Expression of cutaneous lymphocyte-associated antigen and E-selectin ligand by circulating human memory CD4+ T lymphocytes specific for herpes simplex virus type 2

Abstract

Virus-specific memory T lymphocytes traffic to sites of viral infection. Herpes simplex virus (HSV) type 2-specific CD4(+) and CD8(+) T lymphocytes differ with regard to their homing kinetics to infected tissues. We studied the expression of cutaneous lymphocyte-associated antigen (CLA) and E-selectin ligand (ESL) by HSV-2-specific CD4(+) T lymphocytes. Virus-reactive T lymphocytes were identified ex vivo by CD154 or interferon-gamma up-regulation. We detected selective expression of CLA by HSV-2-reactive CD4(+) T lymphocytes, but at levels lower than those we previously observed for CD8(+) T lymphocytes. Short-term HSV-2-reactive CD4(+) lines generated from peripheral-blood mononuclear cells preferentially express CLA, compared with cytomegalovirus- or influenza-specific cells. CLA is expressed by HSV-2-reactive cells that are initially CLA negative before restimulation. Short-term culture-expanded HSV-2-specific CD4(+) T lymphocytes also selectively express ESL. These findings have implications for the optimization of vaccines for HSV and other cutaneous pathogens.

Figure 1

Expression of cutaneous lymphocyte–associated antigen (CLA) by CD4⁺ T lymphocytes after ex vivo restimulation for 6 h. A, Peripheral-blood mononuclear cells (PBMCs) stimulated as indicated were stained with anti-CD4 and isotype control (left) or anti-CD154 monoclonal antibody (right). Gates for CD4⁺CD154⁺ T lymphocytes are shown. B, Expression of CLA by gated CD4⁺CD154⁺ T lymphocytes from dot-plots 6–8 in panel A. Nos. are percentages of positive cells. C, Summary of expression of CLA by CD4⁺ lymphocytes from the 8 subjects seropositive for herpes simplex virus (HSV) type 2 and 6 subjects seropositive for cytomegalovirus (CMV) (detailed in table 1) that were either CD154⁺ or CD154⁻ after the indicated stimuli. D, Expression of interferon (IFN)–γ by PBMCs in response to stimulation with HSV-2 or CMV. CD4⁺ T lymphocytes positive or negative for IFN-γ were analyzed for expression of CLA. Nos. in the dot-plots are percentages of CD4⁺ T lymphocytes that express IFN-γ. Nos. in the histograms are the percentages of the gated cells—either CD4⁺IFN-γ ⁺ or CD4⁺IFN-γ⁻—that express CLA. Mock ag, mock antigen; PE, phycoerythrin; PMA/iono, phorbol myristate acetate plus ionomycin.

Figure 2

Expression of cutaneous lymphocyte–associated antigen (CLA) by CD4⁺ T lymphocytes specific for herpes virus simplex (HSV) type 2 peptides. Peripheral-blood mononuclear cells (PBMCs) from 6 subjects were stimulated with an HLA-appropriate HSV-2 peptide and were stained with a tetramer, anti-CD4, and anti-CLA. A, Example of CLA expression among HSV-2–specific CD4⁺tetramer⁺ or CD4⁺tetramer⁻ T lymphocytes at day 10. B, Summary of data from 6 cultures tested at different time points; data are mean ± SD.

Figure 3

Expression of cutaneous lymphocyte–associated antigen (CLA) by CD4⁺ lymphoblasts responding to whole viral antigens. 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE)–labeled peripheral-blood mononuclear cells (PBMCs) were stimulated for 5 days without exogenous cytokines. A, Examples of forward/side scatter dot-plot (top row, showing gates used for lymphoblasts or monocytes) and CFSE and CD4 staining of gated lymphoblasts (middle row). Stimuli are indicated. Subjects are separated by vertical bars. Subject 1 (columns 1–5) is seropositive for herpes simplex virus (HSV) type 2 and cytomegalovirus (CMV), subject 2 (column 6) is seronegative for HSV-1 and HSV-2, and subject 3 (columns 7 and 8) is seronegative for CMV and seropositive for HSV-2. Row 3 shows CLA expression by gated CD4⁺, CFSE-low lymphoblasts and percentages of positive cells. B, CLA expression of CD4⁺, CFSE-low lymphoblasts from 16 HSV-2–seropositive subjects. Only subjects known to be seropositive for CMV were tested with CMV antigen. Flu, influenza; FS, forward scatter; PHA, phytohemagglutinin; SS, side scatter.

Figure 4

Effect of cutaneous lymphocyte–associated antigen (CLA) depletion on expression of CLA by CD4⁺ T lymphocytes responding to antigens or mitogens. A, Examples of peripheral-blood mononuclear cells (PBMCs) on day 0, both before and after depletion of CLA-expressing cells by use of immunomagnetic beads. Unmanipulated or depleted populations were then stimulated with antigens or mitogens for 5 days. B, Expression of CLA in gated lymphoblasts after in vitro restimulation with the indicated substances. The percentages of CD4⁺ T lymphocytes expressing CLA are shown. Cultures in each column are derived from the starting PBMC populations above them in row A. CMV, cytomegalovirus; HSV, herpes simplex virus; PHA, phytohemagglutinin.

Figure 5

Effect of cutaneous lymphocyte–associated antigen (CLA) depletion on lymphoproliferative responses. Peripheral-blood mononuclear cells (PBMCs) (unmanipulated or depleted of CLA-expressing cells) from 8 herpes simplex virus (HSV)–2 seropositive subjects were costimulated with autologous cells and mitogen or antigen for 5 days, followed by routine [³H]-thymidine incorporation assays. Data are mean ± SD of stimulation indices. PHA, phytohemagglutinin; TT, tetanus toxoid.

Figure 6

Expression of E-selectin ligand (ESL). Peripheral-blood mononuclear cells (PBMCs) were stimulated for 5 days with the indicated antigens or mitogens. Dot-plots are gated on CD4⁺ T lymphoblasts. A, Cells stimulated with UV-killed herpes simplex virus (HSV) type 2 were stained only with phosphatidylethanolamine-F (ab′)₂–labeled goat anti–human Fc (Coulter), the secondary detection reagent for ESL detection. Little staining was noted. B, Chimeric E-selectin–Fc protein was incubated with the same target population and EDTA, followed by incubation with detection reagent and anti-CLA. High CLA expression but little ESL staining was detected. C–E, EDTA was omitted; CLA and ESL were detected for HSV-2 stimulation but not for cytomegalovirus (CMV) or phytohemagglutinin (PHA) stimulation.