Longitudinal analysis of herpes simplex virus-specific CD4+ cell clonotypes in infected tissues and blood

Abstract

Background: Genital infection by herpes simplex virus (HSV)-2 offers a unique model for study of the effects of a remitting and exacerbating infection on the survival and persistence of antigen-specific T cells.

Methods: We used complementarity-determining region 3 (CDR3) length analysis to examine the complete T cell receptor (TCR) beta -chain repertoire in skin-lesion biopsy samples from subjects with genital herpes.

Results: We found that herpetic skin lesions consistently demonstrated oligoclonal CDR3 DNA length distribution, indicating the presence of T cell expansions. Sequence analysis of representative HSV-specific lesional CD4(+) cell clones and TCR beta -variable (TCRBV) sequencing confirmed that the oligoclonal expansions were largely related to HSV-specific T cell proliferation. To assess the persistence of HSV-specific CD4(+) cells that localize to genital lesions, we developed a sensitive and highly specific clonal tracking technique using a combination of TCRBV-specific polymerase chain reaction, followed by liquid hybridization with clonotype-specific probes.

Conclusion: Two different patterns of clonal persistence were observed. Some long-lasting clones appear to home to different epithelia, such as skin and genital mucosa, and to circulate in the peripheral blood, whereas others detected in lesions were absent or very rare in the peripheral blood.

Figure 1

Complementarity-determining region 3 length analysis of peripheral blood mononuclear cells (PBMCs) and a lesion-derived T cell line. Spectratypes from PBMCs (A) and a matched lesion-derived T cell line (B) were generated by stimulation with phytohemagglutinin, as described in Materials and Methods. The autoradiographs were exposed overnight with an intensifying screen. The different T cell receptor β-variable families are indicated above the autoradiographs.

Figure 2

Longitudinal skin lesion repertoire analysis for different T cell receptor β-variable (TCRBV) families. Spectratypes were generated from sequential herpetic skin-lesion biopsies obtained from 1 subject during 2 different recurrences. The peripheral blood mononuclear cell (PBMC)– and lesion-derived cell lines were obtained after stimulation with phytohemagglutinin: lane A, nonlesional skin biopsy; lanes B, C, and D, 1996 skin-lesion biopsies (obtained on 20, 22, and 27 August 1996); lanes E and F, 1995 skin-lesion biopsies (obtained on 28 and 30 November 1995); lane I, autologous PBMC (obtained on 25 September 1996). Herpes simplex virus–specific CD4⁺ cell clones expressing matched TCRBVs were run side by side with the skin-lesion biopsy samples. Clones were established from either the 28 November 1995 skin-lesion biopsy (lane L) or the 25 September 1996 blood draw (lane P).

Figure 3

Herpes simplex virus (HSV)–specific CD4⁺ cell clone tracking by liquid hybridization. Serial 10-fold dilutions of lymphocytes from an HSV-specific CD4⁺ clone were made in a background of allogeneic peripheral blood mononuclear cells (PBMCs, A). A sample without cDNA was used as negative control (C⁻). The 10-cell dilution was repeated 8 times. RNA extractions were performed independently for each sample. The probe was tested by liquid hybridization against all the clones expressing T cell receptor β-variable 2 (B). Hybridization on the corresponding T cell clone is shown as positive control (C⁺).

Figure 4

Spatial distribution of herpes simplex virus–specific reactivity within a lesion. The complete T cell receptor β-variable (TCRBV) gene usage for the glycoprotein gB₂–specific dominant CD4⁺ cell clone (TCRBV12S4J2S3C2; GenBank accession no. AY751304) was shown to be gccagcact at the 3′ end, ctacagagacccgga at the nucleotide sequence covering the clonotypic probe, and acgcagt at the 5′ end. Distribution of the gB₂-specific clonotype among the 4 quadrant biopsies after stimulation with phytohemagglutinin was assessed by liquid hybridization, as described in Materials and Methods. MW, molecular weight; PBMCs, peripheral blood mononuclear cells.

Figure 5

Tissue distribution and temporal persistence of cervix-derived herpes simplex virus (HSV)–specific CD4⁺ cell clonotypes. Clonotype persistence of 2 cervical CD4⁺ cell clones from both subject 1 (A) and subject 2 (B) was assessed by liquid hybridization in cell culture from different tissue specimens. Cell lines established from blood samples (peripheral blood mononuclear cells [PBMCs]), herpetic skin lesions (skin), or cervical cytobrush (cervix) were stimulated with phytohemagglutinin (PHA) or with HSV-2, as described in Materials and Methods. The complete T cell receptor β-variable (TCRBV) gene usage is shown for both cervical CD4⁺ cell clones. Clone Cy2 (TCRBV2S1J2S7C2; GenBank accession no. AY751333) was shown to be cagtgc at the 3′ end, aagaaaggggaggggcg at the nucleotide sequence covering the clonotypic probe, and cctacg at the 5′ end. Clone Cy4 (TCRBV4S1J2S1C2; GenBank accession no. AY751334) was shown to be agcgtt at the 3′ end, cgaggggacagacac at the nucleotide sequence covering the clonotypic probe, and tacgag at the 5′ end. Hybridization on the corresponding T cell clone is shown as the positive control (C⁺). MW, molecular weight.