• Characterization of Plasma Cell Lineages in the Nurse Shark

      Castro, Caitlin Doremus; Flajnik, M. F. (Martin F.) (2013)
      IgM was the first immunoglobulin (Ig) isotype to arise in evolution. Cartilaginous fish, including sharks, skates, and rays, are the oldest animals with an Ig-based adaptive immune system. Sharks express a germline-joined isotype, IgM<sub>1gj<sub>, as well as two forms of IgM, the typical pentameric (19S) form, which is attached to J chain, and a monomeric (7S) form devoid of J chain. Preliminary evidence suggests that 19S IgM consists of low affinity antibodies, whereas the 7S monomers can represent high affinity antibodies that function similar to mammalian IgG. Germline-joined IgM<sub>1gj<sub>, and some 19S IgM are secreted early in ontogeny, while adult sharks produce 19S and 7S IgM. The developmental relationship among the B-cells that produce these different isotypes is unknown. In one model, 19S producers "switch" to secrete 7S IgM by silencing J chain expression after activation by antigen and T cells. In another model, there may be two or more completely separate lineages that give rise to early 19S, late 19S and 7S IgM-secreting B-cells. In order to study the developmental relationship between 19S and 7S IgM-producing B cells in the nurse shark, we examined differences in transcription factor expression, Ig gene usage, heavy/light chain pairings, and sequence signatures from neonatal and adult IgM. In these studies we found a population of Blimp1-negative 19S-secreting cells in neonatal and adult nurse sharks, and unique pairing of a particular IgM H with a σ' L chain in neonatal sharks. In addition to 19S IgM, neonatal nurse sharks express the germline-joined isotype, IgM<sub>1gj<sub>. Therefore, we also examined L chain pairings in IgM<sub>1gj<sub>-secreting cells. Our findings suggest that a κ IgL pairs with IgM<sub>1gj<sub>, and that IgM<sub>1gj<sub> binds to self-molecules in neonatal gill and other tissue. Lastly, we attempted to devise a protocol for the separation of 19S- from 7S-secreting cells in order to examine the IgM repertoire in these populations. These studies provide greater insight into the function of early/innate antibodies in sharks, and we predict that they will also be relevant to mammalian models of early antibody secretion.
    • Differential FOXO1 Localization in SLE and Healthy Human Lymphocyte Subsets

      Hritzo, Molly K.; Golding, Amit; 0000-0001-9614-732X (2018)
      Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by elevated levels of circulating autoantibodies and multi-organ damage. Although SLE is a highly heterogeneous disease, one factor unifies it: lymphocyte hyperactivity driving immunopathogenesis. This involves CD4 helper T cells potentiating autoreactive B cells to produce pathogenic autoantibodies. In healthy individuals, lymphocyte activation is a closely regulated kinetic process controlled by key transcription factors (TF) signaling downstream of the T cell (TCR) and B cell receptor (BCR). Forkhead box O1 (FOXO1) is one such TF that integrates activation and differentiation signals in human lymphocytes. When active, it remains in the nucleus, but upon Akt phosphorylation downstream of TCR or BCR signaling, FOXO1 is inactivated and shuttles to the cytoplasm, linking FOXO1 localization to function. In SLE, both T and B cells are hyperactive, and respond more quickly and strongly to antigen, producing a disproportionate inflammatory response. Thus, we hypothesize that SLE lymphocytes will have altered FOXO1 localization, reflecting altered lymphocyte activation. To address this hypothesis, we first developed a method of examining dynamic native FOXO1 localization in human peripheral lymphocyte subsets using imaging flow cytometry (IFC). IFC combines the quantitative power of flow cytometry with the qualitative images of microscopy and can be performed with many fewer cells than are needed for the more traditional methods. We demonstrated that we can visualize native FOXO1 and detect significant kinetic differences in localization within user-defined subsets of HuT102 cells, a human CD4 T cell line with baseline nuclear FOXO1, as well as primary peripheral human T and B cells. We then used IFC to compare FOXO1 localization in SLE and healthy donor lymphocytes. Interestingly, we found that most T and B cell subsets have nuclear FOXO1 localization in both health and SLE. However, FOXO1 is significantly more cytoplasmic in SLE double negative (DN) atypical memory B cells. Based on our findings, we propose a model by which these DN B cells are highly active in disease flares and may serve as a death-resistant reservoir of autoreactive cells. Future experiments will be aimed at elucidating at how these cells persist in the periphery.
    • Direct Suppression by Healthy and SLE Human Tregs of B cell Immunoglobulin Secretion

      Weingartner, Elizabeth; Golding, Amit (2017)
      Regulatory T cells (Tregs) are characterized by their role in maintaining peripheral self-tolerance and immune homeostasis. One such role is regulation of the humoral response, which is most clearly demonstrated by abundant auto-antibody production in Scurfin mice and IPEX patients, both of which lack functional Tregs. Indirect Treg regulation of the humoral response via their influence on helper T cells, in particular in germinal centers, is well established. Prior demonstration of direct Treg inhibition of B cells has also been demonstrated, primarily in mice, but is mostly attributable to Treg killing of B cells. This regulation may be especially relevant in B cell-mediated diseases, such as systemic lupus erythematosus (SLE). SLE is an extremely heterogeneous, autoimmune disease characterized in part by high titers of autoantibodies. We hypothesize SLE Tregs are deficient in their ability to suppress immunoglobulin secretion by B cells relative to Tregs isolated from healthy donors. We addressed this hypothesis by first developing and characterizing a modified suppression assay in which the only variable is the source of Tregs. In this assay, suppression by human Tregs of a homogenous, germinal center like B cell cell line, Ramos, is measured by ELISA for IgM and flow cytometry for cell death. We demonstrate that human Tregs directly suppress IgM by Ramos B cells through a partially contact- and death-independent mechanism. In addition, we demonstrate that pre-stimulation of the Tregs with αCD3/CD28 increases the suppression of IgM secretion but again, is independent of inducing B cell death. After development and characterization of this modified suppression assay, we evaluated the behavior of Tregs isolated from SLE patients in the assay. Interestingly, we found that the average suppression of IgM secretion by SLE Tregs is not statistically different from the average suppression by Tregs isolated from healthy donors. However, we found that SLE Tregs induce death in the Ramos B cells unlike Tregs isolated from healthy donors. Based on our findings, we offer a model by which Treg-induced B cell death might actually promote more SLE disease by further release of auto-antigens. Future experiments will be aimed at elucidating the mechanism and type of cell death.