Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1875-1876
CORRESPONDENCE
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Letter |
To the editor:
Alloantigen-induced anti-HIV activity occurs prior to
reverse transcription and can be generated by leukocytes from
HIV-infected individuals
Immunization against alloantigens has been proposed as a
possible prophylactic strategy against HIV.1,2 We have
previously reported in Blood that alloantigen-stimulated cell
lines derived from HIV-seronegative donors and their supernatants
inhibit HIV-1 replication of a wide spectrum of isolates by a mechanism
that is independent of IFN-
and of the 
-chemokines MIP-1
,
MIP-1, and RANTES.3 Here we provide further information
on the characterization of allogeneic leukocyte-stimulated anti-HIV
activity that could be relevant for the development of successful
complementary immune-based strategies against HIV infection. We have
found that alloantigen-stimulated anti-HIV activity (1) occurs prior to
reverse transcription, (2) is generated by leukocytes from HIV-infected
individuals, independently of CD4+ T-cell counts, and (3)
circumvents the need for an intact CD28/B7 costimulatory pathway.
To define the molecular mechanism of inhibition of viral replication,
we investigated the effect of anti-HIV supernatants derived from an
alloantigen-stimulated cell line3 on HIV-1 reverse
transcript levels in T-cell blasts infected with
HIV-1BZ167. Levels of long terminal repeat-gag
(LTR-gag) and LTR U3/R reverse transcripts were
measured by quantitative, real-time DNA polymerase chain reaction (PCR)
using primers and probe sequences previously described.4,5
Both late (LTR-gag) (Figure, panel A) and
early (LTR U3/R) (Figure, panel B) reverse transcripts were
significantly decreased following incubation with the
alloantigen-stimulated supernatant (86% and 97% inhibition,
respectively), demonstrating that alloantigen-stimulated anti-HIV
activity occurs prior to reverse transcription. Figure panel C shows
that the alloantigen-stimulated supernatant used in these experiments
had a strong inhibitory effect on HIV-1 replication (93% inhibition),
measured by p24 antigen production. These data suggest that the
antiviral activity mediated by alloantigen-stimulated supernatants is
distinct from the antiviral activity produced by CD8 antiviral factors
(CAF), since CAF does not affect the levels of early or late reverse transcripts.5
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Alloantigen-stimulated anti-HIV activity occurs prior to reverse
transcription (panels A, B, and C) and is generated in the absence of
an intact CD28/B7 costimulatory pathway (panels D, E, and F).
PHA blasts were infected with HIV-1BZ167 (172 TCID50/105 cells) and cultured in the absence
(control) or presence of an HIV-suppressive supernatant derived from an
alloantigen-stimulated cell line (Allo).3 Levels of LTR-gag
(A) and LTR U3/R HIV-1 reverse transcripts (B) in
HIV-1BZ167-infected PHA blasts were determined by
quantitative, real-time DNA PCR.7,8 Results in panels A and
B represent means (number of copies/50 000 ± 10 000 cells) ± standard deviation of 2 independent experiments. HIV-1 p24 antigen
production by HIV-1BZ167-infected PHA blasts (C) was
determined by ELISA. Alloantigen-stimulated supernatants from cultures
performed in the absence ( ) or presence of CTLA4Ig fusion protein (5 µg/mL) added at the beginning of 7-day cultures7 were
assayed for effect on HIV-1BZ167 replication in T-cell
blasts (D) and IL-2 production (E) measured by ELISA.
Alloantigen-specific proliferation (F) was measured 7 days after
primary stimulation. Results are expressed as mean ± SEM of 3 (D), 5 (E), and 6 (F) independent experiments.
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-Chemokines, CAF, and other unidentified soluble
factors, released either by alloantigen-stimulated cell lines or by
primary alloantigenic-stimulated peripheral blood mononuclear cells
(PBMC) from healthy HIV-uninfected individuals immunized in vivo with allogeneic PBMC have been reported to inhibit HIV-1
infection.2,3,6 To determine whether leukocytes from
HIV-infected individuals have the potential to generate anti-HIV
activity after primary alloantigenic stimulation, we analyzed the
effect of supernatants obtained from alloantigen-stimulated PBMC of
HIV-infected patients on HIV-1 replication. The supernatants from the
patients' alloantigen-stimulated cultures inhibited
HIV-1BZ167 and HIV-1Ba-L replication in T-cell blasts to an extent similar to that by supernatants of
alloantigen-stimulated PBMC from healthy individuals
(Table). Furthermore, the fraction of
individuals whose culture supernatants inhibited viral replication greater than 50% was similar in patient and control cultures. Alloantigen-stimulated supernatants from an HIV-infected individual and
healthy control also inhibited HIV-1Ba-L infection in
monocyte-derived macrophages (94% and 64% inhibition of HIV-1
replication). These results demonstrate that alloantigen-mediated
anti-HIV activity acts both in infected T cells and macrophages.
Patient and control alloantigen-stimulated cultures generated similar
amounts of RANTES but undetectable amounts of IFN-. Although these
control cultures produced more IL-2, IFN-, and IL-10 than the
patient cultures, the differences in cytokine production were not
statistically significant (p > .05, Student's t test).
Furthermore, there was no correlation between the levels of these
cytokines and the inhibitory effect on viral replication (r < 0.5).
No significant correlation was observed between patients' CD4 T-cell
counts (range 195-787 cells/µL) and ability of the patients'
leukocytes to generate alloantigen-stimulated HIV-suppressive
activity.
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Inhibition of HIV-1 replication by alloantigen-stimulated
supernatants derived from PBMC cultures of healthy blood bank donors
and HIV-1- infected individuals
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The role of costimulatory requirements in alloantigen-mediated anti-HIV
activity has not been previously addressed. Here we demonstrate that
generation of primary alloantigen-stimulated anti-HIV activity is not
affected by inhibition of CD28/B7 interaction using the CTLA4Ig fusion
protein (Figure, panel D), under conditions in which
alloantigen-specific IL-2 production (Figure, panel E) and
proliferation (Figure, panel F) are significantly inhibited (p < .05).7 These findings suggest that an intact
CD28/B7 costimulatory pathway is not essential for the induction of
alloantigen-stimulated HIV-suppressive activity and could explain the
generation of this activity by alloantigen-stimulated cells from
HIV-infected patients, which have been reported to exhibit immune
costimulatory defects.8,9
These findings contribute to characterization of the molecular
mechanisms and costimulatory requirements for alloantigen-stimulated anti-HIV activity that might be important for the development and
application of immune-based strategies against HIV.
Ligia A. Pinto
Vesna Blazevic
Gene M. Shearer
Experimental Immunology Branch, National Cancer Institute,
National Institutes of Health, Bethesda, MD
Bruce K. Patterson
Laboratory of Viral Pathogenesis, Children's Memorial
Hospital, Northwestern University Medical School Chicago, IL
Matthew J. Dolan
Infectious Disease Service, Wilford Hall USAF Medical
Center, Lackland AFB, TX
The views expressed herein are those of the authors and do not
reflect the official policy of the Department of Defense or other
departments of the US government.
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References |
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