Selective retention of herpes simplex virus-specific T cells in latently infected human trigeminal ganglia

Abstract

Primary infection with herpes simplex virus 1 (HSV-1) and varicella zoster virus (VZV) results in lifelong latent infections of neurons in sensory ganglia such as the trigeminal ganglia (TG). It has been postulated that T cells retained in TG inhibit reactivation of latent virus. The acquisition of TG specimens of individuals within hours after death offered the unique opportunity to characterize the phenotype and specificity of TG-resident T cells in humans. High numbers of activated CD8(+) T cells expressing a late effector memory phenotype were found to reside in latently infected TG. The T cell infiltrate was oligoclonal, and T cells selectively clustered around HSV-1 but not VZV latently infected neurons. Neuronal damage was not observed despite granzyme B expression by the neuron-interacting CD8(+) T cells. The TG-resident T cells, mainly CD8(+) T cells, were directed against HSV-1 and not to VZV, despite neuronal expression of VZV proteins. The results implicate that herpesvirus latency in human TG is associated with a local, persistent T cell response, comprising activated late effector memory CD8(+) T cells that appear to control HSV-1 latency by noncytolytic pathways. In contrast, T cells do not seem to be directly involved in controlling VZV latency in human TG.

Fig. 1.

Viral load in human TG and comparison of the phenotype of TG-resident CD8⁺ T cells to their circulating counterparts. (A) Scatter plot showing the mean viral genome copy number per 10⁵ TG cells. (B) Comparison of the viral copy numbers between the paired left and right TG of individual donors. (C) Percentage of cells expressing CD69, CD27, CD28, or CD45RA on matched PB- and TG-derived CD8⁺ T cells. The percentages shown for the TG-derived CD8⁺ T cells are the averages of the paired left and right TG of individual donors. (D) Comparison of the percentages of CD8⁺ T cells expressing CD69, CD27, CD28, or CD45RA between the paired left and right TG of individual donors. (E) Dot plots from PB- (Upper) and TG- (Lower) derived CD8⁺ T cells of one representative donor. The numbers indicate the percentages of positive cells in the corresponding quadrants. The Mann–Whitney U test (A), Wilcoxon matched-pairs signed-rank test (C), and Spearman correlation test (B and D) were used for statistical analysis.

Fig. 2.

GeneScan analysis of TCRG rearrangements in paired left and right human TG. GeneScanning of TCRG rearrangements in total DNA isolated from a monoclonal human leukemic T cell line (A), the left (B) and right (C) TG of a representative donor and human PBMCs (D). This assay is a two-tube multiplex assay, using the indicated four TCR Vγ-specific and two Jγ-specific primers that are divided over two tubes, coded tube A and tube B. The primer for Jγ-1.1/2.1 was hexachloro-6-carboxyl-fluorescein labeled, whereas the primers for Jγ-1.3/2.3 was 6-carboxyl-fluorescein labeled, yielding a green or blue signal, respectively.

Fig. 3.

Viral and immunological parameters involved in the control of α-herpesvirus latently infected human TG. (A and B) Consecutive slides hybridized with an HSV-1 LAT oligonucleotide (A) or stained for CD3 (B); some LAT⁺ neurons (arrowheads) are not encircled with T cells. (C and D) Consecutive slides stained for VZV ORF62 (C, arrows) or CD3 (D, arrowheads). (E–H) Consecutive slides stained for CD3 (E), CD8 (F), CD4 (G), or CD69 (H). (I and J) Consecutive slides stained for CD8 (I) or granzyme B (J). (K and L) HLA-DR (K) and IL-15 (L) are constitutively expressed by satellite cells. Sections were developed with 5-bromo-4-chloro-3-indolyl-phosphate (A), 3-amino-9-ethylcarbazole chromogen (B–H), or diaminobenzidine (I–L) that resulted in a black, red, or brown color, respectively. All tissue sections were counterstained with hematoxylin, resulting in blue nuclei. (Magnification: A–H and K–L, ×200; I–J, ×400.) Representative photographs of frozen sections (A–H and K–L) and paraffin tissue sections (I–J) are presented from 12 TG donors analyzed.

Fig. 4.

Human TG-derived T cells recognize HSV-1 but not latency-associated VZV proteins. TG-derived T cell lines were cultured for 6 h with autologous BCLCs infected overnight with HSV-1 or rVV expressing four different VZV ORFs. T cell reactivity was determined by an IFN-γ ELISPOT assay. As controls, BCLCs infected with rVV containing no insert (rVV-CTRL) or mock-infected BLCLs were used. Representative results, mean ± SEM, from one of two independent experiments performed in triplicate are presented as the number of spot-forming cells (SFC)/5 × 10³ T cells. The ID numbers of the donors are indicated at the top of each graph.