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Pathogenesis of AIDS-NHL

CD40 Ligand Incorporated into HIV Virions Induces Activation-Induced Cytidine Deaminase (AID) Expression in Human B Lymphocytes M. Epeldegui , D. Thapa , S. Kitchen , J. De La Cruz, J.A. Zack and O. Martínez-Maza UCLA AIDS Institute, Los Angeles, CA, USA AIDS 2010 July 21, 2010

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Pathogenesis of AIDS-NHL

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  1. CD40 Ligand Incorporated into HIV Virions Induces Activation-Induced CytidineDeaminase (AID) Expression in Human B Lymphocytes M. Epeldegui, D. Thapa, S. Kitchen, J. De La Cruz, J.A. Zack and O. Martínez-Maza UCLA AIDS Institute, Los Angeles, CA, USA AIDS 2010 July 21, 2010 Vienna, Austria

  2. Pathogenesis of AIDS-NHL • There are two major mechanisms that are thought to lead to the development of AIDS-associated non-Hodgkin’s lymphoma (AIDS-NHL): • loss of immunoregulatory control of EBV-infected B cells; • molecular lesions (oncogene mutation, MYC:IgH translocations) resulting from DNA modifying events, somatic hypermutation (SHM) and IgH class switch recombination (CSR), which occur during the process of B cell activation. • AID (activation induced cytidine deaminase), is an APOBEC-like DNA-mutating enzyme that mediates both Ig SHM and CSR, and plays a central role in the genesis of B cell lymphomas.

  3. Viruses associated with NHL induce AID expression Several viruses (HIV, EBV, HHV8/KSHV, HCV) have been seen to be associated with NHL. Do these viruses induce AID expression and activity? EBV can induce AID expression and oncogene SHM in B cells(He, et al. J Immunol 171:5215, 2003; Epeldegui, et al. Molecular Immunol44:934, 2007) HCV also induces AID expression in B cells (Machida, et al. PNAS 101:4262, 2004) We have seen that elevated AID expression is seen preceding AIDS-NHL (Epeldegui, et al. AIDS 21:2265, 2007) HIV can directly induce B cell activation (Schnittman, et al. Science 233:1084, 1986) Can HIV induce AID expressionin B cells?

  4. HIV induces AID expression in B cells B cells were isolated from PBMC, exposed to HIV (NL4-3, 100 ng p24), and AID expression measured by qPCR: Infection of B cells was not seen, as there were no changes in p24 levels in HIV exposed B cells after culture, and AZT did not inhibit HIV-driven AID expression by B cells (not shown).

  5. Does HIV induce AID expression via virion-associated T cell-encoded stimulatory molecules? • HIV is known to capture cell surface molecules molecules as it buds from infected T cells: • MHC molecules • complement components • adhesion molecules • immune stimulatory molecules, including CD40L • We concluded that the induction of AID by HIV was likely due to an interaction of stimulatory molecule(s) expressed on the surface of HIV virions with a receptor on B cells: B cell surface: HIV membrane: • CD40 CD40L • DC-SIGN gp120 Martin, et al. J Virol 81:5872, 2007 Martin & Tremblay. Clin Imm 111: 275, 2004 He, et al. J Immunol 176:3931, 2007

  6. Does HIV induce AID expression in B cells via virion-associated CD40L? Transfected 293T cells with: HIV (NL4-3/JR-CSF), CD40L* and/or T147N* (mutant, non-functional CD40L): 293T pNL4-3 or pJR-CSF and pT147N* pNL4-3 or pJR-CSF pNL4-3 or pJR-CSF and pCD40L* NL4-3/JR-CSF expressing CD40L(CD40L+ HIV) NL4-3/JR-CSFexpressingT147N(mutant CD40L+ HIV) NL4-3/JR-CSF (CD40L-negative HIV) * Both pCD40L and pT147N were kindly provided by R. Kornbluth, UCSD

  7. HIV produced in CD40L co-transfected 293T cells contains CD40L HIV produced in 293T cells (and PBMC) was separated using anti-CD40L on magnetic beads, with the amount of CD40L+ HIV (bound virus) assessed by measuring p24: HIV isolated from the plasma of HIV+ subjects also was seen to be CD40L+ (not shown).

  8. CD40L+ HIV (NL4-3), but not CD40L- HIV, induces AID expression nd = AID not detected

  9. CD40L+ HIV (JR-CSF), but not CD40L- HIV, also induces AID expression nd = AID not detected

  10. B cells driven to express AID by exposure to CD40L+ virions display an activated, GC-like phenotype

  11. B cells express elevated levels of several cytokines following exposure to CD40L+ HIV Cytokine levels were measured using multiplexed Luminex-based assays (Milliplex, Millipore)

  12. Conclusions • HIV (both X4 and R5) induces AID expression in human B cells, without infection of these cells. • HIV produced in PBMC, as well as plasma virions, contain CD40L, a B cell-stimulatory molecule expressed on activated CD4 T cells. • CD40L+, but not CD40L-negative, virions made in 293T cells induced AID expression, as well as B cell activation. • These results suggest that HIV may directly contribute to lymphomagenesis by inducing B cell AID expression, resulting in AID-mediated oncogene mutation/translocation. • Certainly, virion-associated cell-encoded activation molecules may play other important roles in HIV pathogenesis: • virion-associated host cell proteins are known to enhance infectivity of HIV (Liao, Roos & Hildreth, AIDS Res Hum Retro 16:355, 2000) • B cell activation driven by stimulatory molecules expressed on HIV virions can promote HIV infection (Martin, et al. J Virol 81:5872, 2007)

  13. cytokine/cytokine-R genetics(promoter SNPs) “high-risk” DNA-modifying activities:IgH CSR;SHM B cell-stimulatory cytokines IgH:oncogene translocations  B cellactivationAID T cells expressing B cell-stimulatory molecules  oncogene mutations NHL antigens EBV infection/transformation (CNS, EBV+ tumors) viruses(EBV, HCV, HIV)

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