Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Tuesday, July 9, 1996

Tu.A.384 -Tu.A.392

Tu.A.384
LOW T CELL REACTIVITY TO COMBINED CD3 PLUS CD28 STIMULATION IS
PREDICTIVE FOR PROGRESSION TO AIDS: CORRELATION WITH DECREASED
CD28 EXPRESSION
Roos, Marijke ThL*, Miedema F*, Dekker L*, Hooibrinlk B*, de Leeuw NASM'. Lange
JMA* *, Coutinho RA***, Schellekens PThA*. *Clin.Viro-lmmuno., Central Lab. Neth. Red
Cross Blood Transf. Serv. and Lab. for Exp. & Clin. Immunol. of the Univ. of Amsterdam;
*Academic Medical Center; ***Municipal Health Service; Amsterdam The Netherlands
Objective and methods: In 219 HIV+ men of the Amsterdam cohort CD4+ F cell counts,
expression of CD28 on CD4+ and CD8+ T cells and T cell responses to CD3 monoclonal
antibodies (mAb) with or without CD28 costimulation were analyzed as parameters for
disease progression within 4 years. In an additional study in the above ientioned cell populations prohlferation an d also apoptosis were measured by staining with either Ki 67 or propidium iodide.
Results: CD28 costimulation considerably enhanced responses with lower coefficients of
variation.fThese responses decreased during HIV-I infection and were a stronger predictive
marker for progression to AIDS than responses to CD3 mAb alone. In a Cox analysis,
besides CD4 T cell counts, reactivity to CD3 plus CD28 mAbs was the only independent
predictive marker with a relative risk of 2.04 and 4.16 for intermediate and low responses.
This independence was confirmed when the group with low CD4+ T cell counts was subdivided into groups with high, intermediate and lowT cell responses. It is generally assumed
that the CD8+CD28 T cells expand during HIV- I infection. We provide evidence, that this
expansion is only relative and mainly due to a decrease in the number of CD8+CD28+ T
cells. CD28 expression on CD4+ and CD8+ T cells correlated with CD28 costimulation.
However CD28 expression failed to reach significance as an independent prognostic marker, Ki-67 expression indicated that the CD8+/CD28- cells proliferated very weakly.The
responses of CD8+CD28+ cells were less impaired than those of the CD4+ cells. After
stimulation apoptosis was clearly demonstrable in the CD8+CD28+Ki-67 population.
Conclusion: Besides CD4+T cell counts onlyT cell reactivity to CD3 plus CD28 mAbs
was independently predictive for disease progressionThe diminished reactivity was mainly
confined to the CD4+ T cell population and correlated weakly with CD28 expression on
CD4+ and CD8+T cells.
MThL Roos, CLB, Plesmanlaan 125, 1066 CX Amsterdam,The Netherlands
Tel: [phone], Fax: + 31 20 512 3310
Tu.A.385
IMMUNOLOGIC AND VIROLOGIC EVALUATION OF INFLUENZA VACCINATION
OF HIV- I INFECTED PATIENTS
Fowke, Keith R., D'Aimicom R*, Chernoff DN#, Pottage J*, Bensen C*, Sha B*, Kessler HA*,
Landay AL*, Shearer GM. National Cancer Institute, Bethesda, MD; * Rush Medical College,
Chicago, IL; # Chiron, Emeryville, CA.
Objective: Evaluation of the effects of influenza vaccination of HIV- I infected patients on
HIV associated immunologic and virologic parameters.
Methods: Forty-six persons (36 HIV infected, 10 uninfected controls) were given influenza
vaccine while on stable combination therapy Blood was collected at the time of vaccination
(week 0) and at visits 2, 4, and I 2 weeks post vaccination. Immunologic evaluation included:
three color flow cytometry of activation (CD38, HLA-DR, CD25, CD71) and maturation
(CD45Ra, CD62L) markers on CD4 and CD8 subsets; detection of peripheral blood
mononuclear cell spontaneous, mitogen and antigen stimulated apoptotic cell death by
microscopic evaluation of nuclear morphology;T helper cell functional analysis by IL-2 production and proliferation; and influenza antibody titre. Quantitative plasma HIV- I RNA was
determined by bDNA.
Results: Fourteen individuals (1 2 HIV infected, 2 controls) have been analyzed through
week 4 post vaccination. Mean CD4 counts at enrollment (HIV-4-, 184.9~187.0; HIV-,
943.0~ 156.9) did not significantly change during the initial four weeks. Flow cytometric
nalysis indicated no change in the relative percentages of cellular phenotypes nor their
ictivation or memory states. Spontaneous, mitogen and antigen induced apoptotic cell
Death was higher in the HIV infected group, compared to controls, but neither group
showed a change over the first four weeks of the study. Plasma HIV RNA was assessed in
28 of the HIV infected group at 0, 2 and 4 weeks post immunization. While there were no
changes in twenty (71.4%) of the participants at week 4, 4 (14.3~%) showed a >0.5 log
increase and 4 (14.3%) a >0.5 log decrease in viral burden.
Conclusions: Immunization of HIV infected patients with influenza vaccine did not result in
alterations in any of the immunologic parameters evaluated.The effects of influenza vaccination on apoptotic cell death in HIV infected people with higher CD4 counts are being further evaluated. Influenza immunization did not result in predictable increases in plasma HIV
RNA which may have been due to continued anti-viral therapy during the study.
Keith R. Fowke, National Cancer Institute, National Institutes of Health, 4BI 7 Bldg. I 0, 9000
Rockville Pike, Bethesda, MD, USA 20892; tel [phone], fax [phone].
Tu.A.390
CD8+ T CELL-MEDIATED SUPPRESSION OF HIV REPLICATION: RELEVANCE OF
KNOWN CHEMOKINES AND OTHER CYTOKINES
Mackewicz Carl E, Barker E, Orque R, Levy JA. Cancer Research Institute, University of
California San Francisco, San Francisco, CA USA
Objective:To determine if the CD8+ cell antiviral factor (CAF) produced by CD8+ cells
from HIV-infected individuals is a known cytokine.
Methods: Levels of the chemokines, RANTES, MIP- I a, MIP- I 3, and MCP- I were measured
by ELISA in CD8+ cell culture fluids that either suppressed (CAF-positive) or had no effect
(CAFnegative) on HIV- I replication in acutely infected CD4+ cells. Recombinant human IL1 3, IL- I6, LIF, and the chemokines, RANTES, MIP- I a, MIP-j3, MCP- I, MCP-3, IP-10, lymphotactin, Gro-, and Gro-j3 at a range of concentrations were tested for inhibitory activity
against HIV replication in naturally and acutely infected CD4+ cells. Finally CAF-positive culture fluids were pretreated with a mixture of neutralizing antibodies specific for RANTES,
MIP- I a, and MIP- I 3, prior to assessing inhibitory effects on HIV replication.
Results: CD8+ T cells from HIV-infected individuals produced levels of certai chemokines
(e.g., that RANTES, MIP- I a, MIP- I 3) maximally reach about 10 ng/mi early after activation
and gradually drop over a two week period in culture. Both CAF-positive and CAF-negative

fluids had levels of RANTES, MIP- I a, and MIP-13 ranging from 0. I to 9.5 ng/mI.The level of
chemokines present in CD8+ cell supernatants did not correlate with the level of their
anti-HIV activity Only recombinant human RANTES, MIP-I 0, MCP-3, and lymphotactin suppressed HIV replication (by 50%) but at higher concentrations (>_100 ng/ml) than produced by CD8+ T cells. No significant antiviral activity was noted with IL- I 3, IL-6, LIF and
the other chemokines. Neutralizing antibodies to RANTES, MIP- I a, and MIP- 13, in combination, had no effect on the ability of CD8+ cell culture fluids to suppress HIV replication.
Conclusions: Some chemokines have anti-HIV activity in vitro at much higher concentrations than found in CD8+ cell culture fluids.The anti-HIV activity detected in CD8+ cell
culture fluids appears to be due to a cellular factor(s) (CAF) distinct from known
chemokines and other cytokines.
Carl E. Mackewicz, Cancer Research Institute, University of California, San Francisco,
San Frar cisco CA 94 143-0128, USA.Tel. (4 I15) 476-407 I; FAX (4 I15) 476-8365
Tu.A.39 I
EFFECT OF MIP- IA, MIP- 1 (AND RANTES ON SUPPRESSION OF HIV REPLICATION
IN T CELL BLASTS AND DENDRITIC CELL/CD4+ T CELL COCULTURES
Rubbert,Andrea,Weissman D., Daucher J., Pettrone K., BarkerT, Combadiere C, Murphy
PM, Fauci AS. National Institutes of Health, Bethesda, MD, USA
Objective:-0 analyze the effect of beta-chemokines in suppressing HIV replication in standard T cell blast and dendritic cell-T cell coculture systems.
Methods: Dendritic cells (DC) are capable of activating CD4+ T cells in the absence of mitogen. In one system, we employed DC and CD4 cells from HIV uninfected donors and the DC
were pulsed with primary or laboratory isolates of HIV-I. In another system, DC and CD4+ T
cells from HtIV infected donors were used. Chemokines, ranging from 0. I to 1000ng/ml, were
added at the initiation of culture and subsequently every second or third day.
Results: Recently, MIP- I a, MIP-I P and RANTES have been identified in supernatants of
HTLV. I transformed CD8+ T cell lines and from stimulated CD8+T cells from HIV-infected individuals; these factors mediated suppressive effects on viral replication in the PM-I
CD4 cell clone or PHA-stimulated PBMC infected with various laboratory and primary isolates of HIV (Cocchi etal, Science 1 995).We have recently described an in vitro model for
studying HIV replication which mimics the cellular interactions that occur at the primary site
of HIV replication, the lymphoid microenvironment. DC were cocultured with autologous
CD4+ 3-cells, which resulted in viral replication in the absence of exogenous stimulation.
CD8+ T-cells f-rom HIV-infected patients were able to inhibit viral replication in these systems. However; MIP-la, MIP- I3 and RANTES did not demonstrate any inhibitory effect on
viral replication in the DC systems, while demonstrating efficient inhibition in T cell blasts.
The addition of neutralizing anti-chemokine antibodies did not abrogate the CD8+ T-cell
mediated suppressor effect. Using functional assays (calcium influx) of the expression and
integrity of 3 chemokine receptors in the two systems, we could not completely explain
the discordance between T cell blasts and DC-CD4+ Tcell systems with regard to 3
chemokine effects.
Conclusions: Our data indicate, that MIP- Ia, MIP-I 3 and RANTES are not responsible for
the suppressive effect mediated by HIV-positive CD8+ cells in the DC/CD4 coculture system. Other mechanisms, in addition to receptor expression, appear to be important in the
divergence in the effect observed between T cell blasts and DC-CD4+ T cell systems.
Andrea Rubbert, M.D. National Institutes of Health, Bethesda, MD, USA.
Tu.A.392
CHEMOKINES REGULTATE ENDOGENOUS HIV REPLICATION: ENHANCEMENT BY
MCP-I AND SUPPRESSION BY RANTES.
Poll Guido *,Biswas P*, Delfanti F*, Sozzani S^, Alfano M*, Moretti GL*, A. Lazzarin*, A.
Mantovani",Vicenzi E*. * San Raffaele Scientific Institute, Milan, Italy; ' "Mario Negri"
Institute for Pharmacological Research Milan, Italy.
Objective:To evaluate the role of chemokines on HIV replication in primary cultures of
infected individuals.
Methods: Chemokine concentrations were determined during HIV isolation from either
total PBMC or CD8-depleted PBMC of long term non progressors (LTNP). CD8-depleted
PBMC cultures from other HIV-infected individuals with CD4 counts between 200 and 500
cells/mm3 were maintained up to 30 days in vitro according to two protocols: I. Cells were
first stimulated by PHA and then maintained in IL-2-enriched medium in the presence or
absence of recombinant (r) chemokines (direct culture); 2. cells were co-cultivated with
allogeneic PHA blasts from seronegative individuals in the presence or absence of rchernokines (Coculture). Supernatants were tested for the presence of either RT activity or
p24 gag Ag by ELISA (Coulter).
Results: Determination of the chemokine contents of supernatants during isolation either
from PBMC or CD8-depleted PBMC of LTNP revealed that several chemokines, including
RANTES, MIP- Il a, MIP- I b, MCP-, and IL-8 are released at concenttioans of ng/ml. No distinctive patterns of chemokine concentrations were observed in the presence or absence of
faction of PBMC.Vir s production from ex-vivo CD-depleted PBMC of IV-infected individuals was demonstrated in a total of 12 cultures or co-cultures. No clear modulatory effects
were observed by addition of different chemokines, including MCP-3, IPl0, and IL-B (100
ng/mI) to the cells. In sharp contrast, the same concentrations of MCP- I and RANTES significantly enhanaced in 50% and suppressed in 80%, respectively, DIV replication compared to
cent-el cultures. MIP- I a consistently caused a partial (50-90%) inhibition of virus production.
Conclusions Chemokines exert important, although opposite effects on DIV replication in
primary CD8-depleted PBMC obtained from infected individuals.These results suggest that
the extent of latent vs actively replicating infected cells may be under the control of different ch~emokines exerting divergent effects on virus expression and spreading.
Guido PoliVia Stamira Dancona N 20-20 I27 Milano, Italy Tel [phone]
Fax: [phone]

'0
0
u
sO
r
0
c0
a)
a)
c
0
U
0
a)
cx
2t
226