HIV-1 induces cytotoxic T lymphocytes in the cervix of infected women

Abstract

Although T lymphocytes are present in the genital mucosa, their function in sexually transmitted diseases is unproven. To determine if cervical T cells mediate HIV-specific cytolysis, mononuclear cells in cytobrush specimens from HIV-1-infected women were stimulated in vitro with antigen. Resultant cell lines lysed autologous targets expressing HIV-1 proteins in 12/19 (63%) subjects, and these responses were detected intermittently on repeated visits. All 8 subjects with blood CD4+ counts > or =500 cells/microl had HIV-1-specific cervical CTL, whereas only 4/11 with counts <500 cells/microl had detectable responses (P = 0.008). Class II MHC-restricted CD4+ CTL clones lysed targets expressing Env gp41 or infected with HIV-1. Class I MHC-restricted CD8+ clones recognized HIV-1 Gag- or Pol-expressing targets, and the epitopes were mapped to within 9-20 amino acids. Comparisons of intra-individual cervical and blood CTL specificities indicate that epitopes recognized by CTL in the cervix were commonly recognized in the blood. These studies provide the first definitive evidence for an MHC-restricted effector function in human cervical lymphocytes.

Figure 1

T lymphocytes in fresh cervical preparations and following clonal expansion. Two-color cytofluorimetric analysis of freshly isolated cervical mononuclear cells (A) and cervical CTL clones (B) obtained from HIV-1-infected women: volunteer 737 (A, upper panels), volunteer 747 (A, lower panels), and volunteer 710 (B, both panels). Left-hand plots represent the two antibody isotype controls (IgG1 FITC and IgG2a PE). (A) Middle plots demonstrate expression of CD3 (FL1) and CD16 (FL2). Surface expression of CD4 (FL1) and CD8 (FL2) are shown in A (right-hand plots) and B (middle plots). TCRαβ (FL1) and TCRγδ (FL2) are shown in B (right-hand plot).

Figure 2

Cervical CTL are detected intermittently in HIV-infected females. The graphs depict responses in six HIV-1-infected adult females (donor number at upper right) tested at 4–8 weekly intervals. Percent specific lysis directed against autologous B-LCL expressing either Env (vPE-16) (open bar), Gag (vDK-1) (black bar), or Pol (vRT) (lined bar) is represented at an E/T ranging from 1:1 to 5:1. Lysis of the control target cells infected with vaccinia alone was <5% in each experiment and is not shown.

Figure 3

CD4⁺ cervical CTL clones from subject 708 lyse HIV1-infected cells (E/T of 5:1). HIV-1 Env-specific clones lyse HIV-1-infected autologous and MHC class II partially mismatched (donor 738) B-LCL but not MHC class II completely mismatched (donors RML and SCHU) B-LCL or HIV-1-uninfected cells. The CTL clones recognize epitopes expressed by the laboratory-adapted strain, HIV-1_LAI, and two primary HIV-1 isolates, no. 1002 and no. 1005.

Figure 4

Cervical CTL clones derived from subject 738 (visit 8) are restricted by HLA-A2 and recognize targets expressing HIV-1 Pol aa 476484 or HIV-1-infected cells. (A) Specific lysis of autologous and MHC class I partially matched allogeneic B-LCL infected with either vRT or the control, vSC-8, at an E/T 3:1. Serological HLA class I typing for the donors are the following: autologous, (A2,74, B58,63, Bw4,w6); allo-1, (A2,28,B44,14,Bw4,w6); allo-2, (A2,B27,44,Bw4); allo-3, (A23,74,B70,57, Bw4,w6). (B) Lysis by clones of autologous B-LCL pulsed with synthetic peptides spanning Pol amino acid region 346-354 (VIYQYMDDL) and 476-484 (ILKEPVHGV) at an E/T of 3:1. C. Lysis at E/T of 3:1 of HIV1-infected and uninfected autologous B-LCL. Targets were infected with either HIV-1_LAI or one of two primary patient isolates, no. 1002 or no. 1005.

Figure 5

Gag-specific CTL clones from the cervix and blood of Subject 710 are HLA class I restricted. Gag-specific clones were tested for lysis of B-LCL infected with vDK-1 (Gag) or vSC-8 (control). Class I MHC-restricted lysis by the clones is evidenced by lysis of the Gagexpressing autologous B-LCL (A24,B14,B27) and B-LCL from donors sharing one class I HLA allele as indicated on the figure. Lysis of the control targets infected with vSC-8 (vac-lac) were <2% for all targets. (A) Three cervical CTL clones from visit 8, tested at an E/T of 3:1. (B) Six cervical CTL clones from visit 11, tested at an E/T of 5:1. (C) Four PBMC-derived CTL clones from visit 8, tested at an E/T of 4:1.