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

XIV International AIDS Conference

Abstracts ThPeA7123-ThPeA7127 365

ThPeA7123 Functional diversity and assay detection of
antigen-specific memory CD4+ T cell subsets
reflect lymph node circulation patterns
programmed by L-selectin (CD62L)
R.L. Hengell, V.B. Thaker', R.A. Lempicki2, M.V. Pavlick3, J.A. Metcalf1,
T. Imamichi2, H.C. Lane1. INIH, Bethesda, MD, United States;2SAIC, Frederick,
United States; 3Georgetown Univ., Washington, United States
Background: HIV-specific CD4+ T cells are thought to be a critical component
of the host immune response to HIV. Most studies examine CD4+ T cell memory
by measuring lymphoproliferation (LPA) or intracellular cytokine (ICC) production
of unfractionated CD4+ T cells. Given conflicting data regarding these assays,
we were interested in determining if they measure the same cells. The purpose
of the present study was to determine if changes in CD4+ T cell subsets alone
(based on CD62L expression) can account for changes in assay measurement of
immunity.
Methods: PBMC were sorted on the basis of CD4+, CD45RA- and CD62L+/expression. With 5d [3H] incorporation and 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) LPA, and 12h ICC (IFN-y), we compared subset memory
responses to tetanus toxoid (TT) and Cytomegalovirus (CMV) (n=9). Cell sizing
(n=6), telomer length, (n=5) CD3 VP sizing (n=5), differential gene expression
(n=8) and confocal microscopy (n=5) were also examined.
Results: LPA to TT was confined to CD62L+ cells (ACPM 82K vs. 5K, p<0.01).
CMV LPA was found in both pools (ACPM 98K vs. 78K, p>0.05) IFN-y expression to TT was negligible, but for CMV it was confined to CD62L- cells. CD62Lcells were larger and more complex (FSC/SSC 371/108 vs. 351/98, p<0.01), had
shorter telomers (ATRF 1003, p<0.01), had less diverse CD3 VP repertoires and
had genes/proteins expressed consistent with effector function and more recent
antigen encounter (all comparisons use medians and rank-sums).
Conclusions: Antigen-specific CD4+ T cells can be separated into functionally
discrete subsets based upon CD62L expression. A functional dichotomy exists
among these cells that correlates with in-vivo circulation patterns. CD62L+ cells,
which circulate to lymph node tissues, contain long-term memory. CD62L- cells,
which circulate to non lymph node tissues, have a more immediate effector phenotype.
Presenting author: Richard Hengel, NIH/LIR/CMRS, MSC 1880, Rm. 11 B05,
Bldg. 10, 9000 Rockville Pike, Bethesda, MD 20892, United States, E-mail: [email protected]
ThPeA7124 CD8 T cells responding to lab-strain HIV recognize
autologous virus-infected targets in asymptomatic
subjects but not in subjects with more advanced
disease
P. Shankar, S.K. Lee, Z. Xu, J. Lieberman. Center for Blood Research, Harvard
Medical School, Boston, MA, United States
Background: Assays that measure CD8 T cell responses against consensus laboratory strain HIV antigens show similar responses in progressors and nonprogressors, suggesting that they may not be representative of the in vivo response
to the evolving autologous virus.
Method: The magnitude of IFNy-producing CD8 T cells responding to autologous
and lab adapted HIVIIIB virus-infected primary CD4 T cells was measured in 11
seropositive subjects at various disease stages. TcR clonotypes of immunomagnetically captured IFNy+ cells were analyzed by anchored PCR and TCRBV gene
sequencing.
Results: The frequency of HIV-specific IFNy-secreting CD8 cells declined with
disease progression. The highest frequencies were seen in CDC stage A subjects (mean 3.87% vs. IIIB and 3.72% vs. autologous HIV) compared to stage B
(mean 3.87% vs. IIIB and 2.2% vs. autologous HIV) and stage C (mean 0.77%
vs. IIIB and 0.33% vs. autologous HIV). The clonal composition of CD8 T cells
responding to the patient's own isolate and HIVIIIe revealed near identical TCRBV
and CDR3 sequences in nonprogressors (LTNP and CDC stage A), whereas in
progressors (CDC Stage B and stage C) the dominant clonotypes responding
to autologous virus were completely different. In accordance with these results,
bulk T cell lines from CD8 T cells responding to IIIB virus lysed autologous HIVinfected targets and vice versa in CDC stage A subjects but not in CDC stage B
and C subjects.
Conclusions: The dominant immune response to autologous virus in advanced
patients becomes type-specific and differs more from the response to lab strain
virus than in early stage patients. In progressors, CD8 T cells responding to consensus epitopes in lab-adapted virus persist from an earlier response, but no
longer recognize the evolved virus.
Presenting author: Premlata Shankar, Center for Blood Research, Harvard
Medical School, 800, Huntington Avenue, Boston, MAO2115, United States, Tel.:
[phone], Fax: [phone], E-mail: [email protected]

ThPeA7125 Strong p24 proliferative responses detected in
HIV-infected children who control viremia
M.E. Feeney, P.J.R. Goulder, E.S. Rosenberg, B.D. Walker. Massachusetts
General Hospital; Harvard Medical School, 2 wellington st #5, United States
Background: Adults with long-term non-progressive HIV infection frequently possess strong HIV-specific T-helper responses. A robust proliferative response to

the p24 antigen has been shown to correlate with maintenance of a low viral load
in HIV-infected adults. To date, the HIV-specific immune responses among children who experience a benign clinical course with a persistently low plasma HIV
burden have not been characterized.
Methods: We examined twelve children with clinical evidence of endogenous
control of viremia. Subjects were chosen who maintained a VL< 5000 RNA
copies per ml while on no antiretroviral therapy, or after prolonged therapy with nucleoside analogues alone. HIV-specific T-helper responses to recombinant HIV-1
protein p24 were assessed using a standard lymphoproliferative assay. The targeted T-helper epitopes were mapped using pools of overlapping 18mer peptides
and CD8-depleted cells in an IFN-y ELISPOT assay.
Results: Six of the twelve children demonstrated strong proliferative responses
(SI >10) to the p24 antigen. Among these subjects, the mean Stimulation Index
was 32.6 (range 14.4 - 63.1). These responses were demonstrated by depletion
assays to be CD4-mediated. Comprehensive screening for recognition of overlapping peptides spanning all HIV proteins (using CD8-depleted cells in an IFN-y
ELISPOT) revealed that most responses targeted Gag epitopes, although RT and
Nef responses were also present in several patients.
Conclusions: Strong p24 proliferative responses are frequently, though not universally, present in children who demonstrate unusually good control of viremia.
An analysis of the precise T-helper and CTL epitopes targeted in these children
is presently underway. These studies should lead to important insights into the
control of HIV in children.
Presenting author: margaret feeney, 2 wellington st. #5, United States, Tel.:
+[phone], E-mail: [email protected]
ThPeA7126 Phenotype and Function of anti-HIV Gag and Pol
IISpecific CD8+ Cytotoxic Lymphocytes in a Cohort
of Untreated Chronically HIV Infected Patients
M. L6pez, J.M. Benito, S. Lozano, J. Gonzalez-Lahoz, V. Soriano. Hospital
Carlos// Ill, Madrid, Spain
Background: Tetrameric complexes have enabled to easily quantify and analyze
different aspects of cytotoxic CD8+ lymphocytes directed against HIV proteins.
An impaired function of these cells could explain its inefficacy to control HIV replication. To explore this hypothesis we have used the tetramer technology in a large
cohort of chronically HIV infected, untreated patients.
Methods: 55 untreated HLA-A0201+ patients were included in the study. In 21
patients a sample after initiating HAART was also available. A sequence specific
primer assay was used for HLA typing. CD8, CD45RA, and CD27 were used to
define the phenotype of HIV specific CTLs in terms of naive (RA+27+), memory (RA-27+ or 27-) and effector (RA+27-) functions. Stimulation of PBMCs with
the peptides was used to measure the abililty ot tetramer(Tet)(+) cells to produce
a-IFN as well as their proliferative potential.
Results: 23(42%) patients had Gag(+) and 6(11%) patients had Pol(+) CD8 cells.
Mean levels of Tet(+) cells were 0.57% and 0.17% for
Gag and Pol respectively No correlation between the level of Tet(+) cells and viral load(VL) was found. Most Gag(+) cells (76%) had a memory phenotype. Only
10% had an effector phenotype. The % of Gag(+) cells producing a-IFN after
stimulation with the peptide was highly variable between subjects (range 11%98%). A significant inverse correlation between the % of a-IFN producing Gag(+)
cells and VL was found (r=-0.63, p<0.05). In vitro stimulation with the peptide
expanded the Gag(+) cells in only 14% of cases. Gag(+) cells decreased with
HAART but, surprisingly, in three patients Pol(+) cells appeared de novo after
controlling VL.
Conclusions: Most Tet(+) circulating cells have a memory phenotype and a variable degree of impairment in their a-IFN production ability and proliferative potential. The percentage of functional Tet(+) cells is inversely correlated with VL.
In some patients, control of viral replication induces de novo appearance of these
cells.
Presenting author: Benito Jose Miguel, Hospital Carlos Ill, Laboratorio de Biologra Molecular, C/ Sinesio Delgado 10, 28029 Madrid., Spain, Tel.: [phone]
(ext 2754), Fax: [phone], E-mail: jbenitol @hotmail.com
ThPeA7127 CD4+ T cell lymphoproliferation and IFN-y
production in response to HIV-1 p24 during primary
infection
J.M. Doisne1, C. Lacabaratz-Porret', A. Urrutia1, C. Deveau2, L. Meyer2,
C. Goujard3, J.F. Delfraissy3, M. Sinet1, A. Venet1. 'INSERM E0109, Le
Kremlin-Bic&tre, France; 2INSERM U292, Le Kremlin-Bic~tre, France; 3Service
de Medecine Interne, Le Kremlin-Bic~tre, France
Background: HIV-1-specific CD4+ T cell responses are weak or absent in the
majority of subjects during chronic HIV infection. Nevertheless, during primary
infection (PI), this response can be detected by lymphoproliferative assay and
evaluation of IFN-g production by ELISPOT or flow cytometry.

Methods: We determined these HIV-1-specific CD4+ T cell responses in subjects
included in the multi-centre French PRIMO cohort and studied longitudinally from
the time of primary HIV infection. We performed lymphoproliferative and IFN-g
ELISPOT assays to compare the frequency and function of virus-specific CD4+ T
cells after stimulation with HIV p24 antigen.
Results: At inclusion, proliferative response to HIV p24 antigen was evidenced in
only 23% (18/77) of the patients, and among the responders, less than a half had