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Depletion of CD4+ T Cells in Semen During HIV Infection and Their Restoration
Following Antiretroviral Therapy
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"In summary, data from this study demonstrate that CD4+ T-cell counts decline
in the male genital tract before they decrease in the peripheral circulation of
HIV-infected men. This provides evidence that genital tract T-cell-dependent
acquired immune functions are impaired in HIV-infected men, perhaps rendering
them more susceptible to concomitant STD infections that can increase HIV
transmission rates. Data from this study also show that treatment with a viral
suppressive ART regimen is associated with significant restoration of CD4+ T
cells in the genital tract, and thus could improve acquired immune function in
the genital tract. Because the relative concentration of seminal macrophages
compared with CD4+ T lymphocytes was high in HIV+ men regardless of disease
stage, macrophages are likely primary HIV host cells in the male genital tract
and vectors of HIV transmission. The numbers of WBCs capable of transmitting HIV
(macrophages and CD4+ T cells) were reduced overall in semen from HIV+ men but
were strikingly elevated in a small subset (2/98 ART- HIV+ men in this study).
It is possible that men such as these with apparent genital immune activation
and elevated HIV titers in semen are highly infectious and may contribute
disproportionately to HIV transmission."....."HIV+ men receiving dual nucleoside
ART had significantly higher concentrations of seminal WBCs including total,
CD8+ and activated T lymphocytes, and macrophages than did ART- HIV+ men (P =
0.0003, 0.0001, 0.0001, and 0.03, respectively). HIV+ men on dual nucleoside ART
also had modestly higher concentrations of seminal CD4+ cells (P = 0.056) and
seminal CD4+ T lymphocytes (P = 0.03) than ART- HIV+ men. Six months after the
addition of indinavir to the ART regimen, substantial increases in CD4+ cell
counts were observed in both semen and blood [seminal CD4+ cells"
JAIDS Journal of Acquired Immune Deficiency Syndromes:Volume 50(3)March 2009pp
283-289
Politch, Joseph A PhD*; Mayer, Kenneth H MD; Anderson, Deborah J PhD*
From the *Division of Reproductive Biology, Department of Obstetrics and
Gynecology, Boston University School of Medicine, Boston, MA; Fearing Research
Laboratory, Department of Obstetrics, Gynecology, and Reproductive Biology,
Brigham and Women's Hospital, Harvard Medical School, Boston, MA; The Fenway
Institute, Fenway Community Health, Boston, MA; and Departments of Medicine and
Community Health, Warren Alpert Medical School of Brown University, Providence,
RI._
Abstract
Background: Information concerning the effects of HIV-1 infection, disease
progression, and antiretroviral therapy (ART) on male genital white blood cell
(WBC) profiles could provide important insight into genital immune defense in
HIV-infected men and seminal HIV transmission mechanisms.
Objective: To compare concentrations of WBC populations in semen from
HIV-1-seronegative (HIV-) and HIV-1-seropositive (HIV+) men and determine
whether HIV disease stage and ART are associated with alterations in seminal WBC
profiles.
Subjects and Methods: Subjects were 102 HIV- men, 98 ART-naive (ART-) HIV+ men,
and 22 HIV+ men on dual nucleoside ART, before and 6 months after addition of
indinavir. Seminal WBCs, macrophages (MO), and T-lymphocyte subpopulations were
enumerated by immunohistology technique.
Results: Seminal CD4+ and CD8+ T-cell populations were severely depleted in most
ART- HIV+ men regardless of peripheral blood CD4+ cell count. Seminal MO counts
were reduced by 50%. HIV+ men on dual nucleoside ART had significantly higher
seminal MO, CD4+, and CD8+ T-cell counts than ART- HIV+ men; addition of
indinavir led to a dramatic (>25-fold, P < 0.001) increase in seminal CD4+
T-cell counts which paralleled an increase in blood CD4+ cell counts. Two
outlier ART- HIV+ men with notably elevated seminal WBC profiles (>20 x 106
WBCs/mL) and infectious cell-associated HIV in semen are described.
Conclusions: HIV infection severely depletes CD4+ T cells in the male genital
tract as it does at other mucosal sites. This provides evidence that ART- HIV+
men have depressed T cell-dependent genital immune defense functions and are
vulnerable to other genital infections that could promote HIV transmission.
Seminal CD4+ T-cell counts rebounded after treatment with a viral-suppressing
ART regimen, indicating that ART may reverse HIV-associated genital
immunosuppression. The relative abundance of seminal MO in HIV+ men suggests
that these cells may be predominant HIV host cells in the male genital tract and
vectors of HIV transmission. A subgroup of HIV+ men with exceptionally elevated
seminal MO and CD4+ T-cell counts and HIV titers may be highly infectious and
contribute disproportionately to HIV transmission.
INTRODUCTION
The human immunodeficiency virus type 1 (HIV-1) is transmitted primarily by
sexual intercourse in the United States and worldwide.1-3 Transmission of HIV is
inefficient relative to many other sexually transmitted disease (STD) pathogens,
and a number of covariates such as concomitant STDs and acute and advanced HIV
disease stage have been associated with elevated titers of HIV-1 in genital
secretions and enhanced HIV transmission.4-6
Male genital tract organs and secretions are populated with white blood cells
(WBCs), which participate in immune defense functions (reviewed in Anderson and
Pudney7). HIV-infected WBCs have been detected in genital organs of
HIV-1-seropositive (HIV+) men.8 HIV-infected CD4+ T lymphocytes and macrophages
migrate from male genital tissues into semen,9 and recent studies have
implicated infected WBCs as vectors of HIV transmission.10-13 Therefore, seminal
WBC profiles provide important information concerning numbers and types of HIV
host cells in the male genital tract, potential cellular vectors of HIV
transmission, and immune defense of the male genital tract.
Mucosal epithelia are populated with memory CD4+ CCR5+ T cells, which are prime
targets of HIV infection.14 Memory CD4+ T cells in the gastrointestinal tract
are dramatically depleted during the early stages of HIV-1 infection before
effects are seen on CD4+ T cells in the peripheral blood.15,16 Several theories
have been presented to explain this effect: (1) memory CD4+ T cells are
preferentially infected and killed by HIV during acute infection because they
express high levels of CCR5 (HIV coreceptor), (2) memory CD4+ T cells are
activated and their lifespan is shortened by HIV infection,17 (3) these cells
are targeted by HIV through an interaction between gp120 and the integrin α4β7,
a mucosal homing receptor for peripheral blood T cells,18 and/or (4) the
migration of memory T cells from peripheral blood to mucosal sites is
disrupted.19 CD4+ T cells are also depleted in the female genital tract after
HIV and simian immunodeficiency virus (SIV) infection.20,21 However, the effects
of HIV infection, disease stage, and antiretroviral therapy (ART) on WBC
profiles in the male genital tract have not been described. The male genital
tract is also populated with memory mucosal T cells,22,23 and this cell
population is targeted by SIV during acute infection in male macaques..24 We
therefore hypothesize that HIV infection leads to depletion of CD4+ T
lymphocytes in the male genital tract. To test this hypothesis, we compared
concentrations of CD4+ T cells, and other WBC populations, in archived semen
samples from HIV-1-seronegative (HIV-) and untreated HIV+ men. Because recent
studies have shown that highly active antiretroviral therapy (HAART) partially
reconstitutes peripheral blood and mucosal CD4+ T-cell populations and immune
function in immunosuppressed HIV-infected subjects,25,26 we also enumerated
seminal CD4+ T cells and other WBC populations in archived semen samples from
HIV+ men on dual nucleoside therapy, and in the same cohort 6 months after
addition of a protease inhibitor (PI) (indinavir) to their ART regimen, to
determine effects of these classes of antiretroviral drugs and HIV suppression
on WBC profiles in semen.
DISCUSSION
Results from this study indicate that CD4+ T cells are depleted in the male
genital tract during HIV infection. Seminal CD4+ T-cell counts were
significantly decreased in ART- HIV+ men but were restored after treatment with
combination ART. These findings are consistent with clinical studies that have
documented depletion of CD4+ T cells at other mucosal sites during early HIV
infection and reconstitution of both peripheral blood and mucosal CD4+ T cells
in HIV+ subjects receiving HAART. A number of reports have documented depletion
of CD4+ T lymphocytes in the gastrointestinal mucosa of HIV-infected
individuals15,35 and their partial restoration after HAART.25,36,37 An earlier
study from our group also documented depletion of WBCs and CD4+ T lymphocytes in
the endocervix of HIV+ women in comparison to uninfected women,20 and Veazey et
al21 reported a similar effect after SIV infection of macaques. Because CD4+ T
cells play an important helper role in both cellular and humoral acquired immune
defense functions, the results of these earlier studies suggest that HIV
infection has a broad suppressive effect on mucosal immune defense functions at
various mucosal sites. A small study performed by Denny et al22 showing a 50%
reduction in the proportion of CD4+ T cells in semen from HIV+ men provided the
first evidence that seminal CD4+ T lymphocytes may be depleted after HIV
infection. The results of the present study showing decreased concentrations of
CD4+ T cells in semen from HIV+ men with high peripheral CD4+ cell counts
provide evidence that selective CD4+ T-cell depletion also occurs in the male
genital tract. These findings suggest that genital T-cell-dependent immune
defense functions may be impaired in HIV-infected men. If so, HIV+ men may be
more vulnerable to genital infections, some of which are cofactors for HIV
transmission.5 Our observation that seminal CD4+ T-cell counts rebound in HIV+
men after treatment with a viral-suppressing ART regimen provides evidence that
genital CD4+ T-cell populations are restored after suppression of viral
replication.
ART- HIV+ men in this study also had reduced seminal CD8+ T-lymphocyte
concentrations, suggesting that HIV infection impairs antiviral cellular immune
defense mechanisms in the male genital tract. This differs from earlier reports
that showed increased concentrations of CD8+ T cells in the endocervix20 and
gastrointestinal mucosa15 after HIV infection. This discrepancy may be related
to the time course of HIV disease and treatment or to differential effects of
HIV infection on the various compartments of the body. The men enrolled in this
study tended to have highly advanced HIV disease and may have initially
manifested increases in mucosal CD8+ cells that declined over time as they
became more immunocompromised. This is supported by data from the present study
indicating that seminal CD8+ T cells were significantly reduced only in men with
peripheral blood CD4+ cell counts less than 500 cells per cubic millimeter. Lim
et al16 found that gut mucosal CD8+ T-lymphocyte counts initially increased
after HIV infection but decreased after depletion of CD4+ blood counts. CD8+
T-cell counts in semen were increased after administration of combination ART
suggesting that ART also reconstitutes CD8-mediated cellular immune functions in
the male genital tract.
The effect of ART on semen WBC populations was most pronounced after the
addition of the PI, indinavir, to combination therapy. Because PIs suppress HIV
viral load in blood and semen,28,38-40 and indinavir penetrates the male genital
tract very effectively,41-43 this observation provides evidence that male
genital tract immune reconstitution occurs when HIV replication is suppressed
and suggests that genital immune depletion is directly related to HIV viral
load. The results of this study are limited by the small sample size, the short
duration of therapy, and the use of a narrow panel of ART drugs. The effects of
longer periods of HAART, and other combinations of antiretroviral drugs on
seminal WBC populations, should be studied.
Peripheral blood CD4+ cell counts in HIV+ men were not associated with CD4+ T
lymphocyte or other WBC concentrations in semen. Although this study did not
include men with acute HIV infection, decreased numbers of seminal CD4+ T cells
in men with high peripheral blood CD4+ cell counts suggest that this cell
population is depleted during early stages of HIV disease and remains so until
viral replication is fully suppressed by ART. Normally, HIV- and HIV+ men have
much higher concentrations of CD4+ cells in blood than semen.44,45 However, 2
men in the ART- HIV+ cohort were extreme outliers. These men, who had very low
peripheral blood CD4+ cell counts (40 and 23 CD4+ cells/mm3 blood), had
extraordinarily high concentrations of seminal CD4+ T cells (6.2 x 106/mL and
2.1 x 106/mL) and macrophages (15.5 x 106/mL and 24..4 x 106/mL). This dramatic
dissociation between CD4+ cell concentrations in blood and genital secretions
can occur because mucosal tissues and especially the male genital tract are
compartments with distinct immunological microenvironments. Regions of the male
genital tract are immunologically privileged sites due to immunological barriers
and high concentrations of immunosuppressive factors,7 and immune cell numbers
and their activation are usually tightly controlled. However, as the 2 outlier
subjects demonstrate, normal immune regulation in the male genital tract can be
disrupted. A number of studies have documented discordance between blood and
semen viral load in some subjects.46-48 Elevated seminal WBC counts are
associated with high seminal HIV viral loads,34,49,50 and the 2 individuals in
this study with dramatically elevated seminal WBC counts were no exception. It
is possible that such men are highly infectious. Factors that have been
associated with elevated concentrations of seminal WBCs and HIV include genital
infections with organisms such as cytomegalovirus50 and Neisseria gonorrhoeae.49
The men in this study with highly elevated seminal WBC counts did not have a
documented symptomatic STD, but it is possible that they had an asymptomatic
genital infection.
In summary, data from this study demonstrate that CD4+ T-cell counts decline in
the male genital tract before they decrease in the peripheral circulation of
HIV-infected men. This provides evidence that genital tract T-cell-dependent
acquired immune functions are impaired in HIV-infected men, perhaps rendering
them more susceptible to concomitant STD infections that can increase HIV
transmission rates. Data from this study also show that treatment with a viral
suppressive ART regimen is associated with significant restoration of CD4+ T
cells in the genital tract, and thus could improve acquired immune function in
the genital tract. Because the relative concentration of seminal macrophages
compared with CD4+ T lymphocytes was high in HIV+ men regardless of disease
stage, macrophages are likely primary HIV host cells in the male genital tract
and vectors of HIV transmission. The numbers of WBCs capable of transmitting HIV
(macrophages and CD4+ T cells) were reduced overall in semen from HIV+ men but
were strikingly elevated in a small subset (2/98 ART- HIV+ men in this study).
It is possible that men such as these with apparent genital immune activation
and elevated HIV titers in semen are highly infectious and may contribute
disproportionately to HIV transmission.
RESULTS
Comparison of Seminal WBC Subpopulations in HIV- and ART- HIV+ Men
ART- HIV+ men had significantly lower concentrations of total WBCs (P =
0..0008), macrophages (P = 0.0026), total T lymphocytes (P = 0.0001), CD4+ cells
(P = 0.0001), CD8+ T lymphocytes (P = 0.0063) and activated (interleukin-2
receptor-α+) T lymphocytes (P = 0.0001) in semen compared with HIV- men (Table
1). Because the anti-CD4 MAb recognizes CD4+ monocytes/macrophages,32,33 seminal
CD4+ T-lymphocyte counts were determined by subtracting the number of CD8+
lymphocytes from the total number of T lymphocytes. The seminal CD4+ cell count
was highly correlated with the seminal CD4+ T-lymphocyte count obtained using
this subtraction method (rho = +0.50, P < 0.0001); as was the case with the
total CD4+ cell count, CD4+ T-lymphocyte counts were also dramatically reduced
in ART- HIV-1-infected men (median counts: 5700/mL in HIV- men vs. 0/mL in ART-
HIV+ men, P = 0.0001).
Peripheral blood CD4+ cell counts did not significantly correlate with seminal
CD4+ T-cell counts or any of the other seminal WBC measures in ART- HIV+ men. To
further elucidate the relationship between peripheral blood CD4+ cell and
seminal WBC counts, ART- HIV+ men were stratified into high (≥500 cells/mm3) and
low (<500 cells/mm3) peripheral blood CD4+ cell groups. ANOVA indicated that
both ART- HIV+ groups had significantly lower concentrations of all seminal WBC
variables, except for CD8+ T lymphocytes, than HIV- men (P < 0.01, Fisher
protected least significant difference tests), and did not differ from each
other (P > 0.10). The results for seminal CD4+ T lymphocytes are shown in Figure
1. The majority of HIV+ men in both groups had undetectable seminal CD4+ T-cell
counts. Although we did not study men during acute HIV infection, 6 of 7
subjects with the highest peripheral blood CD4+ cell counts (>1000 cells/mm3)
had undetectable seminal CD4+ T cells, providing further evidence that genital
T-cell depletion occurs early in HIV disease, before profound reduction in
peripheral CD4+ cell counts. For CD8+ T lymphocytes, although both ART- HIV+
groups had a median of 0, only ART- HIV+ men with peripheral blood CD4+ counts
<500 cells per cubic millimeter showed a significantly lower concentration
compared with HIV- men (P = 0.0004).
Although ART- HIV+ men as a group had lower seminal WBC counts than HIV- men, 2
ART- HIV+ men with advanced disease stage had extremely high seminal WBC
concentrations (Table 2). One ART- HIV+ man with a peripheral blood CD4+ count
of 40 cells per cubic millimeter had a seminal WBC concentration of 55.68 x 106
per milliliter, with 15.5 x 106 per milliliter macrophages and 6.2 x 106 per
milliliter CD4+ T lymphocytes. Another ART- HIV+ subject with a peripheral blood
CD4+ T-lymphocyte count of 23 cells per cubic millimeter had a seminal WBC
concentration of 29.00 x 106 per milliliter, with 24.35 x 106 per milliliter
macrophages and 2.2 x 106 per milliliter CD4+ T lymphocytes. In both of these
cases, seminal WBC concentrations were >100-fold higher than the median values
for HIV- and ART- HIV+ groups. When assessed for HIV-1 using a microculture
technique,34 both samples had high titers of infectious HIV-1 in the cellular
fraction but not in the cell-free seminal plasma fraction. Neither of these men
had recent or concurrent symptomatic STDs as assessed by clinical history and
physical exam.
The Effect of ART on Seminal WBC Profiles in HIV+ Men
HIV+ men receiving dual nucleoside ART had significantly higher concentrations
of seminal WBCs including total, CD8+ and activated T lymphocytes, and
macrophages than did ART- HIV+ men (P = 0.0003, 0.0001, 0.0001, and 0.03,
respectively). HIV+ men on dual nucleoside ART also had modestly higher
concentrations of seminal CD4+ cells (P = 0.056) and seminal CD4+ T lymphocytes
(P = 0.03) than ART- HIV+ men. Six months after the addition of indinavir to the
ART regimen, substantial increases in CD4+ cell counts were observed in both
semen and blood [seminal CD4+ cells, P = 0.03; seminal CD4+ T lymphocytes, P =
0.001; peripheral blood CD4+ cells, P = 0.05 (Table 3)]. As was the case with
ART- HIV+ men, none of the seminal WBC measures were significantly correlated
with peripheral blood CD4+ T-lymphocyte count either before or after addition of
indinavir therapy in the ART cohort.
MATERIALS AND METHODS
Patient Populations
HIV- and ART- HIV+ Men
Subjects were 102 HIV-seronegative and 98 ART-naive (ART-) HIV-seropositive men
who have sex with men, receiving medical care at Fenway Community Health in
Boston, MA, between 1988 and 1993, before the widespread use of ART. Fenway
Community Health is the largest center caring for sexual and gender minority
patients in New England.27 Peripheral blood CD4+ cell counts in the HIV+ men
ranged from undetectable to 1290 cells per cubic millimeter (median = 410).
vART-Treated HIV+ Men
Twenty-two asymptomatic HIV+ men who have sex with men attending Fenway
Community Health (Boston, MA) for primary medical care at the beginning of the
HAART era (1996-1997) provided semen and blood samples for this study after
treatment for a minimum of 6 months with dual nucleoside ART and then again 6
months after addition of a PI (indinavir) to their ART regimen. Details of the
study population and effects of this treatment on seminal and blood HIV-1 levels
are reported elsewhere.28 At the start of the study, 15 participants were
receiving zidovudine/lamivudine; 4 stavudine/lamivudine; and 3
zidovudine/didanosine. After provision of blood and semen specimens for the
first (pre-PI) time point, men received indinavir (800 mg 3 times a day) in
conjunction with dual nucleoside analog therapy. Peripheral blood CD4+ cell
counts in these men before indinavir therapy ranged from 81 to 632 cells per
cubic millimeter (median = 238); 6 months after addition of indinavir, their
CD4+ cell counts ranged from 122 to 672 cells per cubic millimeter (median =
314).
General Methods
Semen Collection and Preliminary Analysis
This study was approved by the Institutional Review Board and was conducted in
accordance with the Helsinki Declaration of 1975, as revised in 2000.. Men
provided written informed consent for participation in the study. Semen was
obtained after a minimum of 48 hours of abstinence by masturbation into sterile
specimen containers. Samples were sent on ice packs immediately to the
laboratory and processed within 2 hours. Semen volume was measured, and
concentration of seminal round cells (a combination of WBCs and immature germ
cells, indistinguishable by phase microscopy29) was assessed microscopically on
a hemocytometer by a trained technician. Semen was diluted 1:1 in sterile
phosphate-buffered saline, and semen cells were pelleted by centrifugation at
400g for 10 minutes. Semen cells were washed 2 times in phosphate-buffered
saline before use in immunohistology assays.
Enumeration of Seminal WBC Populations
The low numbers of WBC subpopulations in semen preclude routine quantitative
analysis by flow cytometry.22 WBCs in semen were enumerated by an
immunohistology assay as previously described30 with slight modifications. The
following monoclonal antibodies (MAbs) were used: anti-HLe-1 (CD45) for
simultaneous identification of all WBCs, anti-Leu 4 + 5b (CD3) for all T cells,
anti-Leu 3a + 3b for detection of CD4+ cells (including T-helper/inducer
lymphocytes, monocytes, and macrophages), anti-Leu-2a for detection of CD8+ T
cytotoxic/suppressor lymphocytes (all from Becton Dickinson, Mountain View, CA),
Dako interleukin-2R for detection of interleukin-2 receptor-α (CD25) on
activated T lymphocytes, and Dako Macrophage for detection of CD68+
monocytes/macrophages (both from Dako Corporation, Santa Barbara, CA).
An aliquot of the washed semen cell fraction (about one fifth of the original
sample) was adjusted to a concentration of 106 round cells per milliliter
saline, and 5 microliters of washed semen cells or peripheral blood mononuclear
cells (positive control) were applied to individual spots of Teflon-coated
multiwell microscope slides (Roboz Surgical Instruments, Washington, DC), dried,
fixed in absolute acetone, and stored at -70°C. For use in the
immunohistology assay, slides were thawed and rehydrated in TRIS buffer (0.05
TRIS, 0.15M NaCl, pH 7.6). MAbs from the panel were applied to individual spots
on the multiwell slides, incubated at 37°C for 30 minutes, and rinsed in
TRIS buffer. Antibody-positive cells were visualized using an alkaline
phosphatase anti-alkaline phosphatase kit (Dako Corporation, Santa Barbara, CA).
All cells reactive with a MAb developed a red precipitate, whereas immature germ
cells, spermatozoa, and other MAb-negative cells appeared blue due to the
hematoxylin counterstain. After microscopically counting both antibody-positive
and antibody-negative round (nonsperm) cells in a minimum of 10 reticle fields,
the cell number was calculated based on the known round cell count determined
previously from the fresh sample. Testing of semen samples was conducted without
knowledge of serostatus, peripheral blood CD4+ cell count, or therapy status.
For HIV-1-infected men, the concentration of peripheral blood CD4+ cells was
determined by flow cytometry at an off-site clinical laboratory certified by the
AIDS Clinical Trial Group.
Statistical Analysis
StatView (version 5.0.1; SAS Institute, Cary, NC) statistical software was
utilized to perform the statistical computations. The various WBC measures did
not satisfy the assumptions of normal distribution and/or homogeneity of
variance.. Therefore, the nonparametric Mann-Whitney U test was performed to
determine differences between 2 independent samples, whereas the nonparametric
Wilcoxon signed rank test was used for comparing 2 related samples. For 3-group
comparisons, 1 factor analysis of variance (ANOVA) was performed on
log-transformed data. Statistically significant ANOVA (P < 0.05) was followed by
Fisher protected least significant difference post hoc tests for pairwise
comparison of groups. Correlations between variables were determined by the
Spearman rank-order correlation coefficient.31
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