• Alkylphosphate and tumor cell recognition by circulating Vgamma2Vdelta2 T cells

      Hebbeler, Andrew Michael; Pauza, C. David (2006)
      The antigen receptor on T cells is a heterodimer consisting of either alpha and beta (alphabeta) or gamma and delta (gammadelta) chains. Mechanisms that control repertoire selection in the alphabeta subset are well known, involving positive and negative selection in the thymus and subsequent regulation by peripheral tolerance. Mechanisms controlling gammadelta T cell repertoire selection are poorly understood, partly because antigen recognition by this receptor is MHC-unrestricted and partly due to our inability to define receptor-binding antigens for the major Vgamma2Vdelta2 subset. Clues to antigen recognition come from the pattern of repertoire selection, including the dominant appearance of the Vgamma2Vdelta2 T cell receptor (TCR) and even more specifically, a strong bias for Vgamma2 chains encoding the Jgamma1.2 segment. In healthy human adults, the majority of circulating gammadelta T cells express the Vgamma2-Jgamma1.2Vdelta2 TCR and respond to stimulation with a variety of phosphoantigens as well as some tumor cells. Analysis of the Vgamma2 chain repertoire during active responses to tumors or phosphoantigens demonstrates a remarkable similarity among individual T cell clones that proliferate in response to either stimulus. Vgamma2 chains responding only to phosphoantigens or tumor cells were possible but infrequent, suggesting that the Vgamma2 repertoire evolved to recognize ligands present on tumor cells or elicited following phosphoantigen treatment. Surprisingly, the repertoire of Vgamma2-Jgamma1.2Vdelta2 T cells that exists in healthy adults is stable over intervals as long as seven years, showing that a population of T cells reactive to self molecules including phosphoantigens, resists negative selection mechanisms of the type controlling MHC-restricted alphabeta T cells and evades peripheral tolerance. Repertoire stability is altered abruptly in the face of HIV infection, wherein we noted a dramatic and specific depletion of the Vgamma2-Jgamma1.2 subset. The loss of Vgamma2-Jgamma1.2Vdelta2 T cells during HIV infection is correlated with an increased susceptibility to tumors and opportunistic pathogens that are characteristic of advanced HIV disease, when the gammadelta T cell loss is most extreme. These studies describe an unconventional T cell subset with an acquired TCR repertoire generated in the absence of MHC restriction that has the capacity for responses to a broad array of pathogens and tumors.
    • HIV envelope glycoprotein-Fc fusion proteins target Fc gamma receptors and elicit effector antibody responses in rhesus macaques

      Shubin, Zhanna; Pauza, C. David (2016)
      A goal for HIV prevention programs is to develop safe and effective vaccines that elicit durable and broadly protective antibodies against both sexual and blood-borne HIV transmission. Many vaccine programs focus on the immune responses to critical epitopes in the HIV envelope glycoprotein (Env) and seek to improve the quality and quantity of antibodies by altering the conformation, oligomerization or glycosylation of Env gp120. As a complementary strategy, we envisioned that attaching an Fc moiety from IgG to immunogens would allow binding to neonatal Fc receptor (FcRn) for FcRn-mediated mucosal delivery. We reasoned that Fc fusion protein immunogens may also mimic immune complexes by binding Fc gamma receptors (Fc?R) on immune cells and would increase the potency of antibody responses. We developed Fc fusion immunogens consisting of either HIVBaL gp120 (Env-Fc) or critical epitopes in the V1V2 region of Env expressed within a carrier protein that provides highly multivalent epitope display (Gag-V1V2-Fc). Fc fusion proteins were attached at their carboxyl terminus to a Gly/Ser linker that is in turn fused to each half of the dimeric Fc domain from rhesus macaque IgG1 (Env-Fc). Env-Fc retained a capacity to bind both cell surface CD4 and Fc?Rs, which led to protein internalization and accumulation in cytoplasmic vesicles. In a rhesus macaque immunization study, Env-Fc was more potent compared to Env (gp120 monomer) in several ways. Env-Fc elicited higher gp120 binding antibody titers with increased breadth, including the capacity for recognizing CD4-induced epitopes, neutralizing activity against Tier 1A HIV pseudotyped viruses, and antibodies mediating antibody-dependent cellular cytotoxicity (ADCC). Serum antibodies produced in Env-Fc immunized macaques had increased durability compared to Env monomer immunization. A Gag-V1V2-Fc fusion protein was constructed to test whether a self-assembling macromolecular structure was a more effective method for directing antibody responses to specific V1V2 regions of the Envelope glycoprotein. This immunogen crossed nasal epithelium barriers more efficiently than HIV Gag (p24 monomer), and elicited V1V2-specific IgG responses. Our work suggests that adding IgG1 Fc to engineered monomeric or oligomeric Env-based immunogens may improve the protective serum antibody response and contribute to the development of a protective HIV vaccine.
    • Interleukin-18 activates Vg9Vd2+ T cells from HIV+ Individuals: Recovering the Response to Phosphoantigen

      Murday, Alanna; Pauza, C. David (2017)
      Gammadelta (gd) T cells, particularly the peripheral blood subset Vg9Vd2, are unconventional lymphocytes that act as strong cytotoxic effectors against both malignant and virus-infected cells. Unlike conventional alpha beta T cells, gd T cells recognize non-peptidic antigens without classical MHC presentation. Most human Vg9Vd2 T cells are stimulated by phosphoantigens related to isopentenyl pyrophosphate (IPP). The Vg9Vd2 subset of human T cells is depleted during HIV disease and not reconstituted after prolonged antiretroviral therapy (ART). Their loss is part of the immunodeficiency syndrome likely linked to increased opportunistic infections or cancer. Our goals are to understand the mechanisms preventing full reconstitution of Vg9Vd2 T cells and discover conditions in HIV+ patients that are limiting the functionality of existing cells. These cells remain incapable of responding to phosphoantigens even while re-gaining responsiveness to aminobisphosphonate drugs including zoledronic acid (Zol). Zol treatment increases stimulatory IPP and promotes secretion of Caspase-1 processed cytokines IL-18 and IL-1β through its effects on the NLRP3 inflammasome. The Vd2 T cell subset was particularly high for IL-18 receptor expression compared to traditional IL-18 targets CD8+ T and natural killer cells. IL-18 stimulation increased proliferation, enhanced the accumulation of effector memory cells, and increased expression of cytotoxic markers in HIV-negative controls. When Vg9Vd2 T cells from HIV+ individuals were stimulated with IPP in the presence of IL-18, there was increased proliferation, accumulation of memory cells, and higher expression of CD56, NKG2D, and CD107a (markers of cytotoxic effector phenotype). IL-18 stimulation specifically expanded the Vg9-JgP+ subset of Vg9Vd2 T cells as is expected for normal responses to phosphoantigen. Interleukin-18 is a potent stimulator of Vg9Vd2 T cell proliferation and effector function. We hope to translate this study to promote recovery of Vd2 T cells in immuno-compromised HIV patients for anti-tumor and anti-viral responses.
    • Non-random expression of CD56 on Vγ2δ2 T cells: Identification of a precursor cytotoxic lymphocyte subset

      Urban, Elizabeth; Pauza, C. David (2010)
      In most lymphocyte subsets, expression of CD56 (neural cell adhesion molecule-1) correlates with cell activation and cytotoxic activity. For T cells bearing the Vgamma2Vdelta2 T cell receptor, isoprenoid pyrophosphate stimulation leads to uniform activation and expansion, but only a fraction of cells express CD56 and display potent cytotoxic activity against tumor cells. We sought to determine whether the development of cytotoxicity among Vgamma2Vdelta2 T cells is random, and therefore a potential property of all cells, or whether it is regulated in a non-random manner, dependent either on antigen recognition properties of specific TCR sequences or the existence of cytotoxic lymphocyte precursors. By tracking the fate of individual cell clones defined by the Vgamma2 chain CDR3 region sequence, we show that CD56 is expressed on precursor cytotoxic T cells already present in the population. The ability to express CD56 was not predicted by TCR sequence, or by the strength of signal received by the TCR, as defined by the proliferation response of individual clones. Although CD56 expression could be induced by cytokine treatment alone, in the absence of expansion, the Vgamma2 repertoire present after cytokine treatment was different from either the ex vivo or phosphoantigen-expanded repertoires, and therefore did not provide a basis for clonotype comparison. For gammadelta T cells, then, cytotoxic effector function requires both proliferative responses to antigen, and a pre-existing capacity for CD56 expression.