Abstract Book Vol. 2 [International Conference on AIDS (14th: 2002: Barcelona, Spain)]

XIV International AIDS Conference

Abstracts WeOrA1347-WePpA2085 5

WeOrAl347 Interleukin 2 is a poor inducer of HIV reactivation,
but enhances viral persistence in lymphocytes
J. Rullas', M.A. Pedraza1, J. Martin-Serrano2, M. Beltran', J. Alcami1. 'Aids
Immunopathogenesis Unit. Centro Nacional de Microbiologfa. Instituto de Salud
Carlos Ill, Ctra. Majadahonda-Pozuelo, km.2, 28220 Majadahonda, Madrid,
Spain; 2Aaron Diamond Aids Center, New York, United States
Background The study of the mechanisms leading to HIV persistence and reactivation represent a major issue in understanding HIV pathogenesis. We have
developed an efficient system that allow transfection of a full length HIV provirus
in resting peripheral blood lymphocytes. In this model we have addressed the
role of IL2 and other cytokines in HIV reactivation from latency and the molecular
mechanisms involved.
Methods Lymphocytes from seronegative donors [transfected by electroporation
with an infectious provirus (NL4.3)] or from HIV-infected patients were activated
with IL2, antiCD3, PMA or TNF-a+1L6. In some experiments resting PBMC were
transfected and stimulated with IL2 or left untreated for 7 days before activation.
HIV replication was assessed by quantifying p24 antigen in supernatants. Cell
proliferation was measured by thymidine incorporation and cell cycle progression
by propidium iodide staining. Induction of Rel/NF-kB family of transcription factors
was analyzed by gel-shift assay.
Results In contrast with high viral replication induced by CD3, IL2 stimulation
resulted in bare replication levels in culture in both HIV-transfected cells and lymphocytes from infected patients. Lack of HIV replication was not due to weak
proliferation of IL2 activated lymphocytes. In agreement with these results NF-kB
was not induced by IL2 treatment. When transfected PBMC were cultured in the
presence of IL2 for 7 days, treatment with PMA, TNF-a or TNFa+IL-6 were able
to induce a strong HIV reactivaton. In contrast, no viral replication was detected
by such stimuli when PBMC were not previously treated with IL2.
Conclusion IL2 was a poor inducer of HIV reactivation from the state of proviral latency despite the induction of robust lymphocyte proliferation in culture. IL2
treatment for seven days increased HIV persistence in resting lymphocytes, suggesting that this cytokine could enhance the stability and/or integration of proviral
DNA.
Presenting author: Joaquin Rullas, Ctra. Majadahonda-Pozuelo, km.2, 28220
Majadahonda, Madrid, Spain, Tel.: [phone], Fax: [phone], E-mail:
[email protected]
WeOrA1l3481 Impact of thymectomy on SIV infection in
macaques
A. Muthukumarl, A. Wozniakowskil, C. Matthews1, D.C. Douek2,
R.P. Johnson3, H.M. McClure4, R.A. Koup2, D.L. Sodoral. 1UTSouthwestern
Medical Center at Dallas, Dallas, Texas -75390-9113, United States; 2NIH,
Vaccine Research Center, Bethesda, MD 20892, United States; 3New England
Regional Primate Center, Southborough MA 01772-9102, United States;
4 Yerkes Regional Primate Center, Atlanta GA, United States
Background: CD4+ T cell depletion is a hallmark of HIV/SIV infection. The thymus is responsible for de novo production of T lymphocytes, and therefore is
important for T cell renewal. HIV infection in humans, as well as SIV infection
of macaques, results in thymic atrophy, reflecting a possible decrease in T cell
production.
Methods: and Results: We assessed the role of the thymus following a
pathogenic SIV infection by comparing thymectomized macaques (n=3) to controls with an intact thymus (n=3). As a quantitative measure of thymic output,
levels of recent thymic emigrants were determined by measuring T cell receptor
excision circles (TREC). Quantification of peripheral blood TREC levels showed
two distinct phases of SIV infection. Phase 1 was marked by a slight increase or
sustained level of TREC, whereas phase 2 was associated with decreasing TREC
levels. For thymectomized macaques phase 1 lasted approximately 12-16 weeks
compared to 16-30 weeks for the control group. During phase 2, TREC levels
declined at similar rates for both groups. Assessment of naive/memory phenotype, CD4/CD8 levels, Ki67 staining and other activation markers suggest that
declining TREC levels results from the contribution of both increased T cell proliferation (expression of Ki67) and decreased thymic output. Examination of the
thymic tissue obtained at necropsy demonstrated characteristic pathology including an increase in the percentage of mature CD8+ T cells and increased levels of
apoptotic/TUNEL+ cells.
Conclusions: We have documented a reduction in the levels of recent thymic
emigrants and distinct thymic pathology following SIV infection, however the impact of thymectomy on disease progression was not dramatic. TREC analysis
permitted the infection to be divided into two distinct phases. Prolonged phase 1
(sustained TREC levels) in macaques with a thymus indicated that the thymus is
important for maintaining the naive T cell pool at early times post-infection.
Presenting author: Donald Sodora, Department of Internal Medicine, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas - 75390 -9113, United States, Tel.: [phone], Fax: [phone], E-mail: donald.

sodora@ utsouthwestern.edu

WeOrAl349 Impact of bone marrow hematopoiesis failure in
T-cell production during SIV/SHIV infection of
macaques
H. Thiebot1, B. Vaslin1, T. de Revel1, S. Derdouch1, G. Gras1, J.M. Bertho2
F. Louache3, W. Vainchenker3, D. Dormont4, R. Le Grand4. 1Service de
Neurovirologie, France, France; 2IPSN, Fontenay-aux-Roses, France; 3lnstitut
Gustave Roussy Villejuif, France; 4Service de Neurovirolgie,
Fontenay-aux-Roses, France
Background: To evaluate, in macaque models of AIDS, the impact of infection
on bone marrow hematopolesis.
Methods: Cynomolgus macaques have been inoculated with 50 MID50 of a SIVmac251 primary isolate or the SHIV89.P chimera which expresses the env gene
of the primary HIV-1 89.6 isolate in a SIVmac239 background. During primary
infection, animals infected with SHIV 89.6P were treated twice a day with either
placebo or with the association of AZT, 3TC and Indinavir (HAART). Virological
and immunological parameters were followed during one year. Purified CD34+
bone marrow cells (over 99% pure) were used for the detection of long term
culture-initiating cells (LTC-IC), colony forming cells (CFC) or generation of T cells
in coculture with thymic stromal cells. A gag PCR was used for the detection the
virus DNA in bone marrow cells.
Results: All the macaques exhibited decreased CFC, starting as early as primary
infection regardless the virus strain used. One year pi., CFC was reduced of approximately 56% of pre-infection values and a dramatic reduction (over 77%) of
primitive long-term culture initiating cells (LTC-IC) was noticed. Virus DNA could
not be evidenced in purified bone marrow cells. Hematopoietic failure could not
be prevented by early HAART, although antiviral treatment during primary infection results in a significant reduction of viral load and prevention from dramatic
circulating CD4+ T cell loss. Generation of T cells in thymic stromal layers was
significantly reduced in all SIV or SHIV infected macaques as early as primary infection. No correlation could be established between impaired progenitor growth
and CD4+ lymphopenia or plasma viremia.
Conclusions: lentiviral infection have a strong impact on myelopofesis and de
novo generation of T-cells, independently of direct virus infection of hematopoletic
cells.
Presenting author: Roger Le Grand, Seervice de Neurovirologie, CEA/DSV/DRM, 60-68 Av
Div Leclerc, 92265, Fontenay-aux-Roses, Cedex, France, Tel.: [phone],
Fax: [phone], E-mail: legrand @ dsvidf.cea.fr
WePpA2085 Loss of memory B lymphocytes during HIV-1
infection: correlation to antigen-specific humoral
immunity and hypergammaglobulinemia
A. De Milito1, R. Thorstesson 2, E. Reizenstein 2, A. S6nnerborg3, F. Chiodi4.
'Karolinska Institutet, Microbiology and Tumor Biology Center, MTC, Karolinska
Institutet, Nobels vIg 16, S 17177 Stockholm, Sweden; 2Swedish Institute for
Infectious Disease Control, Stockholm, Sweden; 3Karolinska Institutet, Huddinge
Hospital, Stockholm, Sweden; 4Karolinska Instituet, Microbiology and Tumor
Biology Center, Stockholm, Sweden
Background: We have reported that loss of memory B cells occurs during HIV infection. Here we evaluated the phenotype of memory and naive B cells in relation
to humoral immunity to common antigens.
Methods: FACS was performed on PBMC from 52 HIV-infected and 40 uninfected subjects for expression of CD19, CD27, CD70, Fas and LAIR-1. Apoptosis
of cultured B cells was analysed by Annexin staining. Antibody titers to measles,
tetanus toxoid (TT) and HIV gp41 were measured with commercial tests.
Results: Memory B cells from patients showed a high degreee of apoptosis compared to controls (35% vs 8%, P<0.001). This was paralled by the upregulation of
Fas expression on memory B cells from patients. Among HIV-infected subjects,
memory B lymphocytes showed a reduced expression of LAIR-1 compared to
donors (53% vs 83%, P<0.05) indicating an activated phenotype, since LAIR1 is lost during terminal differentiation. Interestingly, in HIV-infected subjects we
observed the appearance of a subset of naive B cells with upregulated CD70 expression (4.66%) which is undetectable in normal subjects (1.5%, P=0.01). The
analysis of antibody titers to measles, T and gp41 showed that HIV-infected
subjects with low memory B cells carried significantly lower Abs titers to all 3 Ags
than patients with normal levels of memory B cells. Conversely, the degree of
hypergammaglobulinemia was comparable in the two groups.
Conclusions: Memory B cells during HIV infection display an activated phenotype (Fas high, LAIR-1 low) which may lead to apoptosis. Loss of memory B cells
is accompanied by a decline of antibody titers to common Ags like measles and
tetanus. Conversely, hypergammaglobulinemia may rise as a consequence of IgG
production by activated CD70+ naive B cells. The B cell phenotype responsible
for hypergammaglobulinemia will be further analysed.
Presenting author: Angelo De Milito, MTC, Karolinska Institutet, Nobels vig 16,
S 17177 Stockholm, Sweden, Tel.: [phone], Fax: [phone], E-mail:

Angelo.De.Milito@ mtc.ki.se