Final Program and Oral Abstracts [International Conference on AIDS (8th: 1992: Amsterdam, Netherlands)]

ThA 1537-ThA 1541

TRACK A: BASIC SCIENCE

ThA 1537 POSITIVE SELECTION OF THE VB 5 TCR GENE PRODUCT IN HIV
INFECTED INDIVIDUALS. Wilson Susan E.*, Gompels, Ludlam,
C.A; Coates A.*, Habeshaw,J.* and Dalgleish A.* St.
George' s Hospital;+St Mary's Hospital, *HIVER Ltd, London,
England. AEdinburgh Royal Infirmary, Scotland.
OBJECTIVE: To investigate if HIV infected individuals with normal CD4
counts show selective use of specific VB TCR gene products.
METHODS: PBMC were isolated from both HIV seropositive donors (CDC II)
with normal CD4 counts (>600) and healthy volunteers (controls). The
percentage of CD3+ T cells bearing specific VB TCR was assessed by double
immunofluorescence. PBMC (1 x 106) were labelled with both anti-CD3 (PE)
and a panel of mAbs specific for human TCR VB region epitopes (FITC;
Diversi-T "" aS TCR, T Cell Sciences) for 30 minutes at 40C. Data
aquisition and analysis were performed by flow cytometry. Subsequent
statistical analysis was performed using the Mann-Whitney U-test.
RESULTS: All VB families examined showed slight enhanced expression in
HIV infected donors in accordance with the general immune activation
observed at this stage of disease. However only the VB 5.3 subfamily
(Clone ICI) was significantly increased in HIV seropositive donors
compared to controls (N=16; p<0.01). Furthermore clone W112 which
recognises a subset identified by clone ICI demonstrated borderline
significance.
CONCLUSION: Selective VB usage has been described in a number of
autoimmune conditions and GvH. Thus these results raise the possibility
that the immunopathological mechanism of HIV may be due to the induction
of auto or allo reactivity.
Wilson, Susan. Dept. Medical Microbiology,
St. George's Hospital Medical School, Cranmer Terrace, London
SW17 ORE. England.
Tel: [phone] ext. 56183 Fax: [phone]
ThA 1539 BIASED T CELL RECEPTOR VB REPERTOIRE OF PERIPHERAL BLOOD
DERIVED AND INTESTINAL CD8+ T CELLS IN HIV DISEASE.
K. Densch, I. Dilhne, N. Endres, K. Reich, J. Popinger*, H. Jiger*, M. Classen. II.
Dept. of Medicine, Technical University Munich; Curatorium for Immunodeficiency,
Munich, Germany
The hallmark of HIV disease is a progressive depletion of CD4+ T cells. Apart from
cytolytic effects imposed by viral infection of CD4 cells other mechanisms such as
autoimmune mediated cytotoxicity targeted to gp120 bound to the CD4 molecule or
antiidiotypic cell interactions have been discussed. A striking feature during the course of
HIV disease is an expansion of CD8 + T cells that express activation markers such as MHC
class II and CD45RO molecules in the peripheral blood and other lymphoid organs.
Intrigued by these obversations, we sought to find evidence for antigen driven clonal
expansion of the CD8+ T cell subset. To address this question, we stained CD4+ and
CD8+ T cell subsets isolated from the peripheral blood and the intestinal mucosa
employing a panel of monoclonal antibodies directed to products of various Va of V/I gene
segments. Subsequently, we analyzed the relative frequencies of individual Va and V/I
subsets in HIV patients during various stages of the disease and compared them to those
observed in a healthy control population. As a result we observed a disease coursedependent uneven increase in the fraction of T cells expressing distinct TCR variable gene
segments. Most often Va2 or V/I gene segments were used. Biased TCR V gene segment
usage could be observed in CD4 and CD8 T cells. We conclude from our data that in
HIV disease CD4+ and CD8+ T cells subsets are clonally expanded by hitherto
unidentified antigenic structures. Whether, the latter are virally encoded or, alternatively,
represent autologous antigens remains to be elucidated.
ThA 1541 CONSERVATION OF THE TCR-V-BETA CHAINS REPERTOIRE IN
CD4+ T CELLS DURING PROGRESSION OF HIV INFECTION
G.Gorochov+, B.Autran*, P.Debr6*, F.Sigaux+; + Lab.
Molecular Hematology, H6p. St Louis. * Lab. Cellular
Immunology, H6p. Pitig-Salp6tridre, Paris, France.
Objectives: To study the hypothesis of a selective in-vivo deletion
of T cell receptor (TCR) V-Beta chains as co-factors for CD4+ cell
depletion in HIV disease. We studied the evolution of the usage of
Beta chain repertoire during progression of the disease.
Methods: We performed a PCR analysis of the V-Beta segments with a
series of oligonucleotides specific for the V-Beta families (1-20);
m-RNA were extracted ex-vivo from either total PBL or purified CD4+ T
cells (pCD4) from 11 patients at distinct stages of HIV disease.
Results: The 20 V-Beta families tested were detectable in all
samples, even with CD4+ cells counts <200/mm3. The VB repertoire did
not significantly differ in HIV+ patients and HIV- patients despite
lower expression of some TCR V-Beta families. The relative V-Beta
expression in total PBL and pCD4+ cells were comparable in distinct
patients at early and late stages. A longitudinal analysis was
performed on total PBL and pCD4+ cells in 5 patients at a 12-48
months interval (mean CD4+ counts: 332+128/mm3 and 96+51/mm3 at Ist
and 2nd evaluations). No deletion of particular V-Beta chain usage
was observed along with disease progression and CD4+ cell depletion.
Conclusion: Our results suggest that no preferential V-Beta chain
deletion has occured during disease progression in patients studied.
The CD4+ cell depletion in HIV infection thus appears to occur at
random within the V-Beta repertoire of antigen/superantigen
responding T cells and irrespectively of an hypothetical superantigen
exposure.
Guy Gorochov, Laboratoire d'Immunologie Cellulaire,
URA CNRS 625, CERVI, HNpital PitiA-Salp~tridre, 83,
bld de 1'H6pital 75013, Paris, France, Tel: [phone]; FAX: [phone].

ThA 1538 HIV-1 ACTS AS A SUPERANTIGEN: TCR VB
EXPRESSION REGULATES HIV REPLICATION. Jeffrey Laurence; Hodtsev,
A.S.; Posnett, D.N. Department of Medicine, Cornell University
Medical College, New York, New York 10021 USA
Objectives: Superantigens (SA), molecules recognized by T cells expressing specific Ag
receptor (TCR) VB gene products, bridge MHC and TCR, variously leading to cell stimulation, deletion or anergy. Mammalian retroviruses, as other pathogens, may encode SAs
as a means to block generation of cellular immune reactivity and/or to facilitate replication consequent to direct cell activation. We have identified an HIV-1 SA activity
Methods: Homogeneous, IL-2 dependent CD4+ and CD8+ T cell lines expressing identical
VBs were prepared from HIV-1 seronegative donors using anti-TCR MAb-coated magnetic
beads, then exposed to divergent HIV-1 isolates at 10-1000 TCID-50. Peripheral T cells
from HIV+ individuals were evaluated for VB and HIVexpression by two-color flow cytometry; DNA PCR for HIV 9ag was performed on selected VB subpopulations.
Results: Equivalent levels of HIV-1 infection, quantitated by p24 core Ag assay, were
noted by day 4 post-inoculation, but viral replication consistently varied in a VBrestricted manner by >2 logs by days 10 to 21, a result confirmed by PCR. Vg12+ cells
supported the highest levels of viral production. This did not reflect clonal defects,
for induction of all cells with PMA + PHA led to identical levels of p24 Ag,regardless
of VB type. The phenomenon was MHC-II dependent, but not MHC-II restricted, being reproduced in lines from 8 different donors. It was HIV strain independent, occurring
with TIIIB, BaL, and a direct patient isolate. FACS and DNA PCR of Vg subsets from HIV
infected persons in various clinical stages showed marked enrichment for HIV Env gpl20
expression and HIV provirus, respectively, in the Vg12+CD4+ subsets.
Conclusions: HIV-1 can act as a SA, demonstrating:VB selectivity in vitro and in vivcI
MHC-II dependence but not restriction; lack of requirement for Ag processing; and
viral isolate independence. The Vg12+CD4+ subset may serve as a preferential reservoir
for HIV. While they are not deleted. in vivo, they could be anergic to CD3/TCR signals.
Laurence, Jeffrey, Cornell University Medical College, 411 East
69th Street, New York, NY 10021, USA, Telephone: (l)-212-746-2988,
ThA 1540 T CELL RECEPTOR V GENE EXPRESSION ON CD4+ AND CD8+ PERIPHERAL T
CELLS FROM HIV+ PATIENTS. Hodara. Vida *n**; Jeddi-Tehrani, M.**
Grunewald, J.**; Andersson, R.**; Scarlatti, G.#; Holmberg, V.##; Libonatti, 0.* and
Wigzeil, H**,#. ** Dpt.lmumm oblgy, Karolilnsk I tituick, Swcde. * Dpt.Microbiology, Fr.aediciie, IBA,
Argentnat; # Virology,Nation al Bctriooic l.. laboratriumn. Sweden; ehlmahltm.,South HopitaL,Swede.
Objectives: To analyze T cell receptor (TcR) VB and J1 gene expression in HIV+ patients
and to find out whether there is any relation between the disease progression and the
expression of select TcR genes.
Methods; Peripheral blood was obtained from 2 HIV+ patients at clinical stage CDCII; 5 at
stage III and 5 at stage IV. Mononuclear cells were isolated by Ficoll/Hypaque gradient
centrifugation and T cells by using anti-CD4 and anti-CD8 monoclonal antibodies coupled
to magnetic beads (Dynal Inc., Norway). CD4+ and CD8+ T cell mRNA was obtained after
lysis of 0.5 x 106 cells using oligo dT nucleotides coupled to magnetic beads (Dynal Inc.).
First-strand cDNA was prepared, diluted 1:25 and amplified using polymerase chain
reaction (PCR). 22 VB and one CI specific primers were used. J0/ gene segment usage by
the T cells was studied after blotting PCR products onto nylon membranes and
hybridization with 5'-y-32P-end labeled JIspecific oligonucleotide probes.
Resultsi VB and J gene expression in HIV-infected individuals were found to display
frequent abnormalities. Deletions or reductions in VBI gene expression was noted for
V03, VI 13.1 and V/ 18 in many CD4+ T cell populations whereas in CD8+ T cells only YI3
displayed deletions. In contrast, several individual CD8+ T cell populations displayed
overexpression of certain V/ genes.For instance, VI14 was overexpressed in 5/12 CD8+ T
cells subsets whilst being normal in CD4+ cells. Overexpression of individualJ/ segments
was observed but no association between VI14 and a restricted JI usage was found.
Conclusions: Certain VI genes are selectively deleted or expanded following HIV-1
infection. Such abnormalities tended to increase with progression towards disease,
especially for V/3 deletion. Our findings may help in the understanding of changes in
cell mediated immune reactivity during HIV-infection.
Hodara, Vida; Immunology Dpt., Karolinska Institutet
Box 60400; S-104 01 Stockholm, Sweden
TEL.: [phone] FAX: [phone]
NOTES

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