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Journal of Virology, May 2004, p. 4927-4930, Vol. 78, No. 9
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.9.4927-4930.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Intraepithelial T Cells May Bridge a Gap between Innate Immunity and Acquired Immunity to Herpes Simplex Virus Type 2

Hitoshi Nishimura,¹ Toshiki Yajima,¹ Yoshiko Kagimoto,¹ Mutsuhiro Ohata,² Taketo Watase,² Kenji Kishihara,¹ Fumi Goshima,³ Yukihiro Nishiyama,³ and Yasunobu Yoshikai^1*

Division of Host Defense, Medical Institute for Bioregulation, Kyushu University, Fukuoka 812-8582,¹ Laboratory of Host Defense and Germfree Life,² Laboratory of Virology, Research Institute for Disease Mechanism & Control, Nagoya University School of Medicine, Nagoya 466-8550, Japan³

Received 14 October 2003/ Accepted 6 January 2004

Top Abstract Introduction Accumulation of ifn-{gamma}... Susceptibility of mice depleted... Susceptibility of v{delta}1-/-... References

 
Mice depleted of T cells by in vivo administration of anti-TCR monoclonal antibodies showed susceptibility against an intravaginal infection with herpes simplex virus type 2 (HSV-2). The systemic Th1 response was impaired in the T-cell-depleted mice. Mice deficient in the V1 T subset were susceptible to an intravaginal infection with HSV-2. Intraepithelial T cells bearing V1 may help protect against intravaginal infection with HSV-2 through promoting the systemic Th1 response.

Top Abstract Introduction Accumulation of ifn-{gamma}... Susceptibility of mice depleted... Susceptibility of v{delta}1-/-... References

 
T-cell receptor (TCR) T cells are present only in small numbers in peripheral lymphoid tissues but are abundant in intraepithelial lymphocytes in the skin, intestines, and reproductive organs such as the uterus and vagina (3, 5). The intraepithelial T-cell subsets in the mouse model bear invariant TCRs, including the V1 subset that comprises most of the T cells in the skin and the female reproductive tract (3, 5). Animal models of infection with herpes simplex virus type 2 (HSV-2) have demonstrated that protective mechanisms against primary infection with HSV-2 are mainly mediated by class II-restricted CD4⁺ Th1 cells secreting gamma interferon (IFN-) (7, 10, 11, 14, 17). TCR T cells are reported to play important roles in protection against systemic infection with HSV-1 in mice (15, 16) and to respond to acute vaginal infection with HSV-2 (12, 13). However, the role of the intraepithelial T cells in intravaginal infection with HSV-2 is not yet elucidated. In this study, we examined the susceptibility of mice harboring a mutated TCR chain constant gene or V1 gene to intravaginal infection with HSV-2.

Female C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). All mice were used at 5 to 6 weeks of age. In order to synchronize their estrous cycle at the progesterone-dominant stage, all mice were injected subcutaneously with 0.1 µg of ß-estradiol 17-cypionate (Sigma) 5 days before infection. Mice were injected intravaginally with various doses of the 186 strain of HSV-2 (9, 20) in 20 µl of phosphate-buffered saline. In some experiments, 300 µg of anti-TCR monoclonal antibody (MAb) (UC7-13D5) or isotype control hamster immunoglobulin G was administered to mice 2 days before an intravaginal challenge with HSV-2. Vaginal wash fluids from six mice were individually assayed for virus infectivity. Uterine and vaginal intraepithelial lymphocytes (r-IEL) were prepared as described previously (12, 18). For flow cytometric analysis, r-IEL were stained with biotin-conjugated anti-CD3 MAb, fluorescein isothiocyanate-conjugated anti-TCRß MAb, and phycoerythrin-conjugated anti-TCR MAb (PharMingen, San Diego, Calif.) and then were stained with Red-613-conjugated streptavidin (Life Technologies, Gaithersburg, Md.). The stained cells were analyzed by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.). To determine the V repertoire, total RNA was extracted from r-IEL by the acid-guanidium phenol-chloroform method. cDNA synthesis and PCR were performed by using a cDNA cycle kit (Invitrogen Corp., San Diego, Calif.). RNA was primed with 6.7 pmol of chain C region (C) primers in 20 µl of reaction mixtures for reverse transcription. The C and 5' V primers were described previously (8). PCR products (4 µl) were subjected to electrophoresis on a 1.5% agarose gel (Gibco) and transferred to a Gene Screen Plus filter (New England Nuclear, Boston, Mass.). The Southern blots of PCR products were hybridized with J1 or J2 oligonucleotide probes, which were labeled with [-³²P]ATP by using a Megalabel 5'-labeling kit (Takara Shuzo Co. Ltd., Kyoto, Japan) according to the manufacturer's instructions. The r-IEL were cultured with anti-CD3 MAb (145-2C11; 100 µg/ml) or anti-TCR MAb (100 µg/ml) that had been immobilized on the plates by prior incubation for 1 h. To estimate cytokine production, the supernatants were collected after a 48-h culture. The cytokine activity in the cell-free culture supernatants was assayed by an enzyme-linked immunosorbent assay (ELISA) with mouse IFN- (Genezyme Diagnostics, Cambridge, Mass.). The CD4⁺ T cells (more than 95% purity) were purified from spleen cells by using an autoMACS cell sorter (Miltenyi Biotec, Bergisch Gladbach, Germany) and were cultured in 200 µl of complete culture medium in 96-well flat-bottom plates (Falcon; Becton Dickinson Ltd., Oxford, United Kingdom) at a density of 5 x 10⁵ cells/well, with the same number of mitomycin-treated spleen cells from C57BL/6 mice with or without 2.5 x 10⁴ PFU of heat-inactivated HSV-2 (56°C for 1 h). The mutant mouse (F₂ interbred from 129/Ola x C57BL/6) strain deficient in the V1 gene (V1^–/–) was generated by gene targeting, as described previously (4), and backcrossed with C57BL/6 mice more than eight times. The C57BL/6 background mutant mouse deficient in the TCR chain constant gene (TCR^–/–) was kindly provided by S. Itohara (Riken, Saitama, Japan) (6). The statistical significance of the data was determined by Student's t test, except for lethality data, which were analyzed by the generalized Wilcoxon's test.

Top Abstract Introduction Accumulation of ifn-{gamma}... Susceptibility of mice depleted... Susceptibility of v{delta}1-/-... References

 
We monitored the kinetics of T cells in the uterus and vagina following an intravaginal inoculation with HSV-2. The absolute numbers of T cells in the uterus and vagina increased to a peak on day 3 in C57BL/6 mice after HSV-2 infection (Fig. 1A) (1.5 x 10⁴ ± 0.2 x 10⁴ cells before infection versus 7.2 x 10⁴ ± 0.6 x 10⁴ cells after infection; P < 0.01). Reverse transcription-PCR analysis showed that the T cells on day 3 preferentially used the V1-rearranged J1 gene, similar to those used by the T cells in noninfected control mice (Fig. 1B). We also found V4-rearranged J1, which has been reported for accumulations in the uterus and vagina after infection (13). Early production of IFN- has an important role in determining whether naive CD4⁺ T cells will differentiate into Th1 cells (2, 18). We next examined the IFN- production by T cells accumulated in the uterus and vagina following HSV-2 infection. IFN- production by the T cells in the uterus and vagina was significantly augmented on day 3 in response to immobilized anti-CD3 MAb or anti-TCR MAb (Fig. 1C). There was no interleukin-4 production in response to TCR stimulation (data not shown). Thus, the number of T cells with V1 and with a high ability to produce IFN- increased in the uterus and vagina following HSV-2 infection.

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FIG. 1. T cells in the uterus and vagina in C57BL/6 mice following an intravaginal infection with 250 PFU of HSV-2 strain 186. (A) Flow cytometric analysis of r-IEL on day 3 after HSV-2 infection. The r-IEL were stained with anti-CD3, anti-TCR, and anti-TCRß MAbs. Cells gated on CD3+ cells were analyzed for their expression of TCRß and TCR. Representative data from three independent experiments are shown. (B) V and J usages of r-IEL on day 3 after HSV-2 infection. The total RNA extracted from the r-IEL was reverse transcribed into cDNA and amplified by PCR with primers for C and various V segments. The Southern blot of PCR products was hybridized with an oligonucleotide probe for J1 or J2. The results are representative of three independent experiments. (C) IFN- production by r-IEL on day 3 after HSV-2 infection. The r-IEL were cultured on plates coated with anti-TCR MAb or anti-CD3 MAb for 48 h. IFN- levels in the supernatants were determined by ELISA. Data are means ± standard deviations (SD) for five mice in each group. There was a statistically significant difference from the value for C57BL/6 mice (*, P < 0.05; **, P < 0.01 [by Student's t test]). Representative data from three independent experiments are shown.

Top Abstract Introduction Accumulation of ifn-{gamma}... Susceptibility of mice depleted... Susceptibility of v{delta}1-/-... References

 
To define the roles of T cells in protection against primary intravaginal infection with HSV-2, we examined the effects of in vivo depletion of T cells by anti-TCR MAb on the survival rate of mice after intravaginal inoculation with HSV-2 strain 186. We confirmed that the numbers of T cells were severely reduced in uterine IEL and spleen cells on day 7 after HSV-2 infection when 300 µg of anti-TCR MAb was administered 3 days prior to infection (Fig. 2; data not shown). As shown in Fig. 2A, all mice given anti-TCR MAb died by day 16, and 60% of the mice given control MAb survived beyond 18 days (P < 0.01 by the generalized Wilcoxon's test). We compared virus titers in the organs of control mice and mice given anti-TCR MAb on day 7 after infection with HSV-2 wild-type strain 186 (5 x 10³ PFU), at which time mice began to die. As shown in Fig. 2C, in mice given anti-TCR MAb, larger amounts of infectious virus were detectable in the vaginal wash fluids of mice given anti-TCR MAb (P < 0.01). Thus, these results indicated that mice given anti-TCR MAb were susceptible to HSV-2 infection. To compare the HSV-2-specific Th1 cell responses of control mice and mice depleted of T cells after an intravaginal infection, we examined IFN- production by spleen CD4⁺ T cells in response to heat-inactivated HSV-2 on day 7 after an intravaginal infection with HSV-2. As shown in Fig. 3, IFN- production by CD4⁺ T cells from mice depleted of T cells was significantly lower than that in control mice (P < 0.05).

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FIG. 2. Susceptibility of mice depleted of T cells to intravaginal infection with HSV-2 strain 186. (A) Survival rates of C57BL/6 mice (20 mice in each group) that were injected intraperitoneally with 300 µg of anti-TCR MAb ( T cell-dep) or control hamster immunoglobulin G (control) 3 days before an intravaginal inoculation with 250 PFU of HSV-2 strain 186 and were monitored for survival. Representative data from three independent experiments are shown. *, statistically significant difference from control group (P < 0.01 by the generalized Wilcoxon's test). (B) Flow cytometric analysis of r-IEL from mice depleted of T cells is shown. r-IEL were collected on day 3 after infection with 250 PFU of HSV-2 strain 186 and were stained for expression of TCRß, TCR MAb, and CD3. The dot plot analysis is gated on CD3-positive cells and presented as typical two-dimensional profiles. Numbers represent the percentages of total cells found in each quadrant. (C) Viral titers in vaginal wash fluids of mice depleted of T cells on day 7 after infection with 5,000 PFU of HSV-2 strain 186. Data are means ± SD of five mice in each group. Representative data from three independent experiments are shown. *, statistically significant difference from control group (P < 0.01).

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FIG. 3. IFN- production by spleen CD4+ T cells of mice depleted of T cells in response to HSV-2. CD4+ T cells were obtained from the spleens of T-cell-depleted (-dep.) mice or control mice on day 7 after an intravaginal challenge with 250 PFU of HSV-2 strain 186. The CD4+ T cells were cultured in the presence of mitomycin C-treated spleen cells with or without heat-inactivated HSV-2. IFN- levels in the supernatants were determined by ELISA. Data are means ± SD of five mice in each group. There was a statistically significant difference from the value for the control in response to HSV-2 antigen. (*, P < 0.05 by Student's t test). Representative data from three independent experiments are shown.

Top Abstract Introduction Accumulation of ifn-{gamma}... Susceptibility of mice depleted... Susceptibility of v{delta}1-/-... References

 
We next examined the survival rates of TCR^–/– mice or V1^–/– mice after an intravaginal infection with HSV-2. As shown in Fig. 4, all of the TCR^–/– mice and 90% of the V1^–/– mice died by 11 days, while 40% of their littermates survived beyond 18 days (P < 0.05 by the generalized Wilcoxon's test). Thus, mice deficient in whole T cells and those deficient in V1 cells were equally susceptible to an intravaginal infection with HSV-2.

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FIG. 4. Survival rates of mice deficient in whole T cells or the V1 subset after intravaginal infection with HSV-2 strain 186. TCR–/– or TCR+/+ mice (20 mice in each group) and V1–/– or V1+/+ mice (10 mice in each group) were inoculated with 250 PFU of HSV-2 strain 186 and were monitored for survival. *, statistically significant difference from littermates for each group (P < 0.05 by the generalized Wilcoxon's test).

Protective mechanisms against HSV infection are mediated by two major waves of host responses. The first one, innate immunity, depends mainly on the phagocyte system and NK and NKT cells (1). The second mechanism, acquired immunity, depends on the immune response mediated by T-helper type 1 cells of CD4⁺ ß T cells that are indispensable in protection against systemic HSV infection (6, 14). We also confirmed that mice depleted of TCRß T cells by in vivo treatment with anti-TCRß MAb and TCRß^–/– mice are highly susceptible to an intravaginal infection with HSV-2 (unpublished data). Early production of IFN- has an important role in determining whether naive CD4⁺ T cells will differentiate into Th1 cells (2, 19). The results of the present study showed that epithelial T cells in the uterus and vagina are activated to produce IFN- during the course of HSV-2 infection and that mice deficient in the intraepithelial T cells bearing V1 showed a high susceptibility to the infection. Furthermore, mice depleted of T cells showed an impaired CD4⁺ Th1 response following HSV-2 infection. Thus, these results suggest that the intraepithelial T cells may not only function in the front lines of host defense but also may help protect against HSV-2 infection by promoting a systemic CD4⁺ Th1 response to HSV-2.

 
We thank K. Itano and A. Nishikawa for their excellent technical support. We also thank R. Kubo and J. A. Bluestone for providing MAbs.

This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Japan Society for the Promotion of Science, the Uehara Memorial Foundation, and the Yakult Bioscience Foundation.

 
* Corresponding author. Mailing address: Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Phone: 81-92-642-6962. Fax: 81-92-642-6973. E-mail: yoshikai{at}bioreg.kyushu-u.ac.jp.

Top Abstract Introduction Accumulation of ifn-{gamma}... Susceptibility of mice depleted... Susceptibility of v{delta}1-/-... References

 

1
1. Ashkar, A. A., and K. L. Rosenthal. 2003. Interleukin-15 and natural killer and NKT cells play a critical role in innate protection against genital herpes simplex virus type 2 infection. J. Virol. 77:10168-10171.[Abstract/Free Full Text]
2
2. Constant, S. L., and K. Bottomly. 1997. Induction of Th1 and Th2 CD4⁺T cell responses: the alternative approaches. Annu. Rev. Immunol. 15:297-322.[CrossRef][Medline]
3
3. Haas, W., P. Pereira, and S. Tonegawa. 1993. Gamma/delta cells. Annu. Rev. Immunol. 11:637-685.[Medline]
4
4. Hara, H., K. Kishihara, G. Matsuzaki, H. Takimoto, T. Tsukiyama, R. E. Tigelaar, and K. Nomoto. 2000. Development of dendritic epidermal T cells with a skewed diversity of gamma delta TCRs in Vdelta 1-deficient mice. J. Immunol. 165:3695-3705.[Abstract/Free Full Text]
5
5. Hayday, A., and R. Tigelaar. 2003. Immunoregulation in the tissues by gamma/delta T cells. Nat. Rev. Immunol. 3:233-242.[CrossRef][Medline]
6
6. Itohara, S., P. Mombaerts, J. Lafaille, J. Iacomini, A. Nelson, A. R. Clarke, M. L. Hooper, A. Farr, and S. Tonegawa. 1993. T cell receptor delta gene mutant mice: independent generation of alpha beta T cells and programmed rearrangements of gamma delta TCR genes. Cell 72:337-348.[CrossRef][Medline]
7
7. Manickan, E., R. J. Rouse, Z. Yu, W. S. Wire, and B. T. Rouse. 1995. Genetic immunization against herpes simplex virus. Protection is mediated by CD4⁺ T lymphocytes. J. Immunol. 155:259-265.[Abstract]
8
8. Mokuno, Y., T. Matsuguchi, M. Takano, H. Nishimura, J. Washizu, T. Ogawa, Y. Nimura, and Y. Yoshikai. 2000. Expression of Toll-like receptor 2 on T cells bearing invariant V6/V1 induced by Escherichia coli infection. J. Immunol. 165:931-940.[Abstract/Free Full Text]
9
9. Nishiyama, Y., and F. Rapp. 1981. Repair replication of viral and cellular DNA in herpes simplex virus type 2-infected human embryonic and xeroderma pigmentosum cells. Virology 110:466-475.[CrossRef][Medline]
10
10. Parr, M. B., and E. L. Parr. 1998. Mucosal immunity to herpes simplex virus type 2 infection in the mouse vagina is impaired by in vivo depletion of T lymphocytes. J. Virol. 72:2677-2685.[Abstract/Free Full Text]
11
11. Parr, M. B., and E. L. Parr. 2003. Vaginal immunity in the HSV-2 mouse model. Int. Rev. Immunol. 22:43-63.[CrossRef][Medline]
12
12. Rakasz, E., M. Hagen, M. Sandor, and R. G. Lynch. 1997. T cells of the murine vagina: T cell response in vivo in the absence of the expression of CD2 and CD28 molecules. Int. Immunol. 9:161-167.[Abstract/Free Full Text]
13
13. Rakasz, E., A. Mueller, S. Perlmann, and R. G. Lynch. 1999. Gamma delta T cell responses induced by vaginal herpes simplex 2 infection. Immunol. Lett. 70:89-93.[Medline]
14
14. Schmid, D. S., and B. T. Rouse. 1992. The role of T cell immunity in control of herpes simplex virus. Curr. Top. Microbiol. Immunol. 179:57-74.[Medline]
15
15. Sciammas, R., P. Kodukula, Q. Tang, R. L. Hendricks, and J. A. Bluestone. 1997. T cell receptor-gamma/delta cells protect mice from herpes simplex virus type 1-induced lethal encephalitis. J. Exp. Med. 185:1969-1975.[Abstract/Free Full Text]
16
16. Sciammas, R., and J. A. Bluestone. 1999. TCR and viruses. Microbes Infect. 1:203-212.[CrossRef][Medline]
17
17. Stanberry, L. R. 1992. Pathogenesis of herpes simplex virus infection and animal models for its study. Curr. Top. Microbiol. Immunol. 179:15-30.[Medline]
18
18. Suzuki, T., K. Hiromatu, Y. Ando, Y. Tomoda, and Y. Yoshikai. 1995. Regulatory role of T cells in uterine intraepithelial lymphocytes in maternal anti-fetal immune responses. J. Immunol. 154:4476-4484.[Abstract]
19
19. Szabo, S. J., B. M. Sullivan, S. L. Peng, and L. H. Glimcher. 2003. Molecular mechanisms regulating Th1 immune responses. Annu. Rev. Immunol. 21:713-758.[CrossRef][Medline]
20
20. Tsunobuchi, H., H. Nishimura, F. Goshima, T. Daikoku, Y. Nishiyama, and Y. Yoshikai. 2000. Memory type CD8⁺ T cells protect IL-2R -deficient mice from systemic infection with HSV type 2. J. Immunol. 165:4552-4560.[Abstract/Free Full Text]

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Journal of Virology, May 2004, p. 4927-4930, Vol. 78, No. 9
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.9.4927-4930.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nishimura, H. Articles by Yoshikai, Y. Search for Related Content PubMed PubMed Citation Articles by Nishimura, H. Articles by Yoshikai, Y.