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Departments of
*
Laboratory Medicine and
Medicine, University of Washington, Seattle, WA 98195; and
Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, WA 98109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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22%
of adults are infected with HSV-2, representing a 31% increase over
the last decade (1). After primary infection at mucosal
sites, HSV-2 remains latent in neuronal cells with intermittent HSV
reactivations, resulting in the production of infectious virus with or
without the onset of discernible disease. In immunocompetent persons
infected with HSV-2, viral reactivation is high and occurs on an
average of 20% of days (2). The high rate of HSV-2
shedding at mucosal sites suggests that the immune system is frequently
or even chronically exposed to HSV Ag and likely results in the
intermittent restimulation of HSV-specific T cells. Cellular immune defects are more closely associated with severe HSV disease than humoral immune defects, pointing to a critical role of HSV-specific T cells in the control and resolution of HSV disease. High frequencies of HSV-specific CD4+ and CD8+ T cell precursors are present in PBMC from immunocompetent HSV-seropositive individuals (3, 4), and both cell types infiltrate herpetic lesions (5, 6). In cross-sectional studies, immunosuppressed individuals with severe genital herpes infections (frequent and long-lasting lesions) had significantly lower frequencies of HSV-specific CD8+ CTL precursors than did individuals with mild disease (7). Clearance of HSV from lesions has been associated with the development of local HSV-specific cytotoxic activity; much of the cytolytic activity appears to be due to HSV-specific CD8+ CTL (6).
The high frequency of HSV reactivation despite robust CTL activity raises the question as to whether alterations in T cell specificity occur during the course of infection. Are there deletions of HSV-specific clonotypes and generation of new clonotypes over the course of infection? We sequenced the TCRBV genes utilized by HSV-specific CD8+ CTL clones that were isolated from lymphocytes infiltrating herpetic genital lesions (LIL).3 Clonotype-specific oligonucleotide probes were used to longitudinally track these clonotypes in PBMC and in LIL to determine their longevity and localization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunocompetent individuals with recurrent genital HSV infections were enrolled in an Institutional Review Board-approved protocol at the University of Washington Viral Disease Research Clinic. Subjects were part of our prospective cohort studies of the natural history of HSV-2 infection. We studied three HIV-seronegative individuals (two female, one male) with culture and serologically proven recurrent HSV-2. All three subjects had clinically symptomatic genital herpes for a mean of 19 years before the study and experienced symptomatic recurrences at a mean of 5 per year, a pattern typical of persons with frequent HSV-2 infection (8). Subjects were selected because their recurrences occurred on the buttocks (two individuals) and the thigh (one individual), sites where subjects would allow multiple biopsies.
Viruses
HSV-1 strain E115 and HSV-2 strain 333 were used throughout unless otherwise noted. HSV-1 x HSV-2 intertypic recombinant viruses (IRV) RH1G7, RS1G25, RS1G31, and R7015 (9, 10) containing 0.30–0.46, 0.59–0.73, 0.67–0.73, and 0.82–1.0 HSV-2 map units, respectively, were the kind gift of Bernard Roizman. HSV-1 x HSV-2 IRV Dx1(32), Bx1(13), Bx1(24), and RE6 containing 0.68–0.72, 0.0–0.57 and 0.7–1.0, 0.0–0.57, and 0.78–1.0, and 0.0–0.18 and 0.72–0.84 HSV-2 map units, respectively, were the kind gift of Howard Marsden (11). Clinical isolates 9349 and 9434 from patient (Pt.) 3 were isolated from a swab of an HSV-2 buttock lesion on December 15, 1997, and December 18, 1997, respectively. All viruses were grown and titered on Vero cells as previously described (12).
Cells
PBMC were isolated by Ficoll-Hypaque (Amersham Pharmacia, Piscataway, NJ) density centrifugation. EBV-transformed B cell lines (LCL) were established and maintained as previously described (13).
Lesion-derived HSV-specific CD8+ CTL clones
Lesion-infiltrating T cells were expanded in bulk from vesicle fluid or punch biopsy of HSV-2 culture-positive lesions with 0.8 µg/ml PHA-P (Murex Diagnostics, Dartford, U.K.), 32 U/ml IL-2 (Schiaperelli Biosystems, Columbia, MD), allogeneic irradiated PBMC, and 50 µM acyclovir (5). CD8+ T cells were positively selected using CD8 microbeads and MiniMACS columns (Miltenyi Biotec, Auburn, CA) from bulk lesion cultures containing CD8+ T cells (as determined by flow cytometry) and with cytotoxic activity (as determined by a standard 51Cr release assay). CD8+ T cells were cloned at 1 cell/well with PHA, IL-2, and irradiated autologous PBMC; screened for HSV-specific CTL activity; and expanded (4).
Cytotoxicity assays
Standard 51Cr release assays were performed (4). Briefly, target LCL (autologous or partially/completely mismatched at HLA class I) were mock infected or infected for 18 h with HSV-1, HSV-2, an HSV-1 x HSV-2 IRV, or a clinical HSV-2 isolate at a multiplicity of infection of 10 in the presence of 100 µCi 51Cr; washed; and counted. CD8+ T cell clones and 51Cr-labeled target LCL were cultured together using various E:T ratios. After 4 h at 37°C, 30 µl supernatant were removed and counted in Lumaplates with a Topcount scintillation counter (Packard, Meriden, CT). Percent of specific 51Cr release was calculated as [(mean experimental cpm - mean minimum cpm)/(mean maximum cpm - mean minimum cpm)] x 100%. Spontaneous release (target cells in media alone) was always <30% of maximum release (target cells in Nonidet P-40).
RNA extraction, PCR, and sequencing
Total RNA was extracted from CTL clones (1–2 x 106), PBMC (5–10 x 106), or bulk lesion cultures (3–5 x 106) by the single-step guanidinium method (14), and cDNA was synthesized using random hexamer primers, Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD), and dNTPs (Boehringer Mannheim, Indianapolis, IN). To determine the TCRBV usage of individual clones, cDNA was amplified with 1 of 26 TCRBV family-specific 5'-primers, a 3'-primer from the TCRBC region, dNTPs, and Amplitaq (Perkin-Elmer, Norwalk, CT). Primers (Fred Hutchinson Cancer Research Center, Biotechnology Center, Seattle, WA) were designed using primer sequence data from Ref. 15 . Products were separated on 2% agarose gels, soaked in ethidium bromide, and visualized by UV illumination. TCRAC 5'- and 3'-primers were used as a positive control. Negative controls included no cDNA and cDNA synthesis mixture with no RNA. PCR conditions consisted of 30 s denaturation at 95°C, 30 s annealing at 55°C, and 1 min extension at 72°C for 35 cycles.
Direct sequencing of PCR products was performed using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The TCRBV, TCRBD, and TCRBJ sequence of each clone was aligned relative to other TCRB sequences published in GenBank; and based on this alignment, specific oligonucleotides (15-mers) were designed to span the unique complementarity-determining region 3 of each clone.
Liquid hybridization
PCR product (7 µl) was mixed with 10 mM Tris (pH 8.0), 280 mM NaCl, 100 µM dNTPs, formamide, and 106 cpm 32P-labeled oligonucleotide in a total volume of 25 µl as previously described (16, 17). The amount of formamide used for each probe was adjusted to Tm 34°C. Mixtures were heated to 97°C for 5 min and cooled to room temperature during 15 min; and 10 µl were electrophoresed in 6% acrylamide gels, dried, and exposed to x-ray film.
Southern blotting
PCR products were separated on a 6% acrylamide gel and transferred to a nitrocellulose membrane. Specific DNA sequences were identified by hybridization with 32P-labeled clone-specific oligonucleotide in a mixture containing 5x SSC, 5x Denhardt’s solution, 1% SDS, and 100 µg/ml salmon sperm DNA. After hybridization at 68°C overnight, membranes were washed using high stringency conditions and exposed to x-ray film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HSV-specific CD8+ CTL clones were isolated
from LIL expanded from three immunocompetent HSV-2 infected
individuals. Of the 960 cloning wells, 10, 17, and 20% were positive
for growth from Pts. 1, 2, and 3, respectively; of these, 21, 5, and
10%, respectively, demonstrated cytotoxic activity to HSV-2. All of
the clones that reacted to HSV-2 were subsequently restimulated and
tested for HLA-restricted CTL activity. Table I
displays all the lesion-derived
CD8+ CTL clones from each of the three subjects
that grew and maintained HSV-specific lytic activity after
restimulation. All of the CD8+ CTL clones
specifically lysed autologous HSV-2-infected LCL and not autologous
mock infected LCL or HSV-2-infected allogeneic LCL completely
mismatched at the HLA class I locus (Table I). Interestingly, all the
clones were HSV-2 type specific in that they lysed autologous
HSV-2-infected LCL and not HSV-1-infected LCL (Table I). None of the
clones killed the NK-sensitive K562 cells (data not shown), confirming
the specificity of these clones.
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). Clone 3B.11 lysed LCL matched at A2 and B8 but not LCL
matched at A1 or B45. Clone 3B.11 lost killing activity after two
rounds of restimulation; thus, the HLA-restricting allele could not be
confirmed to either A2 or B8.
Because all lesion-derived clones were HSV-2 type specific, HSV-1
x HSV-2 IRV were utilized to define the antigenic specificity more
closely. Clones 1B.4, 1B.5, 3B.4, and 3B.8 recognized an HSV-2 epitope
contained within 0.0–0.18 map unit (Table I). Clones 2B.1 and 2B.11
recognized an epitope contained within 0.67–0.73 map unit, and clone
3B.22 recognized an epitope contained within 0.68–0.70 map unit. The
region on the HSV-2 genome recognized by 3B.11 could not be determined
because the clone lost killing activity after two rounds of
restimulation. These data indicate that the lesion-derived
CD8+ T cell response among these subjects with
frequently recurring HSV-2 appears, at least by these methods, to be
narrowly focused.
TCRBV usage of lesion-derived HSV-specific CD8+ CTL clones
RT-PCR using 26 TCRBV family-specific primers was performed on the
lesion-derived CD8+ CTL clones followed by DNA
sequencing of the TCRBV, TCRBD, and TCRBJ regions. For each of the
clones, a single dominant band was visualized on ethidium
bromide-stained agarose gels with only 1 of the 26 TCRBV
family-specific primers, a pattern confirming their clonality. All 14
of the clones isolated from Pt.1 utilized TCRBV12J1S1 genes,
and the TCRBV, TCRBD, and TCRBJ regions were identical at the
nucleotide level (Table II). This
suggests that these clones were the progeny of a single progenitor
clone, although whether this clone was expanded within the lesion in
vivo or by in vitro stimulation is unknown. Lesion-derived clones from
Pt.3 (3B.4 and 3B.8) were also identical at the nucleotide level, and
each utilized TCRBV17J1S5 genes. Clone 2B.1 and 2B.11 from
Pt. 2 were sister clones and utilized TCRBV10J2S4 genes,
whereas clones 3B.11 and 3B.22 utilized TCRBV5S2J2S2 and
TCRBV21J2S3, respectively (Table II). Thus, only Pt. 3
demonstrated evidence of more than one unique lesion-derived
CD8+ T cell clonotype from their original biopsy
specimen.
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Liquid hybridization was used to detect the presence of CTL clones
in PBMC and LIL. 32P-labeled clonotype-specific
oligonucleotide probes (15-mers) spanning the CRD3 region were used
(Table II). The concentration of formamide used in each hybridization
reaction reduced the probe Tm to 34°C.
Therefore, the stringency and specificity of each hybridization
reaction were very high. Any single-base pair mismatch in the probe
region would reduce the Tm to 67°C.
To test the specificity of this technique, PCR products from four
lesion-derived HSV-specific TCRBV12-expressing T cells
clones from Pt. 7 (an unrelated HSV-2-seropositive individual) were
mixed with the 1B.5-specific oligonucleotide. Clones from Pt. 7 were
lesion-derived CD4+ HSV-specific T cell clones
that differed by 7 or 8 nucleotides at complementarity-determining
region 3 (Fig. 1A). The 1B.5
oligonucleotide bound specifically to the PCR product from clone 1B.5
and did not hybridize to the PCR products from the
TCRBV12-expressing clones from Pt. 7 (Fig. 1B).
PCR product from all clones could be visualized by ethidium bromide
staining (data not shown), demonstrating that a negative signal in the
liquid hybridization reaction was not due to lack of PCR product. Thus,
even with >50% homology over a 15-nucleotide region, no
cross-hybridization occurred.
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C). A positive signal could be detected with 1 cell
RNA equivalent in a background of RNA from cells not expressing the
particular clonotype. Thus, this technique detected the RNA from 1 cell
expressing a particular TCRBV clonotype. HSV-specific CD8+ CTL clones in PBMC
To determine whether lesion-derived HSV-specific
CD8+ CTL clones were present and persisted in
PBMC from the same individual, we purified RNA from
CD8+ T cells from PBMC obtained at the time of
initial biopsy (if available) and prospectively over time. For these
studies, we utilized RNA from 5–10 x 106
unstimulated PBMC or CD8-selected PBMC. Liquid hybridization was
performed with the clonotype-specific probe. A representative
experiment on Pt. 1 is shown in Fig. 1. A positive signal was detected
in PBMC-derived CD8+ T cells isolated at the time
of the lesion from which clone 1B.4/5 was isolated (February 1995), 1
mo later (March 1995), 6 mo later (Fig. 1E, longer
exposure), and 2 years later (February 1997), but not in PBMC-derived
CD8+ T cells from an unrelated HSV-seropositive
donor (Fig. 1, D and E). Thus, this clonotype was
present simultaneously in a genital HSV lesion and in PBMC. Moreover,
we could detect the persistence of this clone in PBMC for at least 2
years. Similar results were obtained with Pt. 2; clones 2B.1 and 2B.11
were detected in PBMC at the time the clone was isolated from the
lesion (October 1994) and from all subsequent time points
(n = 3) (up to 4.75 years later) but not in PBMC from
an unrelated HSV-2-seropositive individual (Fig. 2A).
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A, and products were
eluted on an acrylamide gel, transferred to nitrocellulose, and probed
with the 32P-labeled 2B.1 clonotype-specific
oligonucleotide. As in Fig. 2A, 2B.1 was detected in PBMC
from Pt. 2 from all time points tested over 28 mo but not in PBMC from
an unrelated HSV-seropositive individual (Fig. 1B). Thus,
both techniques generated identical results.
Each of the three unique clonotypes isolated from Pt. 3 were also found
in PBMC. No PBMC were available from the time the clone was isolated
from a lesion in June 1991. Clones 3B.4 and 3B.8 were detected in PBMC
2 years after they were originally isolated but not detected in PBMC 7
years later (Fig. 3A). In
contrast, clones 3B.11 and 3B.22 were detected in PBMC obtained 7 years
after the clone was initially isolated (May 1998) (Fig. 3, B
and C).
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Once we developed the techniques to detect unique clonotypes in
lesions, we sought to determine whether the same clone infiltrated
subsequent herpetic lesions. For these studies, RT-PCR was performed on
RNA from LIL isolated from different episodes of HSV-2 reactivation.
Pt. 2 agreed to undergo two additional genital lesion biopsies from
recurrences 1 and 4.75 years after initial enrollment. Clone 2B.1 was
detected in the lesion from which it was isolated (October 5, 1994, and
October 7, 1994), from a lesion 1 year later on October 20, 1995 (Fig. 2C) and from a lesion 4.75 years later (data not shown). Pt.
3 agreed to have biopsies of HSV recurrences 6.5 and 7.5 years
postenrollment. Clone 3B.4, isolated from a genital lesion in June
1991, was detected in two subsequent lesions 6.5 (December 1997) and
7.5 (November 1998) years after the clone was initially isolated but
not during a recurrence on 10/98 (Fig. 3A). Clone 3B.11,
also isolated from a lesion in June 1991, was detected in biopsies from
October 1998 and November 1998 (Fig. 2B), and 3B.22 was
detected in the November 1998 biopsy (Fig. 3C). Fig. 3D summarizes the detection of clones from Pt. 3 in PBMC and
lesions. Therefore, lesion-derived HSV-specific
CD8+ CTL clones could infiltrate HSV recurrences
spaced >7 years apart. Pt. 1 did not agree to undergo further
biopsies, precluding study of local long term memory.
HSV-specific clonotypes retain cytotoxic function
To determine whether the clones isolated over time retained
cytolytic function, CD8+ T cell clones isolated
from LIL from Pt. 3 in December 1997 were expanded and tested for CTL
function. One of the CD8+ CTL clones that was
isolated was HSV-2 type specific and restricted to HLA-B45 (Fig. 3E). With the use of the HSV-1 x HSV-2 IRV, this
clone, termed 3B.51, was specific for an HSV-2 epitope contained within
the region 0.0–0.18, the same region as recognized by the earlier
clones 3B.4 and 3B.8. RNA from clone 3B.51 was isolated, and RT-PCR and
liquid hybridization performed with the 3B.4 clonotype-specific
oligonucleotide. The 3B.4 oligonucleotide hybridized with the PCR
product from clone 3B.51 amplified with the TCRBV17 primer (Fig. 3A). Sequencing of this PCR product confirmed that 3B.4 and
3B.51 were identical at the nucleotide level (data not shown), proving
definitively that this clonotype persisted for 7.5 years and retained
cytotoxic activity.
Lesion-derived HSV-specific CD8+ CTL clones lyse autologous HSV isolates obtained 6.5 years later
To determine whether HSV-specific CD8+ CTL
clones exerted selective pressure on HSV-2 resulting in the mutation of
CTL epitopes, we evaluated the lytic activity of CTL clones to the
viral isolate obtained from a buttock recurrence of Pt. 3, 6.5 years
after the CTL clones were initially obtained. Two separate clinical
isolates of HSV-2 obtained from lesion swabs on December 15, 1997
(strain 9349), and December 18, 1997 (strain 9434), were harvested and
used to infect autologous LCL. CTL clones 3B.8 and 3B.22, isolated in
June 1991, were tested for lytic activity against autologous LCL
infected with these 1997 viruses. Both clones lysed autologous LCL and
LCL matched at the appropriate HLA-restricting allele (B45 for 3B.8 and
A2 for 3B.22) infected with either clinical isolate (Fig. 4). This demonstrates that in an
individual with frequently recurring HSV disease, no detectable
mutations in viral genes to which CD8+ T cells
were directed could be identified.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the intriguing aspects of our study was the limited number of unique CD8+ memory T cell clonotypes we were able to derive from LIL and the fact that all the clones were specific for the infecting strain of virus, HSV-2, and were not cross-reactive with HSV-1. We may have skewed our results by the timing of biopsies in that HSV-specific CD8+ T cells infiltrating at times postbiopsy would not be expanded. Although this is a possibility, we did perform multiple biopsies at different stages and time points during lesion evolution. In each of these cases, consistency in the TCRBV usage in LIL was noted. Unfortunately, the total number of T cells that can be isolated from a single biopsy is extremely low, precluding direct analysis of T cells from lesions without expansion. Thus, it is possible that the clones we identified possess a unique stability compared with other clones. Highly restricted TCRBV usage has been observed in lymph nodes draining HSV infection in the murine model, providing further support of a restricted CD8+ T cell response to HSV; the vast majority of T cells recognize a single epitope of glycoprotein B (19, 20). Our data suggest that HSV-specific CD8+ T cell responses appear highly restricted in nature in terms of both TCRBV usage and immunogenic viral epitopes. We studied only persons with frequently recurring HSV, and it is possible that persons with infrequent disease have a more diverse response.
Immune evasion strategies used by HSV are myriad and include latency in immunologically privileged neurons, down-regulation of HLA class I (21, 22, 23), infection and inactivation of immune effector cells (13, 24, 25, 26), and inhibition of complement- and Ab-mediated viral neutralization (reviewed in Ref. 27). Selection of viral mutants that can escape immune surveillance has not been thought to be a mechanism of viral immune evasion utilized by herpesviruses due to the high fidelity of DNA polymerase. However, an example of viral mutation of a CTL epitope in a herpesvirus, namely EBV, has been described (28) although this mechanism does not appear to be universal (29). Few studies have performed detailed sequence analyses of sequential HSV isolates from individuals over time; however, "hot spots" for genetic change in the thymidine kinase gene have been described under drug selection (30). Our study is the first to use in vivo selection of T cell sequences as a mechanism to probe genetic changes in the virus. Our novel albeit limited look at this issue suggests no evidence of viral escape variants. The clinical isolates used to infect autologous target cells were recognized and killed by the CD8+ CTL clones, which is consistent with no mutation(s) of HSV-2 in the genes providing the CTL epitopes. Confirmation of this awaits the identification of the epitope and sequencing of HSV-2 corresponding to this region. However, this does not exclude the possibility that other epitopes had succumbed to immune pressure but that changes were not detected based on our experimental design.
Clonal exhaustion also does not appear to promote viral persistence to this human pathogen in immunocompetent adults. Even in the presence of frequent Ag exposure, shown in certain infections to cause clonal exhaustion or deletion of Ag-specific T cells (reviewed in Ref. 31), all the HSV-specific CD8+ CTL clones that we isolated from our three patients with frequently recurring HSV disease persisted for long periods of time and retained cytolytic function.
Our findings raise several questions related to the complex interaction between the virus and host. Is it the person with frequently recurring disease who has a restricted response whereas those with less frequent disease display a broader response? T cell diversity is likely to be an important component in the establishment and maintenance of a balanced host-virus coexistence. In HIV infection, another persistent human infection, polyclonal HIV-specific CD8+ T cell responses have been associated with better clinical status (32, 33). Do quantitative differences in memory T cell responses make a difference in disease severity in immunocompetent persons? In immunosuppressed persons, severe HSV disease was more closely correlated with low to undetectable numbers of HSV-specific CD8+ memory CTL (7). In the present report, we studied only people with longstanding infection; do the clones isolated at later stages of disease represent clones with more or less avidity/efficacy in vivo than clones isolated earlier in the disease process? Perhaps the major implication of our work is the development of a reproducible tracking assay to define the persistence and localization of a clonotype over time. With the elucidation of specific epitopes recognized by HSV-specific CD8+ CTL, use of the tetramer binding technology (34) will likely be helpful in defining more of the quantitative aspects of viral-host interactions for HSV. However, the novel techniques we outline here provide a useful model for studying the immunobiology of herpesviruses, viruses that persist and in which antigenic variation is not a recognized aspect of their life cycle.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christine Posavad, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, P.O. Box 19024, Room D3-100, Seattle, WA 98109.
3 Abbreviations used in this paper: LIL, lesion-infiltrating lymphocytes; LCL, lymphoblastoid cell line; IRV, HSV-1 x HSV-2 intertypic recombinant virus; Pt., patient.
Received for publication February 23, 2000. Accepted for publication May 8, 2000.
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, nucleotide matches.
B, Liquid hybridization using 1B.5-specific
oligonucleotide with PCR products from 1B.5 and the four TCRBV12 clones
from Pt. 7. C, Serial 10-fold dilutions of RNA isolated
from 1000 1B.5 cells were added to RNA from 3 x 105
CD8+ PBMC from an unrelated HSV-seropositive individual,
cDNA was synthesized, TCRBV transcripts were amplified, and liquid
hybridization was performed. 
