Download Semen of HIV-Infected Individuals Detection of HIV-1

Detection of HIV-1-Specific CTLs in the
Semen of HIV-Infected Individuals
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Alison J. Quayle, Wanda M. P. Coston, Alicja K. Trocha,
Spyros A. Kalams, Kenneth H. Mayer and Deborah J.
Anderson
J Immunol 1998; 161:4406-4410; ;
http://www.jimmunol.org/content/161/8/4406
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Copyright © 1998 by The American Association of
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References
Detection of HIV-1-Specific CTLs in the Semen of
HIV-Infected Individuals1
Alison J. Quayle,2* Wanda M. P. Coston,* Alicja K. Trocha,† Spyros A. Kalams,†
Kenneth H. Mayer,ठand Deborah J. Anderson*
he World Health Organization estimates that .70% of
HIV infections are established as a result of sexual contact (1). HIV in semen is found in both cell-associated and
cell-free forms (2), and HIV variants differ from those found in
blood, which suggests viral compartmentalization (3). The highest
viral loads in semen are associated with peripheral blood CD4
counts of ,200/ml (4, 5), concomitant sexually transmitted infections (6), and asymptomatic genital tract inflammation (4, 5). Conversely, semen virus levels decrease with antiviral therapy and
with treatment of sexually transmitted diseases (4, 6, 7).3 Since
intracellular virus is most readily eliminated by CTLs, the presence
of anti-HIV CTLs in urogenital mucosa would be predicted to
decrease viral load in genital secretions, contain virus locally, and
decrease the chance of transmission to sexual partners.
Vigorous memory CTL responses against all the major HIV
proteins have been demonstrated in the blood of infected individuals (8 –11). HIV-specific CTLs have also been isolated from various sites including the lymph node (12), spleen (12, 13), cerebrospinal fluid (14), and lung (15). Recently, SIV-specific CTLs were
isolated from the vaginal mucosa of chronically infected macaques
inoculated with SIV by the vaginal route (16), and HIV-specific
CTLs were isolated from the cervical mucosa of infected women
(17) indicating that, at least in the female, an antiviral response can
T
* Fearing Laboratory, Department of Obstetrics, Gynecology and Reproductive Biology,
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; †AIDS
Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA
02129; ‡Fenway Community Health Center, Boston, MA 02115; and §Memorial Hospital
of Rhode Island, Pawtucket, RI 02860 and Brown University AIDS Program, Providence,
RI 02903
Received for publication January 22, 1998. Accepted for publication June 5, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AI35564 and by the
Massachusetts Department of Public Health.
2
Address correspondence and reprint requests to Dr. Alison Quayle, Fearing Laboratory. Thorn 217, 75 Francis Street, Brigham and Women’s Hospital, Boston, MA
02115. E-mail address: [email protected]
3
Mayer, K. H., S. L. Boswell, R. S. Goldstein, W. Lo, C. Xu, L. Tucker, M. P.
Pasquale, R. D’Aquila, and D. J. Anderson. 1997. HIV persistence in semen after
adding indinavir to combination antiretroviral therapy. Submitted for publication.
Copyright © 1998 by The American Association of Immunologists
be generated and maintained locally in the genital tract mucosa.
We have recently demonstrated that the semen of HIV-infected
men contains functional T lymphocytes, and that the majority of
these cells are CD81 and express a marker of cytolytic activity
(18). In this study, we sought to determine whether T lymphocytes
from the semen of seropositive men also had anti-HIV cytotoxic
activity.
Materials and Methods
Patients
Five seropositive men were recruited from Fenway Community Health
Center and Massachusetts General Hospital to provide semen and blood
samples. Informed consent was obtained from all individuals. Eligibility
criteria included a previous leukocytic semen specimen (.5 3 104 viable
leukocytes per ejaculate) and a CD4 count of .500/ml. The clinical data
for each individual are summarized in Table I.
Isolation and cloning of effector cells
Semen was obtained by masturbation into sterile specimen cups containing
10 ml of RPMI 1640. Blood was collected in EDTA-treated vacutainers.
Viable semen round cells (SRCs)4 and PBMCs were isolated on density
gradient separation medium (Ficoll-Hypaque, Pharmacia, Piscataway, NJ
and washed three times in HBSS containing 100 mg/ml gentamicin and 1
mg/ml amphotericin B before resuspension in RPMI supplemented with 2
mM L-glutamine, 50 mg/ml gentamicin, 0.25 mg/ml amphotericin B, and
10% FCS (all obtained from Life Technologies, Grand Island, NY)
(cRPMI). Since the isolated SRC fraction contains a large proportion of
immature germ cells, SRCs (10% of sample) and PBMCs were applied to
8-spot slides, allowed to air dry, fixed in acetone, and later immunostained
with Abs to CD3 and CD8 to enumerate exactly the number of T cells (18).
Using published data obtained from leukocytic HIV1 individuals, we ascribed arbitrary values to the proportion of CD3 cells in each SRC population for immediate cloning purposes and adjusted the values after staining
(18). Arbitrary values were 1% for SRCs and 70% for PBMCs. Cells were
seeded in 96-well plates at numbers shown previously to approximate limiting dilution (10 –300 cells/well) in 100-ml volumes containing 1.5 3 106
irradiated (5000 rad) allogeneic PBMCs/ml, 1 mg/ml PHA, and 50 U/ml
IL-2 (Boehringer Mannheim, Indianapolis, IN) in cRPMI. After 2 to 3 wk,
wells exhibiting growth were restimulated with irradiated PBMCs, PHA,
and IL-2.
4
Abbreviations used in this paper: SRC, semen round cell; BLCL, B lymphoblastoid
cell line; IEL, intraepithelial lymphocyte.
0022-1767/98/$02.00
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CTLs play an important role in controlling cell-associated HIV. Since the majority of HIV infections are acquired through sexual
transmission, we investigated whether antiviral CTLs were present in the male urogenital tract using semen as a source of T cells.
We were able to establish anti-HIV cytolytic lines in five of five HIV-infected men with CD4 counts of >500/ml, although cloning
efficiencies were lower than with peripheral blood-derived T cells. CTLs generated from the semen of three men were analyzed
in detail and showed a broadly active response, recognizing gag, env, and pol proteins. Detailed analysis of two gag-specific clones
from one of the individuals demonstrated HLA class I restriction and recognition of the same p24 epitope (EQASQEVKNWMT).
In summary, our results demonstrate the presence of a broad CTL response to HIV in the urogenital tract and provide a rationale
for further studies of local enhancement of genital mucosal responses by anti-HIV immunization. The Journal of Immunology,
1998, 161: 4406 – 4410.
The Journal of Immunology
4407
Table I. HIV-related clinical history of donors
Donor
CD4 Counta
Viral
Loadb
Clinical
Coursec
Drugsd
No. of Years
Infectede
Mode of Transmission
9320
9408
POGO
221L
161J
932
725
.1400
575
.1400
NDf
ND
1.1 3 10c
6.0 3 10d
,400
Aysmp.
Aysmp.
Asymp.
K.S.
Aysmp.
None
None
None
None
None
9
.10
17
.12
.20
Sexual intercourse
Sexual intercourse
Sexual intercourse
Sexual intercourse
Contaminated blood products
Peripheral blood CD41 cell count/ml.
HIV RNA copies/ml plasma.
c
Asymp., asymptomatic; K.S., Kaposi’s sarcoma.
d
HIV-related medication (antiretrovirals, protease inhibitors).
e
Documented positive serological test excluding POGO. POGO documented seropositive from 1996, but single high risk
exposure in 1981 (personal history).
f
ND, not done.
a
b
Immunostaining
B lymphoblastoid cell lines (BLCLs)
Before cloning, BLCLs were established by transformation of peripheral
blood B cells by EBV obtained from the supernatant of the B95-8 cell
line (8).
Recombinant vaccinia viruses and synthetic HIV-1 peptides
Recombinant vaccinia viruses constructed from the HIV IIIB isolate and
expressing the following proteins were used in this study: env, gag, and pol
(Vabt 408); gag (Vabt 401); p17 (Vabt 228); p24 (Vabt 286) (kind gifts of
Therion Biologics, Cambridge, MA); env (PE16); and pol (CF21) (National Institutes of Health AIDS Repository, Rockville, MD). Wild-type vaccinia virus was used as a control (NYCBH, Therion Biologics). Synthetic
p24 HIV peptides (12–21 aa) were synthesized as described previously
(20). Lyophilized peptides were resuspended at 2 mg/ml in 10% DMSO.
Cytotoxicity assay
Cloned T cells were tested for HIV-specific cytolytic activity in a standard
chromium release assay (8). Target cells were autologous BLCLs pulsed
with HIV peptides or infected with vaccinia constructs expressing HIV
proteins (14). Partially MHC-mismatched allogeneic BLCLs were also
used in MHC restriction experiments. In brief, BLCLs to be vaccinia infected were incubated with 3 to 4 multiplicities of infection per cell of
recombinant virus for 16 to 18 h at 37°C. Cells were washed twice, labeled
with 150 mCi Na251Cr for 2 to 3 h, and washed four times. The sensitization of BLCLs with peptides was achieved by incubating cells with 10
mg/ml of peptide with the chromium. Clones and labeled BLCLs were
incubated together in various ratios in 150-ml volumes in 96-well plates at
37°C for 4 to 6 h. Supernatants (100 ml) were subsequently harvested and
counted in a Cobra gamma counter (Packard Instrument, Meriden, CT). The
percentage of specific lysis was calculated from the following formula: 100 3
([experimental release 2 spontaneous release]/[maximum release 2 spontaneous release]). Maximum release values were obtained by the lysis of targets
with 5% Triton X-100. Assays were only evaluated if spontaneous release was
,30% of maximum release. Clones were considered positive if lysis of the
targets was at least three times greater than the lysis of the control vaccinia
target and if the HIV-1-specific lysis was .10% (14, 21).
Results
Isolation, phenotyping, and HIV-specific cytolytic activity of
semen T cells
Our previous studies have shown that viable CD31 cell counts in
HIV1 men range from undetectable to .2.2 3 105 per ejaculate,
HIV-1 env-, gag-, and pol-specific CTLs in semen
To precisely define the HIV cytolytic responses in semen, lines
from three individuals were expanded and subsequently tested for
activity against the individual gag, pol, and env proteins (Table
III). Activity against all three proteins was noted in each donor,
indicating that CTLs derived from the urogenital tract have a
broad-based activity to HIV. With the exception of two lines, all
lines had activity against a single protein. In donor 9320, the predominant protein recognized was pol (43% of all specific activity),
followed by gag (36%) and envelope (21%). In donors POGO and
161J, the predominant protein recognized by CTLs was gag (67
and 57% of specific activities, respectively). In one individual
(9320), we also performed a parallel analysis on a small number of
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T cells were enumerated by a standard indirect immunohistology technique
that has been described previously in detail (18). Primary Abs recognizing
CD3 and CD8 Ags (Dako, Carpinteria, CA) were used, and Ab-positive
cells were visualized using an alkaline phosphatase/anti-alkaline phosphatase kit (Dako). A total of 200 cells were counted per Ab, and the number
of positive cells in the semen sample was calculated from the SRC count.
Clones were phenotyped using similar methodology, with Abs recognizing
the following Ags: CD3, CD8, CD4, CD57 (Dako), TCRab (BF1, a kind
gift of Dr. Michael Brenner, Department of Rheumatology, Brigham and
Women’s Hospital), and TIA-1 (a kind gift of Dr. P. Johnson, Dana-Farber
Cancer Institute, Boston, MA) (19).
with a median value of 9.0 3 103 in men taking antiretroviral
medication and 2.5 3 104 in men who are not on therapy (18).
Since the number of cells recovered from semen is relatively small
and the estimated frequency of HIV-specific T cells in asymptomatic individuals is estimated to range from 1/102 to 104 in PBMCs
(11, 22, 23), we optimized the conditions of this study by recruiting men who had previously had a leukocytic semen sample (defined as .5 3 104 viable mononuclear leukocytes per ejaculate).
A previous study from our laboratory indicates that this is not a
major selection step, since 83% of HIV1 men with peripheral
blood CD4 counts of .200/ml that are not on antiretroviral therapy
are included in this category (18). As shown in Table I, the median
CD4 count of the patients was 932, and the median length of infection was 12 yr. None of the individuals were on antiretroviral
therapy, and all were asymptomatic, with the exception of one man
who had recently developed Kaposi’s lesions.
The numbers of viable round cells recovered from the semen
samples used in this study ranged from 2.5 to 34.0 3 105 cells per
ejaculate (Table II). Immunostaining revealed that between 1.1%
and 14.3% of round cells (median 2.5%) were CD31, with CD81
lymphocytes contributing from 33 to 95% of the T cell population.
Cells were successfully cloned from semen at a limiting dilution
with PHA and IL-2, but cloning efficiencies were considerably
lower than those derived from blood (Fig. 1). Fungal contamination was also observed in some semen-derived cultures, but these
wells were not taken into account when calculating cloning efficiencies. Wells exhibiting proliferation were restimulated, and
those which were successfully expanded were screened for antiHIV cytolytic activity using autologous BLCLs infected with a
trivalent vaccinia vector expressing env, gag, and pol proteins.
HIV-specific cytolytic activity was found in all five donors, with
multiple lines isolated from each donor (Table II).
4408
HIV-SPECIFIC CTLs IN SEMEN
Table II. Enumeration, phenotyping, and assessment of HIV-specific cytolytic activity of semen-derived T
cells
Viable Cells in Semen
Semen CTL Activityd
Peripheral Blood CTL
Activityd
Patient
Round cells
3 105a
CD31 cells
3 103b
CD81 cells
3 103b
Wells exhibiting HIVspecific lysis wells
evaluablec
Wells exhibiting HIVspecific lysis wells
evaluablec
9320
9408
221L
POGO
161J
34.0
0.25
1.60
0.8
2.6
486.2
0.3
21.6
1.8
7.5
387.6
0.1
14.7
1.7
3.9
12/46
3/23
18/66
8/43
8/16
4/90
NDe
32/109
ND
ND
a
Total number of viable cells (mononuclear cells and immature germ cells) recovered from Ficoli density gradient separation
of semen cellular fraction.
b
Cells were phenotyped by standard immunocytochemical staining using an alkaline phosphatase/anti-alkaline phosphatase
detection system. A total of 200 cells were counted per mAb, and the numbers of cells per ejaculate expressing this Ag were
calculated from the total round cell count.
c
Total number of wells exhibiting growth and which could be expanded by one round of restimulation.
d
Lines were considered to have HIV-specific cytolytic activity if lysis of autologous BLCLs infected with the trivalent
vaccinia construct expressing env, gag, and pol was three times the value of BLCLs pulsed with wild-type vaccinia and .10%.
e
ND, not done.
to determine that both of these lines were restricted by HLA B44
(Fig. 3). Phenotyping studies indicated that 100% of the cells in
both lines stained positively for TCRab, CD8, and TIA-1.
MHC restriction and epitope recognition of gag-specific semenderived HIV-1-specific CTLs
Discussion
Two of the gag-specific lines from donor POGO were examined in
greater depth for fine epitope specificity, phenotype, and MHC
restriction. We first used vaccinia constructs expressing the p17
and p24 regions of gag to demonstrate that both gag-specific lines
recognized epitopes within the p24 protein (Fig. 2). When a series
of nested 21-mer peptides covering the p24 sequence in pools of
four (data not shown) were used followed by individual peptides,
data indicated that both lines recognized the gag peptide representing aa 303 to 324. Analysis with shorter (12-mer) peptides
indicated that both recognized the aa sequence 307 to 318. Using
BLCLs partially matched at MHC class I loci, we were also able
FIGURE 1. Relative cloning efficiencies of T cells derived from semen
(E) and from peripheral blood (l). Results are expressed as the percentage
of wells exhibiting growth after 15 to 20 days of culture with irradiated
allogeneic PBMCs, IL-2, and PHA. The number of wells at each cell concentration ranged from 30 to 50.
In this study, we describe a novel method for investigating CTL
activity in the male urogenital tract using semen as a source of
locally derived T cells. We reported previously that the semen of
asymptomatic, seropositive men not taking antiretroviral therapy
contains viable, functional T cells that are predominantly of the
CD81 phenotype and express markers of mucosal derivation and
cytolytic activity (18). Seminal T cells may originate from many
sites within the male genital tract including the rete testis, epididymis, prostate, vas deferens, and urethra (24). Since the potential
antiviral cytolytic activity of male urogenital tract cells has not
been investigated, we optimized study conditions by recruiting individuals who had peripheral blood CD4 counts of .500/ml and a
previous leukocytic ejaculate. We found substantial cytolytic activity in all five individuals examined, although cloning efficiencies were lower than in blood. This may reflect a lower functional
capacity of T cells recovered from semen due to their exposure to
highly immunosuppressive seminal plasma (25) or may reflect innate differences between T cells derived from a mucosal vs a peripheral location. For example, intraepithelial lymphocytes (IELs)
derived from normal gastrointestinal mucosa demonstrate a considerably lower proliferative capacity than their blood counterparts
(26), a phenotypically distinct profile (27), and an oligoclonal T
cell repertoire (28, 29), and may include unique regulatory subsets
of T cells (30).
The presence of HIV-specific CTLs in a mucosal site was first
demonstrated with T cells isolated from bronchoalveolar lavages
(15). The first isolation of HIV- or SIV-specific CTLs from the
genital tract was by Lohman et al. (16). Macaques were vaginally
infected with SIV, acutely and chronically infected animals were
sacrificed, and vaginal IELs were extracted, expanded, and tested
for CTL activity. IELs were characteristically CD81, TCRab1,
and CD21; precursor frequencies were similar to that in macaque
blood; and chronically infected monkeys had greater numbers of
virus-specific cells than those that were acutely infected. More
recently, HIV-specific CTLs were cloned from cervical mononuclear cells that had been extracted from HIV-infected women by
cervical cytobrush collection (17). Although cytobrush sampling
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lines derived from the peripheral blood. Although pol-specific activity dominated in the semen of this individual, only one of four
blood lines was pol-specific.
The Journal of Immunology
4409
Table III. HIV-specific cytolytic activity of semen-derived T cell lines
Specific Lysis (%)a
Donor
tri
gag
pol
env
9320
60-1
60-2
60-3
60-4
60-5
60-6b
100-1
100-2
100-3b
100-4
100-5
100-6
Total
2
2
2
1
2
4
2
2
2
2
3
2
24
19
18
40
82
18
87
84
32
7
8
80
2
1
1
48
8
3
80
79
16
3
5
64
5
38
28
25
3
90
14
3
5
24
5
3
2
6
8
9
1
5
8
20
8
4
5
11
10
4
3
POGO
10.4
30.17
30.35
30.37
30.39
30.42
Total
0
0
0
9
0
0
47
23
34
30
40
36
31
1
0
26
17
15
4
0
0
24
0
0
0
1
NDc
10
ND
ND
ND
ND
1
161J
60.1
60.2b
30.14
30.16
30.12
Total
0
0
0
0
0
56
25
32
46
38
2
57
18
11
14
4
3
22
0
0
0
1
67
54
0
4
0
2
a
Lines were considered cytolytic against HIV env, gag, or pol proteins if lysis of
autologous BLCLs infected with specific HIV vaccinia constructs was .10% and
three times greater than lysis of BLCLs infected with wild-type vaccinia. Cytolytic
clones are denoted in boldface.
b
CTL analysis of lines indicated activity against two or three HIV proteins.
c
ND, not done.
can induce local bleeding, the IEL-like phenotype of these cells
was suggestive of their mucosal derivation. The authors noted
HIV-specific activity in the cervix of 63% of the subjects sampled;
FIGURE 2. Semen-derived, gag-specific clones from donor POGO recognize the gag p24-specific epitope representing aa 307 to 318
(EQASQEVKNWMT). Autologous BLCLs were pulsed with wild-type
vaccinia (control), vaccinia recombinants expressing p17 and p24 gag proteins, the peptide representing aa 303 to 324 (TLRAEQASQEVKNW
MTETLLVQ), and truncations of this peptide. E:T ratios were 5:1.
FIGURE 3. Semen-derived, gag-specific clones from donor POGO are
restricted by HLA class I B44. Autologous (HLA class I A2, A30, B44,
B51, and Bw4) BLCLs or allogeneic BLCLs matched at one locus were
pulsed with the p24 gag peptide representing aa 303 to 324. E:T ratios were
5:1. Results are given as the percentage of HIV-specific cytotoxicity. Background counts (unpulsed BLCLs) were ,1% in all cases.
the highest anti-HIV cervical CTL activity was associated with a
peripheral blood CD4 count of .500 cells/ml. A comparison between small numbers of blood and cervix-derived gag-specific
lines in one donor indicated that CTLs derived from the two sites
recognized common epitopes. We did not undertake a comparison
of epitope specificities in the paired lines that we derived from one
of our donors, but we did note a predominance of pol specificity in
semen that was not seen in the blood of this individual. Clearly, it
still remains to be established whether blood and mucosal CTLs
exhibit different CTL repertoires, whether these will reflect differences in TCR repertoires, where and how Ag is presented to these
cells, or whether distinct HIV variants are being recognized (3).
The isolation of HIV-specific cytotoxic T cells from the female and
male urogenital tracts indicates that virus-specific lymphocytes are
recruited to the urogenital mucosa of HIV-infected individuals. The
presence of these effector cells in the genital tract may be critical in
controlling local HIV infection, thus reducing the viral load in mucosal secretions and the chance of transmission to partners during
sexual intercourse. A study looking at the correlation of CTL activity
with viral load in semen is currently underway. Indications that a local
immune response may prevent dissemination of virus come from a
study which found that some seronegative women with seropositive
partners and a history of multiple unprotected sexual exposures had
HIV-specific secretory IgA but not IgG in their vaginal secretions
(31). We presently hypothesize that an appropriately immunized individual with a strong, local, antiviral mucosal immune response
should be able to eradicate or to locally contain HIV following genital
transmission. If partners are concordant for MHC class I molecules,
an immunized individual may also be able to specifically kill donor
HIV-infected cells derived from the genital secretions of their partner
(32, 33). In contrast, MHC discordance may result in a host vs donor
immune response, and this allogeneic recognition may result in an
alternative mechanism of eradicating virally infected cells. Whether
MHC discordance between sexual partners would decrease the chance
of virus transmission or concordance would increase sexual transmission, as has been recently shown in a perinatal transmission study, has
yet to be investigated (34).
Clearly it would be advantageous to develop an anti-HIV vaccine that would elicit HIV-specific mucosal CTLs. Although some
studies indicate that immunization at a mucosal site is not a prerequisite for induction of a mucosal immune response (35, 36),
there is a substantial amount of evidence to indicate that mucosal
immunization strategies are more effective at generating a local
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wt
4410
response (37). In addition, long-lived antiviral CTLs can be detected in mucosal tissues after mucosal but not systemic immunization, suggesting that the maintenance of memory cells is also
dependent upon the chosen route of immunization (38). Of further
note is the observation that inoculation at proximal mucosal sites
appears to be more effective at inducing measurable immune responses than inoculation at distant mucosal sites. For example,
recent studies have indicated the effectiveness of a vaginal or rectal
immunization protocol in inducing a genital tract immune response
(39 – 41). Consequently, we plan to address in our future studies
whether antiviral CTL responses can be induced in the male urogenital tract by vaccination and, if so, which routes of immunization are the most effective at inducing and maintaining a local
immune response.
Acknowledgments
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We thank the individuals who donated samples for this study. We also
thank Dr. Eric Rosenberg at Massachusetts General Hospital and Robert
Goldstein and Judy Erdman at Fenway Community Health Center for recruiting patients, Lidiya Shver and Lynne Tucker for excellent technical
assistance, and Dr. Gail Mazzara at Therion Biologics for providing recombinant vaccinia viruses.
HIV-SPECIFIC CTLs IN SEMEN