• Analysis of in vivo expression and function of Src-family protein tyrosine kinases during lymphocyte development

      Longo, Nancy S.; Abraham, Kristin M. (1999)
      Src-family protein tyrosine kinases (PTKs) are critical components of T and B lymphocyte signaling cascades. To examine the relationship between Src-PTK expression and lymphocyte development, we defined quantitative and qualitative expression patterns of Src-PTKs during lymphocyte ontogeny. A quantitative RT-PCR assay was designed to distinguish transcripts derived from the lck proximal (Type I) vs distal (Type II) promoter elements, and fynT vs fynB isoforms. These assays were then used to measure lck and fyn expression in normal fetal and adult tissue. Changes in Type I and Type II promoter utilization correlate with pre-TCR and alphabetaTCR expression suggesting that as receptor complexes develop they couple to different intracellular signaling cascades that differentially regulate lck expression. The fynT isoform, not fynB, is detected in both lineages, including pre-T and pre-B cells, suggesting a role for Fyn in lymphocyte development before TCRalphabeta or IgM expression. In spite of much lower levels of fynT and lck transcripts in the B cell lineage, compared to lck transcripts in thymocytes; accumulation of lck and fynT correlates temporally with acquisition of immune receptor expression in both lineages. In addition to immune receptors, Src family PTKs are activated by receptors regulating growth and differentiation in thymic stroma which is required for lymphocyte development. To examine the impact of Src-PTK function in thymic stroma on lymphocyte development, a transgenic model system was developed in which the expression of polyoma virus middle T antigen (PymT), a molecule that activates Src-PTKs, was directed to thymic subcapsular epithelial cells using a mutant Class II promoter element. PymT expression constitutively activated endogenous Src-PTKs which phosphorylated PymT, inducing a PymT association with p85, the regulatory subunit of PI 3-kinase. Src-PTK activation was associated with changes in tyrosine phosphorylation of cellular proteins. Transgene expression in subcapsular stroma resulted in upregulation of epidermal growth factor (EGF) receptor and 4 to 5-fold thymic lymphoid hyperplasia. However, normal thymic architecture, proliferation and apoptotic responses, thymocyte differentiation, repertoire selection, and thymic involution were maintained. These findings suggest that the level and/or persistence of signals generated via Src-PTKs in thymic subcapsular epithelium serve as major defining factors influencing homeostatic control of thymic organ size and cellularity.
    • Applying MyD88 signaling in CD8+ T cells to enhance anti-tumor immunity

      Kaczanowska, Sabina; Davila, Eduardo, Ph.D.; 0000-0003-0191-6371 (2016)
      T cell immunotherapy is a promising strategy for the treatment of patients with advanced cancers. Despite promising results in patients with hematological malignancies, response rates to solid tumors remain low. One of the biggest challenges is effectively activating and sustaining anti-tumor T cell responses in the context of an immunosuppressive tumor environment. Several approaches are currently being explored to improve the efficacy of adoptive T cell transfer, including the activation of co-stimulatory signaling pathways in T cells. Toll-like receptor (TLR) engagement on T cells is a potent co-stimulatory signal that increases anti-tumor activity by enhancing T cell proliferation, effector function, and T cell survival. We developed and tested two genetic engineering strategies to exploit the co-stimulatory effects of TLR signaling in CD8+ T cells. First, CD8+ T cells were modified to express and secrete the TLR5 ligand (TLR5L), flagellin, as a means to deliver this immune adjuvant to the tumor for enhanced anti-tumor activity. TLR5L-secreting T cells exhibited improved proliferation, cytokine secretion, and anti-tumor activity in both xenogeneic and syngeneic models of melanoma. The anti-tumor activity of TLR5L-secreting T cells was associated with decreased numbers of phenotypically exhausted T cells and fewer myeloid-derived suppressor cells. Second, we designed and characterized a synthetic fusion protein composed of the T cell co-receptor CD8α and the TLR adaptor protein MyD88, termed CD8α:MyD88. The expression of CD8α:MyD88 on T cells increased T cell responses to low concentrations of tumor antigens as well as augmented the expression of effector molecules and co-stimulatory proteins. These effects were antigen-dependent and accompanied by elevated levels of TLR signaling-related proteins. The enhanced anti-tumor activity of CD8α:MyD88-expressing T cells in tumor-bearing mice was associated with a unique tumor cytokine/chemokine signature, improved T cell infiltration, elevated levels of antigen presentation, and fewer macrophages with an immunosuppressive phenotype. The use of T cells as vehicle to deliver TLR5L to the tumor site and the co-stimulation of T cells through a synthetic CD8α:MyD88 receptor represent two novel and versatile approaches for modulating the tumor microenvironment to enhance anti-tumor immunity.
    • 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.
    • Dissecting the protective memory CD4 T cell response to influenza virus infection

      Teijaro, John; Farber, Donna L. (2009)
      An important distinguishing feature of the adaptive immune response is the de-velopment of immunologic memory, which provides protective immunity against repeat pathogen encounters. T lymphocytes, including the CD4 and CD8 T cell subsets, are both known to persist as memory. Despite increasing knowledge of the mechanisms un-derlying T cell activation and function, the generation, maintenance and protective effi-cacy of T cell memory remains poorly defined. At present, there are no existing vaccine strategies to generate memory T cell mediated protective immunity. Furthermore, the qualities that define a protective memory T cell response are incompletely understood. Our laboratory has identified heterogeneous populations of influenza hemaggluti-nin (HA) specific memory CD4 T cells, with respect to phenotype, function, and migra-tion to both secondary lymphoid and peripheral tissues. However, how these diverse memory CD4 T cell subsets mediate protection to influenza virus infection remains un-explored. In order to investigate protection mediated my memory CD4 T cells, we established complementary models using TCR-transgenic and polyclonal influenza-specific T cells. Herein we deomonstrate that the presence of memory CD4 T cells in BALB/c mice resulted in enhanced viral clearance in the context of slightly increased morbidity as measured by weight loss and physical appearance. In addition, reduction in viral loads by memory CD4 T cells was not affected in the absence of B cells or CD8 T cells, and ap-peared to be IFN-γ dependent. In addition, we demonstrate that lung-derived memory preferentially migrate to and are retained within the lung tissue and facilitate rapid clear-ance of virus compared to spleen-derived memory CD4 T cells, which provided no bene-ficial protection compared to naive controls. Moreover, viral clearance in mice receiving lung memory CD4 T cells occurred in the context of reduced weight loss and physical morbidity. Finally, we demonstrate that the physiological outcome of a memory CD4 T cell-mediated secondary response to influenza can be significantly improved by targeting the CD28 pathway with the costimulation modulator CTLA4Ig. While CTLA4Ig is sup-pressive for primary immune responses to influenza, leading to increased viral loads, re-duced lung function and increased morbidity. In contrast, CTLA4Ig treatment of mem-ory CD4 T cell-mediated secondary responses to influenza is remarkably curative, result-ing in less morbidity and immunopathology, improved lung function, and enhanced re-covery. The optimized secondary response induced by CTLA4Ig treatment is character-ized by reduced lymphoid memory responses and recruitment to the lung, with intact in situ lung memory T cell responses and effector function. Our results reveal the dual na-ture of memory T-cell-mediated secondary responses and identify protective subsets, and suggest costimulation modulation as a novel strategy to optimize anti-viral immunity by limiting the memory T-cell response to its protective capacities.
    • Isolation and characterization of activation-induced T-cell membrane domains

      Parsey, Merdad Vaziri; Lewis, George K., Ph.D. (1992)
      T cells interacting with antigen presenting cells (APCs) form polarized adhesion zones between the cells. T cell surface molecules involved in both adhesion and signaling, including the T cell antigen receptor/CD3 complex (CD3/Ti) and cytoskeletal elements, redistribute to the region of contact. However, little is known about the structure and composition of the membrane at this site. Using immobilized anti-CD3 antibodies to mimic T cell-APC interactions and stimulate T cells, we have characterized the site of T cell attachment. Two sets of studies were carried out. First, we found that several anti-CD3 antibodies induced characteristic changes in attachment site shape, microfilament morphology, and the apparent molecular weight of p56{dollar}\sp{lcub}lck{rcub}{dollar}, a protein tyrosine kinase (PTK), during the first 30 minutes of activation. After 12-96 hours, anti-CD3 stimulated cells exhibited growth arrest. These changes were not elicited by immobilized anti-CD7, -CD11a or -CD71 antibodies, and were inhibited by the microfilament disrupting agent, cytochalasin D. Second, the attachment site was isolated using the detergent saponin, and shearing. The substrate attached material (SAM) represented 4.8 {dollar}\pm{dollar} 0.8% of the total cell protein and consisted of a patch of membrane, oriented with its cytoplasmic surface exposed to the medium. Biochemical analysis of SAM revealed the CD3/Ti complex only when stimulatory anti-CD3 (CD3-SAM) antibodies were used. This association was dependent on time, temperature, and antibody specificity, arguing for its selectivity. CD3-SAM also showed rapid and sustained enrichment in p56{dollar}\sp{lcub}lck{rcub}{dollar} and several phosphotyrosinylated proteins. Non-stimulatory antibodies failed to induce localization of these molecules to the SAM. Stimulation of cells with PMA and ionomycin prior to attachment with non-stimulatory antibodies did induce the localization of p56{dollar}\sp{lcub}lck{rcub}{dollar}, but not of any other PTK substrates or the CD3/Ti complex, to the SAM. These are the first studies showing the selective association of tyrosine phosphorylated proteins to a region of the membrane associated with CD3/Ti mediated signaling. Thus, T cell activation involves the active reorganization of the cytoskeleton, the CD3/Ti complex and specific tyrosine phosphorylated proteins to the site of signaling.
    • Monomeric IL-12p40 binds partner proteins to modulate immune cell function

      Gerber, Allison; Singh, Nevil; 0000-0001-9442-3255 (2021)
      Cytokines are critical mediators used by immune cells to communicate as well as protect. The IL-12 family of cytokines are made up of  and  subunits typically assembled within one cell and secreted as a heterodimer. IL-12p40 is the shared β-subunit for both IL-12 (paring with IL-12p35) and IL-23 (with IL-23p19). However, the IL-12p40 monomer is often secreted in excess during infections, but its biological role was not known. In this thesis we investigated the function of secreted IL-12p40 monomer in vivo with the central hypothesis that the monomer combines with multiple α-subunits in vivo to generate IL-12 as well as other heterodimeric cytokines. Consistent with this hypothesis, in chimeric mice containing mixtures of cells that can only express either IL-12p40 or IL-12p35, but not both together, we found that functionally active IL-12 was generated. This alternate two-cell pathway requires IL-12p40 from hematopoietic cells to extracellularly associate with IL-12p35 from radiation-resistant cells. The two-cell mechanism was sufficient to influence local T cell differentiation in sites distal to the initial infection and helped control systemic dissemination of a pathogen although not parasite burden at the site of infection. Broadly, this suggests that early secretion of IL-12p40 monomers by sentinel cells at the infection site may help prepare distal host tissues for potential pathogen arrival. In addition to this role in generating IL-12 through two-cell assembly, we found that IL-12p40 has a novel partner protein, CD5L. This novel heterodimer was present in the serum of uninfected mice, with differences in the basal levels between B6 and Balb/c animals, with Balb/c having higher amounts of p40-CD5L. Functionally, we found that treatment with p40-CD5L leads to IL-4 and IL-10 production by T cells. Taken together, this thesis offers at least two major fundamental advances in cytokine biology – one the concept of a two-cell assembled cytokine and second the identity of a novel TH2-promoting heterodimeric cytokine. The first has significance in immunotherapy and understanding immunity to tissue-specific modulation of immune responses. The second is expected to drive significant research on allergy, responses to parasites and immune deviation.