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The majority of new human immunodeficiency virus type 1 (HIV-1) infections are acquired through sexual activity (15). Cell-free and cell-associated virus particles are shed in semen (11, 17) and cervicovaginal secretions (13, 41). Animal studies suggest that this virus crosses intact genital mucosae in sexually exposed individuals and infects antigen-presenting cells, such as dendritic cells and macrophages, in the mucosa of the rectum, the penile foreskin, and the distal urethra of the male and in the cervix and vagina of the female (24, 25, 36). Local nonspecific immune mechanisms, such as proteases, defensins, cytokines, low pH, and H₂O₂-producing bacteria (3, 4), likely influence the efficiency of sexual transmission. Virus-specific immune defenses in the genital tract have also been documented. Anti-HIV-1 antibodies have been detected in genital secretions from men and women (5, 6), and virus-specific cytotoxic T lymphocytes (CTL) have been demonstrated in cervical specimens (28) and semen (32).

Accumulating evidence has implicated CTL in containing the systemic spread of HIV-1 in infected individuals (21). The emergence of HIV-1-specific CTL has been correlated temporally with the damping of viral replication during primary infection (7). In addition, a stable clinical status in HIV-1-infected individuals has been associated with high, persistent levels of circulating CTL (34). The presence of HIV-1-specific CTL in the reproductive tract suggests that these cells might play a role in limiting sexual transmission (28, 32).

An animal model that closely parallels the pathogenesis of AIDS is critical to the elucidation of mucosal events in AIDS immunopathogenesis and the development of vaccine strategies that enhance mucosal immune responses against venereally transmitted HIV-1. Simian immunodeficiency virus (SIV)- and chimeric simian/human immunodeficiency virus-infected macaques develop an AIDS-like illness characterized by CD4⁺-T-lymphocyte loss and the development of opportunistic infections and tumors (22, 23). The evaluation of AIDS virus-specific CTL responses in these models has been facilitated by the definition of CTL epitopes and their restricting major histocompatibility complex (MHC) class I alleles in rhesus monkeys (2, 27). However, characterization of mucosal CTL has proven to be difficult because of the limited cell yields from sampled mucosal sites. Our laboratory (18) has recently shown that SIVmac-specific CTL can be detected in relatively small samples of rhesus monkey cells by utilizing a tetrameric MHC class I-peptide complex constructed with Mamu-A*01 and the dominant CTL epitope 9-amino-acid fragment of SIVmac Gag, which we refer to as p11C, C-M (amino acids 181 to 189). With this technology, we have characterized virus-specific CTL in the blood and lymph nodes of rhesus monkeys during primary and chronic SIVmac infection (19, 20) and in naive monkeys immunized with a recombinant modified vaccinia virus Ankara-SIV gag-pol construct (35). In the studies described in this report, we sought to characterize SIVmac-specific CD8⁺ T lymphocytes with cytotoxic activity in male genital fluid by using the tetrameric Mamu-A*01-p11C, C-M complex.

We first screened multiple semen specimens from five uninfected control and four SIVmac-infected adult (7- to 10-year-old) rhesus monkeys (Macaca mulatta) for the presence of lymphocytes by flow cytometry and microscopy. Animals were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (29). All nine monkeys shared the MHC class I allele Mamu-A*01, as determined by a PCR-based technique described elsewhere (16, 18). Infected monkeys received 20 animal infectious doses of uncloned SIVmac strain 251 by intravenous inoculation at least 1 year prior to the initiation of these experiments. All uninfected monkeys and three infected monkeys were clinically asymptomatic at the time of this study. Infected monkey 575 exhibited chronic wasting and diarrhea consistent with AIDS.

Ejaculates were obtained by electrostimulation of anesthetized monkeys via a rectal probe method (39). Reproductive performance in rhesus monkeys can be influenced by environmental factors, such as season and housing (12, 38). Furthermore, unlike the semen of most mammalian species, the semen of rhesus monkeys often contains a small fluid fraction in association with a firm coagulum that resists liquefaction (14). For these reasons, monkeys were chosen for this study on the basis of the production of adequate quantities of seminal fluid during electrostimulation. Seminal cells were washed with RPMI 1640 supplemented with penicillin (100 U/ml)-streptomycin (100 µg/ml) and 2% fetal bovine serum. Flow-cytometric analyses were conducted with a Coulter EPICS Elite ESP flow cytometer (Beckman Coulter, Inc.). Data analysis was performed with EPICS Elite software, version 4.02 (Beckman Coulter, Inc.). Data presentation was performed with WINMDI software, version 2.7 (Joseph Trotter, La Jolla, Calif.) and Power Point software, version 4.0c (Microsoft Corp.).

Mononuclear-cell numbers in freshly collected, unfractionated ejaculates were enumerated by using a hemacytometer and ranged from 0 to 6 × 10⁵ per ejaculate for the uninfected controls and from 0 to 9.6 × 10⁶ per ejaculate for the SIVmac-infected monkeys. On flow-cytometric analysis of multiple specimens, a distinct cell population with forward- and side-scattering properties consistent with mononuclear leukocytes was evident in the semen from only one of the five uninfected control monkeys (data not shown). The presence of lymphocytes and macrophages was confirmed by microscopic examination of cytologic preparations of semen. In contrast to what was observed in the uninfected animals, ejaculates from all four of the SIVmac-infected monkeys contained lymphocytes and macrophages during at least one collection period. Semen from one of the infected monkeys (no. 575) contained an additional cell population exhibiting the high forward- and side-scattering characteristics of granulocytic cells; microscopic examination confirmed the presence of neutrophils, in addition to lymphocytes and macrophages, in this specimen (data not shown).

We next analyzed those ejaculates with sufficient numbers of lymphocytes, as well as whole blood anticoagulated with EDTA, for the presence of T-cell subpopulations by flow cytometry. Mononuclear-cell populations were gated on the basis of forward and side light scattering properties and evaluated for binding of the allophycocyanin (APC)-coupled monoclonal antibody (MAb) FN18, which recognizes rhesus monkey CD3 (a gift from D. M. Neville, Jr., National Institutes of Health, Bethesda, Md.), and for the expression of CD4 by using anti-CD4 (OKT4; Ortho Diagnostics Systems, Inc.). Specimens were incubated with antibodies for 15 min before being washed with phosphate-buffered saline-2% fetal bovine serum and then fixed with formaldehyde. The percentages of CD4⁺ T cells in semen were lower than those in whole blood for specimens from all monkeys examined, including the uninfected control monkey with leukocytic semen (61.0 and 38.5% for blood and semen, respectively) and three of the SIVmac-infected monkeys, 579 (38.4 and 6.4%), 575 (60.0 and 6.4%), and 403 (32.0 and 25.0%). These findings are in accordance with observations in humans that have shown low concentrations of CD4⁺ T cells relative to the concentrations of CD8⁺ T cells in semen from healthy, fertile individuals (40) and in HIV-seropositive men (33).

To determine whether T-cell populations in semen from SIVmac-infected monkeys contain a virus-specific CD8⁺ subset, unfractionated seminal cells from SIVmac-infected Mamu-A*01⁺ rhesus monkeys were analyzed for the presence of tetrameric Mamu-A*01-p11C, C-M complex-binding CD8⁺ T cells (Fig. 1). Previous work has shown that this tetramer complex primarily binds CD3⁺ CD8/⁺ cells (18). Therefore, Mamu-A*01-p11C, C-M complex-binding cells were evaluated in gated CD3⁺ CD8/⁺ cell populations. Tricolor flow-cytometric analyses of freshly collected, unfractionated seminal cells (containing 1 × 10⁵ to 6 × 10⁵ spermatozoa) and 100 µl of fresh whole blood were performed with the soluble phycoerythrin (PE)-coupled tetrameric Mamu-A*01-p11C, C-M complex as described by Seth et al. (35). The tetramer was used in conjunction with the MAbs anti-CD3 (FN18)-APC and anti-CD8/ (2ST8-5H7)-PE-Texas red (ECD; Beckman Coulter, Inc.). Specimens were incubated with the tetramer on ice for 20 min; then antibodies were added. After a 15-min incubation on ice, the specimens were washed and then fixed in formaldehyde. Tetramer-binding T cells were undetectable in the peripheral blood and semen from the uninfected control monkey with leukocytic semen (Fig. 1). The percentages of tetramer-positive CD8⁺ T cells in blood and semen from infected monkeys ranged from 0.4 to 16% and from 5.9 to 22.0%, respectively. These values are in the same ranges as those observed in peripheral blood and lymph nodes of chronically infected monkeys (20). Interestingly, the percentage of tetramer-positive CD8⁺ T cells was higher in semen than in blood for three of the evaluated monkeys (Fig. 1).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Percentages of CD3+ CD8/+ cells binding tetrameric Mamu-A*01-p11C, C-M complex in peripheral blood leukocytes (PBL) and freshly collected semen of an uninfected Mamu-A*01+ control rhesus monkey with leukocytic semen (SIV) and SIVmac-infected, Mamu-A*01+ monkeys (no. 579, 138, 403, and 575) as determined by tricolor flow cytometry.

The low mononuclear-cell yields from these ejaculates precluded the analysis of cytotoxic activity in freshly collected specimens. Therefore, we assessed antigen- or mitogen-stimulated seminal lymphocytes in three infected monkeys for their ability to recognize and lyse autologous target cells expressing SIVmac gene products. Seminal lymphocytes were obtained by density gradient centrifugation (Ficoll-Hypaque) and expanded for 12 days in interleukin-2-containing (20 U/ml) medium, after stimulation with either p11C, C-M-pulsed, irradiated, autologous peripheral blood mononuclear cells (monkeys 579 and 138) or concanavalin A (5 µg/ml; Sigma Chemical Co.) (monkey 403) as described elsewhere (18). Lymphocytes were then assessed as effector cells in a standard ⁵¹Cr release assay by using U-bottomed microtiter plates containing 10⁴ target cells with various concentrations of effector cells. Autologous B-lymphoblastoid cell lines were used as target cells and were incubated with 1 µg of p11C, C-M (CTPYDINQM)/ml or 1 µg of the negative-control peptide p11B (ALSEGCTPYDIN)/ml for 90 min during ⁵¹Cr labeling. Plates were incubated in a humidified incubator at 37°C for 4 h. Specific release was calculated as follows: [(experimental release  spontaneous release)/(maximum release  spontaneous release)] × 100. Spontaneous release was <20% of the maximal release with detergent (2% Triton X-100) in all assays. Lysis was assessed at six effector/target ratios.

Total seminal mononuclear-cell numbers increased approximately two to fivefold upon stimulation in vitro. As shown in Fig. 2 (top), flow-cytometric analysis demonstrated the presence of tetrameric Mamu-A*01-p11C, C-M complex-binding CD8/⁺ T cells after cultivation. Gag epitope-specific cytolytic activity was confirmed in all specimens after in vitro expansion, as determined by a functional assay (Fig. 2, bottom). Compared with that in freshly collected seminal cells (Fig. 1), the proportion of tetramer-positive cells doubled in cultured cells from monkey 579, decreased in cultured cells from monkey 138, and remained unchanged in cultured cells from monkey 403 (Fig. 2). In a study of semen from HIV-1-infected humans, the cloning efficiencies of seminal mononuclear cells were found to be lower than those observed in blood from the same individual (32). In vitro growth characteristics of antigen-specific lymphocyte populations in ejaculates may be influenced, in part, by inhibitory factors in semen (1, 37) or the absence of sufficient concentrations of functional antigen-presenting cells. Alternatively, seminal T lymphocytes may represent end-stage cell populations with a limited capacity to proliferate.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   (Top) The percentages of tetramer-binding CD3+ CD8/+ cells in aliquots of cultured seminal lymphocytes from SIVmac-infected Mamu-A*01+ monkeys, assessed by flow cytometry as described in the text. (Bottom) SIVmac Gag epitope p11C, C-M-specific lytic activity in in vitro-expanded seminal lymphocytes from these monkeys. E/T, effector/target.

Previous work has shown that epitope-specific CD8⁺ T cells in peripheral blood and lymph nodes of SIVmac-infected rhesus macaques express cell surface molecules associated with memory and activation (18, 20). To determine whether tetramer-positive CD8⁺ T cells in semen have a similar phenotype, CD3⁺ CD8/⁺ tetramer-binding cells from the ejaculate of an SIVmac-infected, Mamu-A*01⁺ rhesus monkey (no. 579) were analyzed by four-color flow cytometry. Analysis was performed on gated CD3⁺ CD8/⁺ T cells in freshly collected, unfractionated seminal cell specimens by using biotinylated tetrameric Mamu-A*01-p11C, C-M complex coupled to Alexa 488-labeled NeutrAvidin (Molecular Probes), anti-CD8-ECD, anti-rhesus monkey CD3-APC, and either anti-CD11a (25.3.1)-PE, anti-CD45RA (2H4)-PE, anti-HLA-DR (MHC class II; 13)-PE (Beckman Coulter, Inc.), anti-CD62L (Leu8)-PE (Becton Dickinson), or anti-CD95 (Fas; DX2)-PE (Caltag).

Similar to circulating and lymph node CTL (18, 20), seminal tetramer-positive CD8⁺ T cells expressed the activation-associated molecules CD11a, CD95, and MCH class II (Fig. 3). Most cells failed to express CD62L and CD45RA, molecules associated with a naive phenotype (Fig. 3). (No anti-CD45RO MAb is currently available for staining rhesus monkey lymphocytes.) A similar array of cell surface molecules was also expressed on tetramer-negative CD3⁺ CD8/⁺ T cells from this monkey (Fig. 3). Although the concentration of lymphocytes available from the uninfected control monkey with leukocytic semen was insufficient for analysis of all of these markers, approximately 75% of the CD3⁺ cells in ejaculates from this animal expressed MHC class II (data not shown). A predominantly memory-associated phenotype (CD45RO⁺) has been observed in CD4⁺ cell populations in semen from both HIV-seronegative and -seropositive men (9). Thus, T lymphocytes that are shed or migrate into the seminal compartment most likely represent activated, memory populations that can include virus-specific cells.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Phenotypic characterization of tetrameric Mamu-A*01-p11C, C-M complex-negative (Tetramer) and complex-positive (Tetramer+) CD8/+ T cells in freshly collected semen from an SIVmac-infected Mamu-A*01+ monkey (no. 579). Flow cytometric analysis was performed on gated CD3+ CD8/+ T cells.

The source of antigen-specific CD8⁺ T cells in semen is uncertain. Like humans (10, 31), macaques have T-lymphocyte populations in the mucosa and submucosa throughout the male reproductive tract, with the exception of the germ cell compartment (26). The Gag-specific CD8⁺ T lymphocytes detected in ejaculates from these monkeys were phenotypically similar to CTL previously examined in peripheral blood and lymph nodes (18, 20). However, the discordance between the percentages of tetramer-positive CD8⁺ T cells in semen and blood in the same monkeys suggests that genital tract CD8⁺-T-cell populations may not necessarily be influenced by the same factors that regulate systemic cell-mediated immune responses. The suggestion that HIV-1 replication is to some extent compartmentalized in the reproductive tract is supported by data demonstrating differences in virus loads and viral genotypes in genital secretions and blood (8, 30, 42). Thus, seminal CTL activity may reflect regional differences in virus replication, as well as unique environmental factors such as immunosuppressive constituents, prostaglandins, hormones, endogenous microorganisms, or local inflammation.

These results provide evidence that cytolytically active, virus-specific lymphocytes are a component of the regional immune response in the male reproductive tract during chronic SIVmac infection. Obtaining sufficient numbers of viable lymphocytes from genital secretions for functional characterization can be problematic. Therefore, recent studies of HIV-1-specific CTL in the reproductive tract have relied on expanding cells through cloning (28, 32). The present experiments illustrate the power and efficiency of the tetramer technology for directly evaluating freshly collected, antigen-specific CD8⁺ lymphocytes in vivo, particularly in specimens with limited cell yields.