found that they exhibit the typical morphological characteristics of terminally differentiated PCs. Transfer experiments demonstrated that BM nIgM PCs arise from a progenitor in the peritoneal cavity (PerC), but not isolated PerC B1a, B1b, or B2 cells. Immunoglobulin (Ig) gene sequence analysis and examination of B1-8i mice, which carry an Ig knockin that prohibits fetal B-cell development, indicated that nIgM PCs differentiate from fetal-lineage B cells. BrdU uptake experiments showed that the nIgM ASC compartment contains a substantial fraction of long-lived plasma cells (LLPCs). Finally, I demonstrated that nIgM PCs occupy a survival niche distinct from that used by IgG PCs.

In the second part of this dissertation, I characterized the unique survival niche of nIgM LLPCs, which maintain constitutive high titers of nIgM in the serum. By using genetically deficient or Ab-depleted mice, I found that neither T cells, type 2 innate lymphoid cells, nor mast cells, the three major hematopoietic producers of IL-5, were required for nIgM PC survival in the BM. However, IgM PCs associate strongly with IL-5-expressing BM stromal cells, which support their survival in vitro when stimulated. In vivo neutralization of IL-5 revealed that, like individual survival factors for IgG PCs, IL-5 is not the sole supporter of IgM PCs, but is likely one of several redundant molecules that together ensure uninterrupted signaling. Thus, the long-lived nIgM PC niche is not composed of hematopoietic sources of IL-5, but a stromal cell microenvironment that provides multiple redundant survival signals.

In the final part of my study, I identified and characterized the precursor of nIgM PCs, which I found in the first project to be resident in the PerC, but not a B1a, B1b, or B2 cell. By transferring PerC cells sorted based on expression of CD19, CD5, and CD11b, I found that only the CD19+CD5+CD11b- population contained cells capable of differentiating into nIgM PCs. Transfer of decreasing numbers of unfractionated PerC cells into Rag1 knockouts revealed an order-of-magnitude drop in the rate of serum IgM reconstitution between stochastically sampled pools of 106 and 3x105 PerC cells, suggesting that the CD19+CD5+CD11b- compartment comprises two cell types, and that interaction between the two necessary for nIgM-PC differentiation. By transferring neonatal liver, I determined that the early hematopoietic environment is required for nIgM PC precursors to develop. Using mice carrying a mutation that disturbs cKit expression, I also found that cKit appears to be required at a critical point near birth for the proper development of nIgM PC precursors.

The collective results of these studies demonstrate that nIgM is the product of BM-resident PCs, which differentiate from a PerC B cell precursor distinct from B1a cells, and survive long-term in a unique survival niche created by stromal cells. My work creates a new paradigm by which to understand nIgM, B1 cell, and PC biology.

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.

The chromatin architecture of antigen receptor loci has been hypothesized to facilitate the assembly of variable (V), diversity (D), and joining (J) gene segments during lymphocyte development. The 1.6 megabase Tcra/Tcrd locus is unique since it undergoes highly divergent Tcrd and Tcra recombination programs in CD4–CD8– double negative (DN) thymocytes and CD4+CD8+ double positive (DP) thymocytes, respectively. In this dissertation, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus by using three-dimensional fluorescence in situ hybridization (3D-FISH) and chromosome conformation capture (3C).

Using 3D-FISH, we found the 3' portion of the locus is contracted in both DN and DP thymocytes as compared to B cells. Remarkably, the 5' portion of the locus is contracted in DN thymocytes, but is decontracted in DP thymocytes. We propose that the fully contracted conformation in DN thymocytes allows Tcrd rearrangements involving Vα gene segments distributed over one megabase, whereas the unique 3'-contracted, 5'-decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3' of the available Vα gene segments. This would maintain a large pool of distal Vα gene segments for subsequent rounds of recombination.

To study the conformational changes at the molecular level, we used 3C to detect interactions between different sites spanning 400kb in the contracted 3' portion of the locus. The Tcra enhancer (Eα) is known to activate Vα and Jα segment promoters and to stimulate Vα-to-Jα recombination in DP thymocytes. We detected various pair-wise interactions between elements essential for initial Tcra recombination, including proximal Vα segments, TEA promoter, 5' Jα array and Eα. Notably, these interactions occur specifically in DP thymocytes and all are Eα-dependent. We proposed that in addition to regulating transcriptional activity, Eα promotes synapsis of RSSs by tethering proximal Vα and 5'Jα segments together to facilitate initial Tcra recombination.

We also asked whether a known chromatin organizer, CTCF, regulates the formation of the DP stage-specific, Eα-dependent chromatin hub. Using ChIP-seq, we identified CTCF binding sites at Eα, TEA promoter, and many Vα promoters in DN and DP thymocytes. Loss of CTCF in DP thymocytes resulted in impaired primary Vα-to-Jα recombination, reduced Vα and TEA germline transcription, and reduced interactions between Eα and Tcra genes. Strikingly, we also observed aberrantly increased Tcrd gene transcription and interactions between Eα and Tcrd gene segments in CTCF-deficient DP thymocytes. Our data suggest that CTCF helps Eα to organize a DP stage-specific chromatin hub that sets the stage for synapsis and recombination of proximal Vα and 5' Jα segments in DP thymocytes.

The adaptive immune system, comprised of B and T lymphocytes, responds with unique specificity to antigens. Specificity is imparted by the process of V(D)J recombination. During V(D)J recombination, gene segments spread across genomic space are assembled to generate unique antigen receptors by a cut-and-paste reaction in somatic cells. The mechanisms that regulate V(D)J recombination remain unclear.In T cells, two receptor classes are possible. These TCRs, the TCR and TCR, are assembled in part from genes sharing a single genetic locus. This single locus for both the T cell receptor and T cell receptor  (Tcra-Tcrd) chains has a unique nested structure that makes its regulation and recombination particularly interesting. In the Tcra-Tcrd locus, Vsegments are interspersed among the V segments. Rearrangement of Tcrd precedes Tcra recombination, and since these events are on the same locus, Tcrd recombination was hypothesized to diversify the starting point for primary Tcra recombinations, which are the first of a series of sequential recombination events possible for functional TCRchain generation. We generated a novel mouse model genetically modified to be incapable of Tcrd recombination. Using this model, we have determined that Tcrd recombination indeed diversifies the Tcra repertoire. Particularly, primary rearrangements are skewed toward more proximal V segments, while later rounds of rearrangement proceed as expected from the restricted primary V repertoire. We also investigated the specific impact of rearrangement to two relatively distal V segments of particular interest, Trav15d-1-dv6d-1 and Trav15-1-dv6-1, on the Tcra repertoire. To understand the significance of rearrangements to these V segments, we used two lines of mice lacking each of these segments. We found that primary Tcra recombinations utilizing V segments immediately upstream of each deleted gene segment were reduced. In the case of deletion of Trav15d-1-dv6d-1, the most distal V segment, we observed a reduction in primary recombinations using all upstream V segments. In the case of Trav15-1-dv6-1 deletion, primary recombinations upstream of this segment were reduced, but utilization of V segments 5′ of the most proximal upstream V segment was not disrupted. Unexpectedly, we also observed a reduction in secondary recombinations to V segments immediately upstream of either Trav15d-1-dv6-1 or Trav15-1-dv6-1 when either segment was deleted. These gene segments or their surrounding regions appear to be important in promoting diversity of the Tcra repertoire not only by their use as Tcrd recombination substrates, but also by a mechanism that remains to be explored. The structure of the Tcra-Tcrd locus also plays a role in recombination. Early in thymocyte development, the Tcra-Tcrd locus is contracted. During this period, Tcrd recombinations are capable of assembling segments distal in linear space. Later in development, when Tcra recombines, the locus is decontracted, and rearrangements generally proceed using the most proximal available segments. We and others have previously demonstrated that chromatin topology has a major impact on transcription and V(D)J recombination. Control of locus conformation is heavily influenced by contacts between CCCTC binding factor (CTCF)-bound elements. How CTCF regulates recombination at the Tcra-Tcrd locus is only partially understood. Herein, we used a novel mouse model lacking an intergenic CTCF binding element (CBE) in the Tcra-Tcrd locus (‘INT1’) to determine the impact of this specific CBE on Tcrd recombination. We find that loss of INT1 skews Tcrd rearrangements toward utilization of proximal V segments. These results suggest that INT1 serves as an important structural feature facilitating varied Tcrd recombination.

T lymphocytes of the adaptive immune system recognize antigens using T cell receptors, with each lymphocyte bearing a receptor of unique specificity. T cell receptors are generated through V(D)J recombination, a process in which gene segments are assembled for the generation of a functional receptor protein via somatic recombination.

The T cell receptor β (Tcrb) locus encodes the β chain of the T cell receptor αβ heterodimer. Among antigen receptor loci, the Tcrb locus is unique in that it frequently associates with the nuclear periphery during the developmental stage in which the locus undergoes recombination. Previous work showed that the association of the Tcrb locus with the nuclear periphery was a stochastic process and that the recombination of Tcrb alleles at the nuclear periphery was suppressed compared to Tcrb alleles in the interior of the nucleus. However, the mechanisms that instruct the frequent association of Tcrb alleles with the nuclear periphery remained unknown.

We characterized the association of the Tcrb locus with the nuclear lamina (NL) at high resolution using DamID. DamID analysis revealed that the association of the Tcrb locus with the NL was heterogeneous, and that a lamina-associated domain (LAD) border within the locus segregated the Tcrb recombination center (RC) from RC-proximal chromatin. This LAD border constrains the activity of the Tcrb enhancer (Eβ) to the RC. Accordingly, deletion of the LAD border caused the Eβ-dependent activation of RC-proximal chromatin, resulting in a loss of NL association, increased chromatin looping to the RC, and increased transcription and histone acetylation of genes within the affected 300 kb region. Activated gene segments underwent recombination at higher frequencies, causing a substantial alteration of the Tcrb repertoire. Therefore, our studies identified a LAD border in the Tcrb locus that served to limit the Eβ-dependent activation of RC-proximal gene segments in order to maintain the diversity of the Tcrb repertoire.

Diversity of T cell receptors (TCR) and immunoglobulins (Ig) is generated by V(D)J recombination of antigen receptor (AgR) loci. The Tcra-Tcrd locus is of particular interest because it displays a nested organization of Tcrd and Tcra gene segments and V(D)J recombination follows an intricate developmental program to assemble both TCRδ and TCRα repertoires. However, the mechanisms that dictate the developmental regulation of V(D)J recombination of the Tcra-Tcrd locus remain unclear.

We have previously shown that CCCTC-binding factor (CTCF) regulates Tcra gene transcription and rearrangement through organizing chromatin looping between CTCF- binding elements (CBEs). This study is one of many showing that CTCF functions as a chromatin organizer and transcriptional regulator genome-wide. However, detailed understanding of the impact of specific CBEs is needed to fully comprehend the biological function of CTCF and how CTCF influences the generation of the TCR repertoire during thymocyte development. Thus, we generated several mouse models with genetically modified CBEs to gain insight into the CTCF-dependent regulation of the Tcra-Tcrd locus. We revealed a CTCF-dependent chromatin interaction network at the Tcra-Tcrd locus in double-negative thymocytes. Disruption of a discrete chromatin loop encompassing Dδ, Jδ and Cδ gene segments allowed a single Vδ segment to frequently contact and rearrange to diversity and joining gene segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity. In addition, we showed that insertion of an ectopic CBE can modify chromatin interactions and disrupt the rearrangement of particular Vδ gene segments. Finally, we investigated the role of YY1 in early T cell development by conditionally deleting YY1 in developing thymocytes. We found that early ablation of YY1 caused severe developmental defects in the DN compartment due to a dramatic increase in DN thymocyte apoptosis. Furthermore, late ablation of YY1 resulted in increased apoptosis of DP thymocytes and a restricted TCRα repertoire. Mechanistically, we showed that p53 was upregulated in both DN and DP YY1-deficient thymocytes. Eliminating p53 in YY1-deficient thymocytes rescued the survival and developmental defects, indicating that these YY1-dependent defects were p53-mediated. We conclude that YY1 is required to maintain cell viability during thymocyte development by thwarting the accumulation of p53.

Overall, this thesis work has shown that CTCF-dependent looping provides a central framework for lineage- and developmental stage-specific regulation of Tcra-Tcrd gene expression and rearrangements. In addition, we identified YY1 as a novel regulator of thymocyte viability.

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.

As members of the adaptive immune response, T- and B- cells express unique antigen receptors generated from antigen receptor loci. These loci encode multiple Variable (V), Diversity (D), and Joining (J) gene segments. Through a process known as V(D)J recombination, genomic rearrangements occur to generate a unique antigen receptor proteins. During each stage of lymphocyte development, antigen receptor loci are epigenetically regulated. The epigenetic regulation promotes and inhibits V(D)J recombination through different mechanisms. To generate an antigen receptor protein, the substrates for rearrangement (recombination signal sequences, RSSs) must be made accessible to the recombination machinery. Moreover, once an antigen receptor locus has rearranged and produced a successful in-frame protein, a mechanism known as allelic exclusion prevents further recombination.

The nucleosome can positively and negatively regulate V(D)J recombination. Therefore, we defined the in vivo nucleosome organization of accessible and inaccessible RSSs on the Tcr loci. We used Tcrb and Tcra alleles which lack various cis-elements (e.g. enhancers and promoters) and terminate transcription. By comparing nucleosome organization and histone octamer occupancy, we found that accessible alleles are characterized by lower histone octamer occupancy and in some cases movement of nucleosomes. Also, we found that some these changes are mediated by transcription through the RSS. We concluded that one mechanism by which cis-elements epigenetically regulate RSS accessibility is by histone octamer loss and nucleosome repositioning and that some of these changes are mediated by transcription.

In addition, we further investigated how allelic exclusion prevents Tcrb locus recombination in CD4, CD8 double positive (DP) thymocytes. A previous study had introduced the Tcra enhancer (Eα) into the middle of the Tcrb locus to test if allelic exclusion was mediated solely by RSS accessibility. That study found that Eα could force RSS accessibility in DP thymocytes, but Vβ RSS accessibility did not overcome additional mechanisms involved in allelic exclusion. One potential mechanism that has been suggested in the literature is changes in locus conformation. Thus, we tested if RSS accessibility and locus conformation together mediate allelic exclusion. We generated two alleles that overcome changes in RSS accessibility, due to the presence of Eα and that overcome changes in locus conformation, due to a decrease in distance between Vβ and DJβ RSSs. We found that both alleles are accessible in DP thymocytes and we detected Vβ to DJβ recombination in DP thymocytes. Therefore, the epigenetic mechanisms that regulate Tcrb allelic exclusion consists of changes in RSS accessibility and changes in locus conformation.

Recombination of Tcrb gene segments in DN thymocytes is subject to allelic exclusion, such that only a single functional V_β - DJ_β rearrangement is generated per T cell. For Tcrb to be allelically excluded the two alleles must initiate recombination asynchronously and once a β-protein is selected, feedback signals must suppress further recombination. Earlier studies of antigen-receptor loci implicated directed monoallelic association with pericentromeric heterochromatin in the initiation or maintenance of allelic exclusion. In this study we used three-dimensional fluorescent in situ hybridization to directly visualize the nuclear localization of Tcra and Tcrb, pericentromeric heterochromatin, and the nuclear lamina. Here we provide evidence for a fundamentally different basis for Tcrb allelic exclusion. We demonstrate that Tcrb is highly associated with pericentromeric heterochromatin and the nuclear lamina in pro-B cells and in DN and DP thymocytes. We also find that Tcrb does not associate with peri-centromeric heterochromatin and the nuclear lamina in a strict monoallelic fashion. Rather, Tcrb alleles independently associate with the two compartments, leading to a stochastic distribution of nuclei containing both, one, or neither allele associated. In the subset of DN thymocyte nuclei with monoallelically associated Tcrb alleles, the non-rearranged allele is most often associated with repressive compartments. This suggests that association with these compartments inhibits recombination prior to β-selection. This inhibition occurs without altering the conformation of the locus. Moreover, the introduction of an ectopic enhancer into Tcrb, led to both a repositioning of Tcrb away from these repressive compartments. This repositioning was correlated with an increase in the frequency of recombination and a break in allelic exclusion. These data lead us to propose that stochastic rather than directed interactions of Tcrb alleles with repressive nuclear compartments bias initial Tcrb recombination to be monoallelic in developing thymocytes and that such interactions are essential for Tcrb allelic exclusion.

T cells and B cells each express unique antigen receptors used to identify, eliminate, and remember pathogens. These receptors are generated through a process known as V(D)J recombination, in which T cell receptor and B cell receptor gene loci undergo genomic recombination. Interestingly, recombination at certain genes is regulated so that a single in-frame rearrangement is present on only one allele per cell. This phenomenon, termed allelic exclusion, requires two steps. First, recombination can occur only on one allele at a time. In the second step, additional recombination must be prevented. Though the mechanism of the second step is well-understood, the first step remains poorly understood.

The first step of recombination necessitates that alleles rearrange one at a time. This could be achieved either through inefficient recombination or by halting further recombination in the presence of recombination. To separate these mechanisms, we analyzed recombination in nuclei unable to complete recombination. We found that rearrangement events accumulated at antigen receptor loci, suggesting that the presence of recombination does not stop additional rearrangements and asynchronous recombination likely results from inefficient recombination at both alleles.

Association with repressive subnuclear compartments has been proposed to reduce the recombination efficiency of allelically excluded antigen receptor loci. Of the alleleically excluded loci, Tcrb alleles are uniquely regulated during development. Other allelically excluded alleles are positioned at the transcriptionally-repressive nuclear periphery prior to recombination, and relocate to the nuclear interior at the stage in which they recombine. However Tcrb alleles remain highly associated with the nuclear periphery during rearrangement. Here we provide evidence that this peripheral subnuclear positioning of Tcrb alleles does suppress recombination. We go on to suggest that peripheral localization mediates the first step of allelic exclusion.

In search of the mechanism by which recombination is suppressed on peripheral Tcrb alleles, we investigated the subnuclear localization of a recombinase protein. Two recombinase proteins are required for recombination, one of which is recruited to actively transcribing (and more centrally located) DNA. Here we demonstrate that one recombinase protein is unable to localize to peripheral Tcrb alleles, potentially serving as the mechanism by which recombination is suppressed on peripheral alleles.

Throughout their development in the thymus and during their maintenance and the immunological response in the periphery, T cells rely on the regulation of classical apoptotic pathways to promote cell survival or death. Several proteins of the Bcl-2 family have been shown to be critical in thymocyte and T cell survival and consequently, in T cell function. Among these proteins, the antiapoptotic proteins Bcl-2 and Mcl-1 are critical for promoting T cell survival at multiple stages of the T cell "life cycle." While these proteins have been reported to interact with several of the proapoptotic members of the Bcl-2 family, the specific interactions by which Mcl-1 in particular promotes T cell survival in vivo were not well understood. Further, how different stimuli (for example, cytokine signaling and T cell activation) modulate the specific functions of Mcl-1 had also not been thoroughly explored.

We utilized mouse models to dissect the roles of Mcl-1 at multiple stages of T cell development and function. We utilized conditional knockout and double knockout strategies to build genetic pathways for Mcl-1 activity during thymocyte development and in peripheral T cells under a variety of conditions. In the thymus, the major role of Mcl-1 is to inhibit the activity of proapoptotic Bak because the loss of Bak, but not the loss of Bax or Bim, rescued the survival of Mcl-1-deficient thymocytes at both the double negative and single positive stages. Further, we concluded that this role is not shared with Bcl-2 because overexpression of Bcl-2 did not rescue DN or SP survival.

In peripheral T cells, the loss of Bak rescued T cell survival in the presence of IL 7, but not during conditions of cytokine withdrawal. Interestingly, the overexpression of Bcl-2 or the loss of Bim partially rescued the survival of T cells during cytokine withdrawal, indicating that Mcl-1 has dual roles in T cells: cytokine-dependent and cytokine independent. Additionally, we found that cytokines of the common gamma chain family have different effects on the activity of Mcl-1 due to the differential regulation of other proteins of the Bcl-2 family, most notably Bim.

Finally, we utilized a Bcl-2 reporter mouse model to examine the role of Bcl-2 in the establishment of CD8+ T cell memory to infection. Although it is known that Bcl-2 is dynamically regulated in response to activation, the importance of this regulation in the establishment of T cell memory is not yet clear. We show that a subset of effector T cells within a previously defined memory precursor population retained high Bcl-2 expression at the peak of the immune response. Using adoptive transfer of sorted effector T cells, we provide preliminary evidence that the cells with memory potential lie within a strict range of Bcl-2 expression. These studies indicate that the regulation of Bcl 2 is likely critical in establishing T cell memory and provide a platform for the future study of the factors that influence T cell memory.