Antigen Drives Regulatory B10 Cell Development and Function

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

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

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

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

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

Antineoplastic Cytotoxicity and Immune Adjuvancy of a Recombinant Oncolytic Poliovirus

Our group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.

Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.

IL-10-producing Regulatory B Cell Development in Human Autoimmune Disease

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.

Morphing low-affinity ligands into high-avidity nanoparticles by thermally triggered self-assembly of a genetically encoded polymer.

Multivalency is the increase in avidity resulting from the simultaneous interaction of multiple ligands with multiple receptors. This phenomenon, seen in antibody-antigen and virus-cell membrane interactions, is useful in designing bioinspired materials for targeted delivery of drugs or imaging agents. While increased avidity offered by multivalent targeting is attractive, it can also promote nonspecific receptor interaction in nontarget tissues, reducing the effectiveness of multivalent targeting. Here, we present a thermal targeting strategy--dynamic affinity modulation (DAM)--using elastin-like polypeptide diblock copolymers (ELP(BC)s) that self-assemble from a low-affinity to high-avidity state by a tunable thermal "switch", thereby restricting activity to the desired site of action. We used an in vitro cell binding assay to investigate the effect of the thermally triggered self-assembly of these ELP(BC)s on their receptor-mediated binding and cellular uptake. The data presented herein show that (1) ligand presentation does not disrupt ELP(BC) self-assembly; (2) both multivalent ligand presentation and upregulated receptor expression are needed for receptor-mediated interaction; (3) increased size of the hydrophobic segment of the block copolymer promotes multivalent interaction with membrane receptors, potentially due to changes in the nanoscale architecture of the micelle; and (4) nanoscale presentation of the ligand is important, as presentation of the ligand by micrometer-sized aggregates of an ELP showed a low level of binding/uptake by receptor-positive cells compared to its presentation on the corona of a micelle. These data validate the concept of thermally triggered DAM and provide rational design parameters for future applications of this technology for targeted drug delivery.

Visual memory-deficit amnesia: a distinct amnesic presentation and etiology.

We describe a form of amnesia, which we have called visual memory-deficit amnesia, that is caused by damage to areas of the visual system that store visual information. Because it is caused by a deficit in access to stored visual material and not by an impaired ability to encode or retrieve new material, it has the otherwise infrequent properties of a more severe retrograde than anterograde amnesia with no temporal gradient in the retrograde amnesia. Of the 11 cases of long-term visual memory loss found in the literature, all had amnesia extending beyond a loss of visual memory, often including a near total loss of pretraumatic episodic memory. Of the 6 cases in which both the severity of retrograde and anterograde amnesia and the temporal gradient of the retrograde amnesia were noted, 4 had a more severe retrograde amnesia with no temporal gradient and 2 had a less severe retrograde amnesia with a temporal gradient.

Impact of Leukocyte Function-Associated Antigen-1 Blockade on Endogenous Allospecific T Cells to Multiple Minor Histocompatibility Antigen Mismatched Cardiac Allograft.

BACKGROUND: Blocking leukocyte function-associated antigen (LFA)-1 in organ transplant recipients prolongs allograft survival. However, the precise mechanisms underlying the therapeutic potential of LFA-1 blockade in preventing chronic rejection are not fully elucidated. Cardiac allograft vasculopathy (CAV) is the preeminent cause of late cardiac allograft failure characterized histologically by concentric intimal hyperplasia. METHODS: Anti-LFA-1 monoclonal antibody was used in a multiple minor antigen-mismatched, BALB.B (H-2B) to C57BL/6 (H-2B), cardiac allograft model. Endogenous donor-specific CD8 T cells were tracked down using major histocompatibility complex multimers against the immunodominant H4, H7, H13, H28, and H60 minor Ags. RESULTS: The LFA-1 blockade prevented acute rejection and preserved palpable beating quality with reduced CD8 T-cell graft infiltration. Interestingly, less CD8 T cell infiltration was secondary to reduction of T-cell expansion rather than less trafficking. The LFA-1 blockade significantly suppressed the clonal expansion of minor histocompatibility antigen-specific CD8 T cells during the expansion and contraction phase. The CAV development was evaluated with morphometric analysis at postoperation day 100. The LFA-1 blockade profoundly attenuated neointimal hyperplasia (61.6 vs 23.8%; P < 0.05), CAV-affected vessel number (55.3 vs 15.9%; P < 0.05), and myocardial fibrosis (grade 3.29 vs 1.8; P < 0.05). Finally, short-term LFA-1 blockade promoted long-term donor-specific regulation, which resulted in attenuated transplant arteriosclerosis. CONCLUSIONS: Taken together, LFA-1 blockade inhibits initial endogenous alloreactive T-cell expansion and induces more regulation. Such a mechanism supports a pulse tolerance induction strategy with anti-LFA-1 rather than long-term treatment.