Peripheral blood CD4 T-cell and plasmacytoid dendritic cell (pDC) reactivity to herpes simplex virus 2 and pDC number do not correlate with the clinical or virologic severity of recurrent genital herpes

Abstract

Leukocytes participate in the immune control of herpes simplex virus (HSV). Data from HIV coinfections, germ line mutations, and case reports suggest involvement of CD4 T cells and plasmacytoid dendritic cells (pDC). We investigated the relationships between these cells and recurrent genital herpes disease severity in the general population. Circulating CD4 T-cell responses to HSV-2 were measured in specimens from 67 immunocompetent individuals with measured genital lesion and HSV shedding rates. Similarly, pDC number and functional responses to HSV-2 were analyzed in 40 persons. CD4 responses and pDC concentrations and responses ranged as much as 100-fold between persons while displaying moderate within-person consistency over time. No correlations were observed between these immune response parameters and genital HSV-2 severity. Cytomegalovirus (CMV) coinfection was not correlated with differences in HSV-2-specific CD4 T-cell responses. The CD4 T-cell response to HSV-2 was much more polyfunctional than was the response to CMV. These data suggest that other immune cell subsets with alternate phenotypes or anatomical locations may be responsible for genital herpes control in chronically infected individuals.

Fig 1

Representative flow cytometry data. (A) Gating scheme for CD4 T-cell intracellular cytokine cytometry. FSC-A, forward scatter A; SS, side scatter. Black polygons indicate gated cell subsets. (B) Responses of PBMC from an HSV-2-seropositive individual. At left, cells were stimulated with UV-HSV-2 and stained with isotype control MAb. The remaining panels show cells treated with the indicated stimuli and stained with the anticytokine MAb. The numbers are the percentages of gated CD4 T cells in the respective quadrants. (C) pDC enumeration analysis for a representative subject. Black polygons in the left 2 dotplots show cell gating. The right 3 dotplots show the percentages of gated cells positive for the indicated markers enclosed in the small polygons. (D) Gating scheme for DC ICC analyses. The third dotplot shows boxed CD123^high CD11c^neg cells. The fourth dotplot shows the final pDC gate of HLA-DR^pos lineage^neg cells. (E) Responses of gated pDC to media, live HSV-2 at MOI 100, and TLR agonists or controls. Numbers in quadrants are percentages of gated cells expressing the indicated patterns of cytokines.

Fig 2

Scatter plots of all subjects' genital herpes shedding (A) and lesion (B) rates versus the net percentages of circulating CD4 T cells expressing IFN-γ after in vitro challenge with UV-killed whole-cell-associated HSV-2.

Fig 3

Relationships between CMV-specific and HSV-2-specific CD4 T cells in coinfected persons. P values for Pearson correlations are indicated. (A) Proportions of circulating CD4 T cells responsive with HSV-2 (x axis) and CMV (y axis) in 46 coinfected persons. (B) The proportions of CMV-specific CD4 T cells that express two or three cytokines are indicated on the y axis. The x axis shows the proportion of HSV-2-specific CD4 T cells with polyfunctionality.

Fig 4

Intracellular cytokine cytometry results for circulating CD4 T-cell expression of IFN-γ for specimens collected during and after symptomatic genital HSV recurrences in five subjects. Peripheral blood mononuclear cells (PBMC) were collected between 3 and 13 times per participant over a course of 5 to 122 days. Results are expressed as percentage of circulating live CD3⁺ CD8⁻ CD4⁺ T cells expressing IFN-γ. PBMC were collected and frozen, and then all samples for each participant were run on the same day. Red data points are times at which HSV-2 was detected by PCR from genital specimens.

Fig 5

(A) Range and stability of pDC concentrations expressed as cells/μl of blood within individuals over a period of repeated sampling. Lines connect results from the same individuals. The x axis is ordered by mean cell concentration measured over 1 to 3 visits per person among 40 HSV-2-infected and 8 HSV-seronegative subjects. HSV-seronegative subjects are shown in gray. (B) Range and stability of pDC reactivity to live HSV-2 expressed as the percentage of cells expressing IFN-α in response to HSV-2 at MOI 100. Lines connect results from the same individuals. The x axis is ordered similarly to that described for panel A. HSV-seronegative subjects are shown in gray.

Fig 6

(A) Mean pDC number and function and genital herpes severity among HSV-2-infected persons. Each data point represents the average pDC data value for one person for 1 to 3 visits. The relationships between pDC number per microliter of blood and genital HSV shedding and between pDC number and genital lesion rate are shown in the upper panels. The relationships between the percentage of circulating pDC accumulating intracellular IFN-α in response to live HSV-2 at MOI 100 and genital HSV shedding and between pDC IFN-α reactivity and genital lesion rate are shown in the lower panels. (B) pDC IFN-α reactivity to HSV-2 correlates with reactivity to a synthetic TLR 9 agonist (left) but not as strongly with a synthetic agonist of TLR7 and TLR8 (right). Each data point represents the average pDC values for one person for 1 to 3 visits. HSV-2-uninfected and -infected individuals are included.