CELL INTERACTIONS IN THREE-DIMENSIONAL TISSUE CULTURE
     
Leonid B. Margolis, Ph.D., Principal Investigator
Jean-Charles Grivel, Ph.D., Staff Scientist
Yoshinori Ito, Ph.D., Postdoctoral Fellow
Nina Malkevitch, Predoctoral Fellow
Philip M. Murphy, M.D., Collaborator, LHD, NIAID
Chad Womack, Ph.D., Collaborator, IMS, NIAID
Anthoni Fauci, M.D., Collaborator, NIAID
Ronald Collman., M.D., Collaborator, School of Medicine, University of Pennsylvania, Philadelphia PA
Erik De Clercq., M.D., Collaborator, Rega Institute for Medical Research, Katholieke Universiteit, Leuven, Belgium
Domenique Schols, M.D., Collaborator, Rega Institute for Medical Research, Katholieke Universiteit, Leuven, Belgium
Frank Kirchhoff., Ph.D., Collaborator, Department of Virology, University of Ulm, Germany
Leonid Margolis
 
Critical events in HIV disease occur in lymphoid tissue where HIV kills infected CD4+ cells as well as many noninfected cells, disrupting a complex system of cell-cell interactions that mediate normal immune response. The general aim of the project is to determine the underlying mechanism(s) of HIV-1-mediated immunopathogenesis by using our system of ex vivo-infected human tonsils. In vivo, the ability of HIV-1 to use CXCR4 coreceptor exclusively or in addition to CCR5 coreceptor is often associated with massive loss of CD4+ T cells and rapid disease progression.

We are currently testing the working hypothesis that the HIV-1 env gene that determines the usage of CXCR4, exclusively or in addition to CCR5, is sufficient to induce T cell depletion and immunosuppression. In addition, we are investigating the contribution of HIV accessory genes, in particular that of the nef gene, to viral tissue cytopathicity. Earlier, we showed that coreceptor specificity determines cytopathicity of monotropic HIV-1 variants of B subtype.

Ex vivo Cytopathicity of R5 and X4 HIV-1 Variants of Non–B Subtypes
Margolis, Malkevitch, Grivel, Womack, Fauci
To investigate the ex vivo cytopathicity of R5 and X4 HIV-1 variants of non–B subtypes, we used primary isolates of subtypes A, E, and C and prototypic subtype B variants. We observed no relationship between the maximal level of viral replication and either coreceptor usage or viral subtype. However, we did observe a strong correlation between viral coreceptor usage and CD4+ T lymphocyte depletion across all subtypes tested. All R5 HIV-1 infections resulted in mild depletion of CD4+ T cells, whereas all X4 HIV-1 infections caused severe depletion of CD4+ T cells irrespective of their subtype. Thus, non–B HIV-1 subtypes are similar to HIV-1 subtype B in that coreceptor specificity determines cytopathicity in human lymphoid tissue infected ex vivo.

Relation of Differential Cytopathicity of Dual-Tropic R5X4 HIV-1 Variants of B Subtype to Coreceptor Usage
Malkevitch, Glushkova, Margolis, in collaboration with Collman, de Clercq, Schols, Murphy
To determine whether differential cytopathicity of R5X4 viruses is also related to the viruses’ preferential use of a particular coreceptor, we infected CCR5 delta32 tissue with three R5X4 HIV-1 variants: 89.6, 89-v345.FL, and 89-v345.SF; the latter two are isogenic to 89.6 except for the V3-V5 region of gp120. Variant 89.6 severely depletes tissue of CD4+ T cells, whereas 89-v345.SF depletes only mildly. Variant 89-v345.FL was an intermediate depletor. Both in CCR5 delta 32 and normal lymphoid tissue, 89.6 and 89-v345.FL replicated with similar kinetics. In contrast, 89-v345.SF did not replicate in lymphoid tissue lacking CCR5. In agreement with these results, replication of 89.6 was completely inhibited by an CXCR4 ligand AMD3100 but was insensitive to a CCR5 ligand RANTES, whereas 89-v345.SF replication was almost completely inhibited by RANTES but not by AMD3100. Replication of 89-v345.FL was reduced both by AMD3100 and RANTES. Among two studied primary isolates, which were dual-tropic in transfected cell lines, one was not inhibited by RANTES but was almost completely blocked by AMD3100. Thus, in lymphoid tissues, some R5X4 isolates appear to use CXCR4 exclusively, whereas others use CCR5 exclusively. R5X4 HIV variants that use CXCR4 preferentially are highly cytopathic, whereas those preferentially using CCR5 deplete CD4+ T cells mildly. Hence, preferential coreceptor usage determines cytopathicity of R5X4 variants.

Effect of HIV Accessory Gene nef to HIV Pathogenesis Mediated by Modulation of Cell Surface Receptors
Glushakova, Margolis, in collaboration with Kirchhoff
To test the hypothesis that cell surface receptors mediate the effects of an accessory gene nef on HIV infection, we infected tissues with NL4-3 clones bearing nef alleles from sequential patients’ isolates as well as with clones bearing recombinant alleles. The HIV-1 variants containing the nef alleles isolated at later time points, showing two- to-three-fold higher levels of replication than those containing the nef alleles initially isolated at the early time point. Higher levels of replication correlated with efficient depletion of CD4+ T cells. A significant correlation occurred between Nef activity in CD4 down-regulation and the efficiency of virus replication in ex vivo-infected tissue, suggesting a link between Nef-mediated CD4 down-regulation, viral replication, and disease progression. In summary, coreceptor preference of HIV-1 variants is a sufficient determinant of viral cytopathicity for monotropic viruses of B and non–B subtypes as well as for dual-tropic viruses; and down-modulation of cell surface receptors may mediate nef effects on HIV-1 infection in lymphoid tissue.
 

PUBLICATIONS

  1. Glushakova S, Munch J, Carl S, Greenough TC, Sullivan JL, Margolis L, Kirchhoff F. CD4 down-modulation by human immunodeficiency virus type 1 nef correlates with the efficiency of viral replication and with CD4(+) T-cell depletion in human lymphoid tissue ex vivo. J Virol 2001;75:10113-10117.
  2. Grivel JC, Malkevitch N, Margolis L. Human immunodeficiency virus type 1 induces apoptosis in CD4+ but not in CD8+ T cells in ex vivo-infected human lymphoid tissue. J Virol 2000;74:8077-8084.
  3. Grivel JC, Santoro F, Faga G, Malnati M, Ito Y, Fitzgerald W, Lusso P, Margolis L. Suppression of CCR5- but not CXCR4-tropic HIV-1 in lymphoid tissue by human herpesvirus 6. Nat Med 2001;7:1232-1235.
  4. Malkevich N, Womack C, Pandya P, Grivel JC, Fauci AS, Margolis L. Human Immunodeficiency Virus Type 1 (HIV-1) non-B subtypes are similar to HIV-1 subtype B in that coreceptor specificity is a determinant of cytopathicity in human lymphoid tissue infected ex vivo. J Virol 2001;75:10520-10522.
  5. Malkevitch N, McDermott DH, Yi Y, Grivel JC, Schols D, De Clercq E, Murphy PM, Glushakova S, Collman RG, Margolis L. Coreceptor choice and T cell depletion by R5, X4, and R5X4 HIV-1 variants in CCR5-deficient (CCR5delta32) and normal human lymphoid tissue. Virology 2001;281:239-247.
  6. Margolis L, Glushakova S, Chougnet C, Shearer G, Markham P, Robert-Guroff M, Benveniste R, Miller CJ, Cranage M, Hirsch V, Franchini G. Replication of simian immunodeficiency virus (SIV) in ex vivo lymph nodes as a means to assess susceptibility of macaques in vivo. Virology 2000;275:391-397.