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*
Departments of Medical Microbiology, University of Nairobi, Nairobi, Kenya;
Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom;
Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada; and
§
Division of Infectious Diseases, Department of Medicine, University of Toronto, Toronto, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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release in response to known class I
HLA-restricted CTL epitope peptides using effector cells from the blood
and cervix of HIV-1-resistant and -infected sex workers and from
lower-risk uninfected controls. Eleven of 16 resistant sex workers had
HIV-specific CD8+ T cells in the cervix, and a similar
number had detectable responses in blood. Where both blood and cervical
responses were detected in the same individual, the specificity of the
responses was similar. Neither cervical nor blood responses were
detected in lower-risk control donors. HIV-specific CD8+ T
cell frequencies in the cervix of HIV-resistant sex workers were
slightly higher than in blood, while in HIV-infected donor cervical
response frequencies were markedly lower than blood, so that there was
relative enrichment of cervical responses in HIV-resistant compared
with HIV-infected donors. HIV-specific CD8+ T cell
responses in the absence of detectable HIV infection in the genital
mucosa of HIV-1-resistant sex workers may be playing an important part
in protective immunity against heterosexual HIV-1
transmission. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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32) are rendered relatively resistant to sexual
acquisition of HIV-1 (2). However, the CCR532 mutation
is largely confined to Caucasian populations (3), while
over 80% of new HIV-1 infections occur in the developing world
(4). Because heterosexual transmission accounts for
70–80% of infections in these populations (5), initial
host-virus interactions are likely to occur at the genital mucosa. Recent interest has focused on local HIV-specific immune responses in the genital tract of individuals who remain uninfected despite repeated sexual exposure to HIV-1 (highly exposed persistently seronegative, HEPS3). Mucosal HIV-1-specific IgA is found in many HEPS subjects (6, 7, 8), but it is thought that HIV-specific humoral responses alone may be insufficient to protect against infection (1). HIV-1-specific Th and CTL immune responses have been detected in the blood of HEPS individuals (8, 9, 10, 11, 12, 13, 14, 15). In addition, a stepwise increase in the frequency of bulk CTL recognizing HIV-1 env has been seen with increasing levels of prior HIV exposure in HEPS prostitutes, suggesting that HIV-specific CTL may be causally associated with protection against HIV-1 infection (16). However, although CD8+ lymphocytes from HEPS donors can protect against systemic HIV-1 challenge in a SCID/beige mouse model (17), other murine experiments have shown that mucosal rather than systemic (splenic) HIV-specific CTL are necessary to confer resistance to mucosal viral transmission (18). Recent work has shown that transient infection of the colonic mucosa in rhesus macaques can induce class I HLA-restricted CTL recognizing SIV env and that the presence of mucosal CTL correlates absolutely with protection against subsequent colonic viral challenge (19).
These studies suggest that while HIV-specific CTL are likely to be
important in mediating protective immunity, these responses may need to
be present in the genital tract to prevent heterosexual HIV-1
acquisition. Although HIV-1-specific CTL have been demonstrated in the
genital tract of HIV-1-infected women (20), there are no
previous data regarding mucosal CTL responses in the genital tract of
exposed, uninfected populations. This may be partly due to the
difficulties inherent in demonstrating low-frequency CTL using samples
obtained from a site with a rich microbial flora and that contain
relatively few T lymphocytes. The IFN- enzyme-linked immunospot
(ELISPOT) assay, which has been previously used to document
HIV-specific CD8+ responses in the blood of both
HIV-1-infected and HEPS donors (15, 21), provides an
estimate of Ag-specific CD8+ lymphocyte
frequencies that correlates closely with those measured by either
tetrameric MHC-peptide complexes or limiting dilution CTL assays
(22). ELISPOT offers the added advantages of being an
overnight assay, thereby minimizing the effect of bacterial
contamination, and of requiring fewer input cells than standard bulk
CTL assays (15, 22, 23). For these reasons, we have used
the IFN- ELISPOT to estimate CTL frequencies in the cervix and blood
of HEPS sex workers.
In an observational cohort study of the epidemiology and immunology of
HIV-1 in sex workers in a slum area of Nairobi (24, 25),
we have identified a subgroup of women who exhibit relative resistance
to HIV-1 infection (26). It is estimated that these women
have over 60 unprotected sexual exposures to HIV-1 per year, despite
behavioral counseling and provision of condoms; their exposure is
uniformly through vaginal intercourse. Resistance in this subgroup is
associated with systemic HIV-1-specific Th and CTLs (7, 15), mucosal HIV-1-specific IgA responses (7), and
certain MHC class I/II alleles (27), but not with
polymorphisms in CCR5 or altered cellular susceptibility to HIV-1
(3). We now report the occurrence and specificity of
HIV-1-specific, IFN--secreting, CD8+ T
lymphocytes in both cervix and blood of these HIV-1-resistant sex
workers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Women were enrolled through a dedicated sex worker clinic in the Pumwani district of Nairobi, Kenya, and were classified as HIV-1 resistant if they were seronegative on enrollment and remained both seronegative and PCR negative for at least 3 years of continuing sex work (26). We studied systemic and cervical responses to previously defined HIV-1 CTL epitopes in a subgroup of HIV-1-resistant and HIV-1-infected sex workers attending the annual clinic resurvey between October 1998 and February 1999. The influence of coexistent sexually transmitted infections (STI) on immune responses in the genital tract mucosa is unknown, and for this reason any women with clinical or laboratory evidence of STI were excluded.
Lower-risk HIV-1-uninfected control women were enrolled from a mother-child health care clinic in the Pumwani district of Nairobi and from an infertility clinic in Nairobi’s Kenyatta National Hospital. A physical examination was performed, and blood was drawn for HIV-1 and syphilis (rapid plasma reagin) serology. In both groups, women with clinical or laboratory evidence of cervicitis were excluded, as were those with any history of commercial sex work.
Informed consent was obtained from all study participants, and the study conformed to ethical guidelines from the University of Manitoba and the University of Nairobi.
General laboratory methods
Molecular HLA typing was performed on all study subjects using amplification refractory mutation system-PCR with sequence-specific primers, as previously described (28). HIV-1 serological testing employed a synthetic peptide enzyme immunoassay (Detect HIV, Biochem ImmunoSystems, Montreal, Canada), and positive tests were confirmed using a recombinant Ag enzyme immunoassay (Recombigen HIV-1/2 EIA, Cambridge Biotech Corporation, Galway, Ireland). All HIV-1-seronegative sex workers were confirmed to be HIV-1-uninfected employing a PCR system that uses primers for env, nef, and vif HIV-1 provirus genes (26, 29), which have been specifically adapted to detect African clades.
Flow cytometry analysis
Peripheral blood T lymphocyte subset analysis was performed using anti-CD4 FITC/CD8 PE (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Cervical mononuclear cell subpopulations were characterized from cryopreserved specimens using anti-CD3 FITC, anti-CD4 Tri-color, and anti-CD8 PE. After incubation with mAbs for 30 min at 4°C, 105 cells were washed in PBS with 1% FBS, fixed, and analyzed using a FACS flow cytometer (Becton Dickinson Immunocytometry Systems) with CellQuest software. Gating was used to select the lymphocyte fraction.
HIV-1 CTL epitope selection and peptide synthesis
HIV-1 peptides were selected from a panel of previously defined A, B, and D clade CTL epitopes. Epitope selection was based on 1) the class I HLA haplotype of the donor; and 2) where possible, on the results of previous systemic HIV-1-specific CTL assays in this study population (15). Peptides were synthesized by F-moc chemistry using a Zinnser Analytical synthesizer (Advanced Chemtech, Louisville, KY), and purity was established by HPLC.
Sampling and transport
Blood was drawn into 12-ml tubes containing the anticoagulant acid citrate dextrose. Cervical samples were obtained using a cytobrush (Histobrush; Spectrum Labs, Dallas, TX), which was inserted into the cervical os, gently rotated through 360°, and transferred immediately into 5 ml of RPMI 1640. To avoid contamination with blood, the cytobrush specimen was obtained before other sampling (STI cultures, etc.), was not collected from women who were actively menstruating, and was rejected if it contained visible blood. All samples were transported to the laboratory within 2 h. The cytobrush was vigorously agitated and discarded, and the remaining cell suspension was agitated to loosen any mucus clumps. Both cervical mononuclear cells (CMC) and PBMC were then isolated by Ficoll-Hypaque gradient centrifugation, washed, and resuspended in RPMI 1640 with 10% FCS (R/10).
IFN- ELISPOT assays
A modified ELISPOT assay was used to detect peptide-specific
IFN- release by either freshly separated or cryopreserved PBMC, and
by freshly separated CMC, as previously described (15).
First, 96-well nitrocellulose plates were precoated with a first layer
IFN- mAb, 1-DIK (MABTECH, Nacka, Sweden). PBMC or CMC were then
added in duplicate wells, either with predefined HIV-1 class
I-restricted peptide epitopes at a concentration of 20 µM or with no
peptide (negative control) or in 1:100 PHA (Murex Biotech, Dartford,
U.K.; positive control). PBMC assays were run at 2 x
105 and 5 x 104/well,
while CMC assays were frequently run at a single input concentration
due to low cell numbers. Plates were incubated overnight at 37°C in
5% CO2, then the cells were discarded, and the
plate incubated at room temperature for 3 h with a second
biotinylated anti-IFN- mAb (7-B6-1 biotin; MABTECH), followed by
streptavidin-conjugated alkaline phosphatase (MABTECH) for 2 h.
Individual IFN--producing cells were detected as dark blue spots
using an alkaline phosphatase-conjugate substrate kit (Bio-Rad,
Hercules, CA). The spots were counted by eye by an unblinded study
investigator, and the numbers were confirmed using a dissecting
microscope (magnification, x40).
HIV-1-specific IFN- responses were reported as number of
spot-forming units (SFU)/106 mononuclear cells,
after subtracting background rates of spontaneous IFN- secretion.
The background rate of IFN- secretion was defined as the number of
SFU/106 mononuclear cells incubated in media
alone, without peptide stimulation. Values reported generally represent
the HIV-1 responses quantified at the highest cell input numbers. An
HIV-1-specific ELISPOT response was defined as follows: 1) IFN-
release seen in response to 1:100 PHA (criterion for an adequate
assay); 2) 
20 HIV-1-specific SFU/106
mononuclear cells; 3) IFN- release in HIV-1 peptide wells exceeded
background (spontaneous) rates of IFN- release by a factor of at
least 2; and 4) if serial dilutions had been established, a titratable
response was required.
CD8 lymphocyte depletion assays
CD8+ lymphocyte depletion was performed
using anti-CD8+ Ab-coated immunomagnetic
beads (Dynabeads HLA cell prep I; Dynal, Lake Success, NY), according
to manufacturer’s instructions. Significant reduction of an ELISPOT
response was defined as a 50% reduction in HIV-1-specific IFN-
release after CD8+ lymphocyte depletion.
Data analysis
Statistical analysis used the SPSS for Windows Rel. 9.0.0 1998 package (SPSS, Chicago, IL). Comparison of means between study groups was performed by one-way ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In total, 26 HIV-1-resistant sex workers and 16 HIV-1-infected sex workers were enrolled between October 1998 and February 1999. The cervical samples from 10 HIV-1-resistant and 5 HIV-1-infected sex workers were inadequate for analysis (no response to PHA), so simultaneous cervical and systemic responses could be studied for 16 resistant and 11 HIV-1-infected sex workers. The mean cytobrush yield for adequate cervical assays was 4.3 x 105 CMC (range, 8 x 104 –1.2 x 106 CMC), and for inadequate assays was 6 x 104 CMC. The cell yields of adequate cervical samples did not differ between positive and negative assays (3.7 x 105 and 6.5 x 105 cells, respectively; p = 0.1). HIV-1 infection status did not influence cell yield from cervical cytobrushes (4.6 x 105 CMC for HIV-1-infected and 4.1 x 105 for HIV-1-resistant sex workers; p = 0.7). HIV-1-infected subjects had a mean blood CD4+ lymphocyte count of 416/mm3 (160–780/mm3) and CD8+ count of 1164/mm3 (770–2730/mm3). HIV-1-resistant sex workers had a mean CD4+ count of 975/mm3 (660–1602/mm3) and CD8+ count of 894/mm3 (320–1360/mm3).
Phenotypic analysis of CMC
Phenotypic analysis was performed on CMC specimens from three
HIV-1-resistant sex workers (representative example shown in Fig. 1
), and two HIV-1-infected sex workers. A
well-defined CD8+ lymphocyte population was
present in all samples, with CD8+ lymphocytes
comprising 11.8–24.1% of the gated lymphocyte subpopulation
(0.1–0.4% of total events). No differences were noted in
CD8+ lymphocyte frequencies according to HIV-1
infection status.
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HIV-1-specific IFN- responses were found in the cervix and
blood of 11 of 16 (69%) HIV-1-resistant sex workers (Table I; Fig. 2)
and in the cervix and blood of 8 of 11 (73%) HIV-1-infected sex
workers (Table I). HIV-1-specific responses in the cervix were
associated with a systemic response to the same HIV-1 CTL epitope in 9
of 11 (82%) resistant and 7 of 8 (88%) infected sex workers.
HIV-1-specific responses were localized to the blood in three subjects
(2 of 11 HIV-resistant and 1 of 8 HIV-infected subjects) and to the
cervix in three subjects (2 of 11 resistant and 1 of 8 infected
subjects). The ELISPOT responses of HIV-1-infected sex workers tended
to be greater in magnitude than those in resistant women, both in blood
(606.4 vs 62.2 SFU/106 PBMC; p =
0.01) and cervix (189.4 vs 78.4 SFU/106 CMC;
p = 0.2). Rates of background (spontaneous) IFN-
release did not vary between subjects with or without positive
HIV-1-specific responses, either in the blood (27.8 vs 22.1
SFU/106 PBMC, respectively; p =
0.4) or in the cervix (73.2 vs 67.2 SFU/106 CMC,
respectively; p = 0.8).
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Follow-up cervical specimens were obtained after a positive ELISPOT assay for one HIV-1-infected and four HIV-1-resistant sex workers. Persistent HIV-1-specific responses were found in the cervix of one of one HIV-1-infected and three of four HIV-1-resistant sex workers after an interval of 5–21 wk. The intensity of cervical responses had increased from baseline in two resistant sex workers (ML889, 20–150 SFU/106 CMC; ML1792, 25–67 SFU/106 CMC), had decreased in one (ML1622, 200–50 SFU/106 CMC), and were no longer detectable in one (ML1589). Data were not available concerning changes in sexual behavior over the intervening period.
HIV-1-specific IFN- responses are enhanced in the cervix of
HIV-1-resistant sex workers
To look at the relative intensity of systemic and cervical responses, a cervical-systemic differential was calculated for each peptide epitope by subtracting the peptide-specific systemic (PBMC) response from the cervical (CMC) response. This showed that the cervical responses were relatively enriched in the HIV-1-resistant group, compared with systemic (mean differential, +13.5 SFU/106; p = NS), while in the HIV-1-infected group systemic responses were considerably more intense than cervical (mean differential, -320.2 SFU/106; p = 0.008 between groups).
HIV-1-specific systemic and genital tract IFN- responses are
diminished or abrogated by CD8+ lymphocyte depletion
CD8+ depletion assays were performed using
PBMC from 10 HIV-1-resistant and 5 HIV-1-infected sex workers and using
cervical specimens from 3 resistant and 3 infected sex workers. In
general, PBMC depletion assays were run using cryopreserved samples
obtained at the same point in time as the original sample. Due to
limited cell numbers in cryopreserved mucosal specimens, cervical
CD8+ depletions were run using follow-up samples
obtained at a later point in time. Bead depletion resulted in a mean
depletion of CD8+ lymphocytes of 77%
(62.2–92.1%) in PBMC assays and of 82% in cervical assays
(74.9–89.5%). Systemic responses were diminished or abrogated after
CD8+ lymphocyte depletion in 8 of 10 (80%)
resistant (Fig. 3A) and 5 of 5
(100%) HIV-1-infected (Fig. 3B) sex workers; cervical
responses were diminished or abrogated in all HIV-1-resistant and
HIV-1-infected individuals (Fig. 4).
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Responses to class I-restricted HIV-1 CTL epitopes were studied in
the blood and cervix of seven HIV-1-uninfected, lower-risk
Kenyan women (Table I
), largely using peptides to which
responses had been detected in at least one HIV-1-resistant sex worker.
No HIV-1-specific cellular responses were found in the blood or cervix
of these lower-risk controls (Table I; p = 0.02 for
both comparisons).
HIV-1-resistant women do not respond to HLA-mismatched HIV-1 peptides
To confirm that these responses were HLA restricted, PBMC or CMC from HIV-1-resistant sex workers were incubated with HLA class I-mismatched CTL epitopes. The majority of HIV-1 peptides selected had been recognized by at least one HIV-1-resistant woman in HLA-matched assays, including: DTVLEDINL (A*6802, clade A pol); VSFEPIPIHY (A29, clade B gp120); LSPRTLNAW (B57/58, clade A gag); ATPQDLNMML (B53, clade A p24); DLNMMLNIV (B14, clade A p24); and VPLRPMTY (B35, clade B nef). No responses were seen to these HLA-mismatched epitopes in six of six PBMC and two of two CMC specimens from HIV-1-resistant sex workers (data not shown).
Associations with HIV-1-specific ELISPOT responses
No association was found between cervical or systemic responses
and total CD4+ or CD8+
lymphocyte counts in either HIV-1-resistant or HIV-1-infected subjects
(data not shown). Neither systemic nor mucosal HIV-1-specific IFN-
responses were associated with sexual risk behaviors as reported by
study subjects at the time of study enrollment, including number of
clients per day, duration of sex work, or frequency of condom use (data
not shown). Because women with STI were excluded from the study, no
data was available to examine the possible influence of concurrent STI
on systemic or mucosal HIV-1-specific responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The exact mechanism by which these mucosal CTL could function is not yet clear. HIV-1 can cross into the submucosa by transcytosis across a tight epithelial barrier, a process that can be inhibited by HIV-specific IgA (30). Furthermore, CD4+ and CCR5+ cell populations, which are susceptible to productive HIV-1 infection, are present in this region (31, 32). The cervical submucosa is well-supplied with CD8+ T lymphocytes, both of a memory (CD45RO+) and effector (perforin+) phenotype (33), and CD3+ cytolytic activity is present in the genital tract throughout the menstrual cycle (31, 32, 33). Therefore, we hypothesize that HIV-1-specific cervical CTL in the submucosa may target susceptible host cells, which have undergone productive infection by HIV-1 after viral transcytosis across the epithelial tight membrane.
HIV-specific systemic (blood) responses have been previously described in our study cohort of HIV-1-resistant sex workers (15, 16); however, the vast majority of HIV-1 transmission in this population occurs across the genital mucosa during heterosexual intercourse (25). Therefore, HIV-specific CTL in the cervix might play a more direct role than do systemic responses in immune-mediated protection against sexually acquired HIV-1 infection. If these mucosal HIV-specific CTL in the genital tract play a critical role in protection against HIV-1 infection in this prostitute population, then enrichment of HIV-1-specific CD8+ responses in the cervix might be predicted, because this is the site of viral exposure. In keeping with this hypothesis, we found that cervical responses were slightly enhanced relative to systemic in HIV-resistant prostitutes, while HIV-infected women showed considerable enrichment of HIV-specific responses in the blood. Although overall responses were weaker in HIV-resistant than HIV-infected prostitutes, their relative enhancement at the site of repeated viral exposure is consistent with the hypothesis that they play a major role in protection against heterosexual transmission of HIV-1.
Musey and colleagues have demonstrated that HIV-1-specific CTL (involving both CD4+ and CD8+ cells) could be generated from cervical specimens in HIV-1-infected women (20). In these HIV-1-infected women, comparisons of intraindividual cervical and blood CTL specificities also indicated that epitopes recognized by CTL in the cervix were commonly recognized in the blood, although relative frequencies of CTL in cervix and blood were not examined. The importance of these responses in the genital tract of HIV-1-infected individuals is not clear, although they could potentially play a part in reducing HIV transmission to sexual partners.
This study used an IFN- ELISPOT assay to demonstrate
CD8+ lymphocyte-mediated HIV-1-specific responses
from cervical and blood specimens. ELISPOT has been previously used to
document HIV-specific responses in both HIV-1-infected and HEPS donors
(15, 21). Estimates of Ag-specific
CD8+ lymphocyte frequencies using ELISPOT
correlate well with those measured by either tetrameric MHC-peptide
complexes or limiting dilution CTL assays (22). However,
the ELISPOT technique has the advantage over tetramer staining of
increased sensitivity at low precursor frequencies (1/50,000 as opposed
to 1/5,000 (23)), and ELISPOT also allows rapid screening
of responses against a wide variety of HIV-1 CTL epitopes, many of
which are not currently available as MHC-peptide tetramers. In
comparison to limiting dilution assays, the ELISPOT is a relatively
rapid assay and so is less subject to overgrowth by genital tract
flora. In addition, ELISPOT requires fewer effector cells than limiting
dilution assays, making it better suited to analysis of samples with
relatively few T lymphocytes.
Previous studies of HIV-specific immune responses in the genital tract
of HEPS subjects have been confined to the detection of HIV-1-specific
IgA, both in this cohort of HIV-1-resistant Kenyan sex workers
(7) and other exposed uninfected groups (6, 8). Eight of the 16 (50%) HIV-1-resistant subjects in the
present study have been enrolled in previous studies of genital tract
IgA (7, 34); either HIV-1-specific IgA or HIV-neutralizing
mucosal IgA was found in eight of eight (Table II), including three subjects who had no
demonstrable CD8+ lymphocyte responses in our
study. Several study subjects have also been tested for systemic HIV-1
env-specific Th responses (detected in five of seven)
(7); for systemic CTL responses against an HIV-1
env/vaccinia construct (detected in five of nine)
(16); and for systemic CTL responses against class I
HLA-restricted HIV-1 peptide epitopes (detected in three of three)
(15) (Table II). However, many of these data were only
available for a small subset of study subjects, and the data were not
collected at the same time as those in the current study. Because
HIV-specific immune response frequencies may increase or wane over time
(10), analysis of the association between various
responses based on these data may therefore not be warranted.
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The B57-restricted HIV-1 CTL epitope LSPRTLNAW, derived from the p24 region of A clade HIV-1, was recognized in several B58+/B57- women from both HIV-1-infected and -resistant sex worker groups. This is not unexpected, given that cross-presentation of B57-restricted HIV-1 epitopes by the structurally related B*5801 molecule (which is more common in African populations) has been well described (37, 38). Similarly, the B7 restricted epitopes TPGPGVRYPL (HIV-1 nef) and SPRTLNAWV (HIV-1 p24) were recognized in resistant sex worker ML851, who has B*8101 rather than B7; this is also likely to be due to cross-presentation by the closely related African B*8101 molecule (39, 40).
In summary, we have demonstrated CD8+
lymphocyte-mediated IFN- responses to HIV-1 CTL peptide epitopes in
the cervix of highly exposed, uninfected Kenyan sex workers. The
specificity of these responses was generally mirrored by systemic
(PBMC) responses. These HIV-1-specific responses were enhanced in the
genital tract at the likely site of repeated viral exposure and
persisted in some subjects for up to 5 mo. These responses were shown
to be mediated by CD8+ T cells using depletion
experiments in both HIV-resistant and HIV-infected subjects and were
not found in lower-risk control donors. These data suggest that
HIV-1-specific CD8+ responses in the genital
tract may play a role in protection against heterosexual HIV-1
infection in exposed, uninfected individuals.
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Acknowledgments |
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Footnotes |
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2 Addresss correspondence and reprint requests to Dr. Rupert Kaul, Institute of Molecular Medicine, Molecular Immunology Group, John Radcliffe Hospital, Headington, Oxford, OX3.9DS, United Kingdom. E-mail address:
3 Abbreviations used in this paper: HEPS, highly exposed persistently seronegative; ELISPOT, enzyme-linked immunospot; STI, sexually transmitted infection; CMC, cervical mononuclear cell; SFU, spot-forming unit.
Received for publication August 24, 1999. Accepted for publication November 16, 1999.
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