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Comparative Study
. 2018 Oct 15;201(8):2315-2330.
doi: 10.4049/jimmunol.1800725. Epub 2018 Sep 10.

Phenotypic and Functional Signatures of Herpes Simplex Virus-Specific Effector Memory CD73 + CD45RA high CCR7 low CD8 + T EMRA and CD73 + CD45RA low CCR7 low CD8 + T EM Cells Are Associated With Asymptomatic Ocular Herpes

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Free PMC article
Comparative Study

Phenotypic and Functional Signatures of Herpes Simplex Virus-Specific Effector Memory CD73 + CD45RA high CCR7 low CD8 + T EMRA and CD73 + CD45RA low CCR7 low CD8 + T EM Cells Are Associated With Asymptomatic Ocular Herpes

Ruchi Srivastava et al. J Immunol. .
Free PMC article

Abstract

HSV type 1 (HSV-1)-specific CD8+ T cells protect from herpes infection and disease. However, the nature of protective CD8+ T cells in HSV-1 seropositive healthy asymptomatic (ASYMP) individuals (with no history of clinical herpes disease) remains to be determined. In this study, we compared the phenotype and function of HSV-specific CD8+ T cells from HLA-A*02:01-positive ASYMP and symptomatic (SYMP) individuals (with a documented history of numerous episodes of recurrent ocular herpetic disease). We report that although SYMP and ASYMP individuals have similar frequencies of HSV-specific CD8+ T cells, the "naturally" protected ASYMP individuals have a significantly higher proportion of multifunctional HSV-specific effector memory CD8+ T cells (CD73+CD45RAhighCCR7lowCD8+ effector memory RA (TEMRA) and CD73+CD45RAlowCCR7lowCD8+ effector memory (TEM) as compared with SYMP individuals. Similar to humans, HSV-1-infected ASYMP B6 mice had frequent multifunctional HSV-specific CD73+CD8+ T cells in the cornea, as compared with SYMP mice. Moreover, in contrast to wild type B6, CD73-/- deficient mice infected ocularly with HSV-1 developed more recurrent corneal herpetic infection and disease. This was associated with less functional CD8+ T cells in the cornea and trigeminal ganglia, the sites of acute and latent infection. The phenotypic and functional characteristics of HSV-specific circulating and in situ CD73+CD8+ T cells, demonstrated in both ASYMP humans and mice, suggest a positive role for effector memory CD8+ T cells expressing the CD73 costimulatory molecule in the protection against ocular herpes infection and disease. These findings are important for the development of safe and effective T cell-based herpes immunotherapy.

Conflict of interest statement

Conflict of Interest: The authors have declared that no conflicts of interest exist.

Figures

Figure 1:
Figure 1:. Frequent HSV-1 VP11/12220–228 epitope-specific CD73+CD8+ memory T cells detected in ASYMP individuals compared to SYMP individuals:
The frequency of CD73+CD8+ T cells specific to the VP220–228 peptide/tetramer complex was analyzed in HLA-A*02:01 positive HSV-1 seropositive ASYMP and SYMP individuals. (A) Representative FACS data of the frequencies of CD73+CD8+ T cells, specific to VP11/12220–228 epitope, detected in PBMCs from one HLA-A*02:01 positive HSV-1 seropositive ASYMP individual (left two panels) and one HLA-A*02:01 positive HSV-1 seropositive SYMP individual (right two panels). (B) Average frequencies of PBMC-derived CD8+ T cells, specific to VP11/12220–228 epitope, detected from nine HLA-A*02:01 positive HSV-1 seropositive ASYMP individuals compared to nine HLA-A*02:01 positive HSV-seropositive SYMP individuals. The CD73 molecule expression level on CD8+ T cells specific to VP220–228 epitope was analyzed in HLA-A*02:01 positive HSV-1 seropositive ASYMP and SYMP individuals. (C) Representative FACS data of the CD73 expression level on CD8+ T cells specific to VP220–228 epitope detected from one HLA-A*02:01 positive HSV-1 seropositive ASYMP individual (left panels) and one HLA-A*02:01 positive HSV-1 seropositive SYMP individual (right panels). (D) Average CD73 expression level on CD8+ T cells, specific to VP11/12220–228 epitope, detected from eight HLA-A*02:01 positive HSV-1 seropositive ASYMP individuals compared to six HLA-A*02:01 positive HSV-seropositive SYMP individuals. The frequency of A2AR+CD8+ T cells specific to VP220–228 epitopes complex was analyzed in ASYMP and SYMP individuals. (E) Representative FACS data of the frequencies of A2AR+CD8+ T cells detected from one ASYMP (left panel) and one SYMP individual (right panel). (F) Average frequencies of A2AR+CD8+ T cells from nine ASYMP compared to nine SYMP individuals. The results are representative of two independent experiments in each individual. The indicated P values, calculated using unpaired t test, show statistical significance between SYMP and ASYMP individuals.
Figure 2.
Figure 2.. Frequent HSV-1 VP11/12220–228 epitope-specific CD8+ T cells with effector memory phenotype (CD73+CD8+ TEMRA and CD73+CD8+ TEM cells) detected in ASYMP individuals compared to SYMP individuals:
The CD8+ T cells specific to VP11/12220–228 peptide/tetramer representing shown in Fig. 1 were analyzed in terms of TNAIVE, TCM, TEMRA and TEM phenotypes. Representative FACS data of the frequencies of (A) CD45RAhighCCR7highCD8+ TNAIVE cells, (C) CD45RAlowCCR7highCD8+ TCM cells, (E) CD45RAhighCCR7lowCD8+ TEMRA cells, and (G) and CD45RAlowCCR7lowCD8+ TEM cells detected in one ASYMP individual and one SYMP individual. Representative FACS data of the frequencies of (B) CD45RAhighCCR7highCD8+ TNAIVE cells, (D) CD45RAlowCCR7highCD8+ TCM cells, (F) CD45RAhighCCR7lowCD8+ TEMRA cells and (H) CD45RAlowCCR7lowCD8+ TEM cells detected from 10 ASYMP and 10 SYMP individuals. The results are representative of two independent experiments in each individual. The indicated P values, calculated using unpaired t test, show statistical significance between SYMP and ASYMP individuals.
Figure 3.
Figure 3.. Frequent HSV-1 VP11/12220–228 epitope-specific CD73+CD8+ polyfunctional T cells detected in symptomatic individuals:
(A) VP11/12220–228 epitope-primed CD8+ T cells from ASYMP individuals were divided into CD73+CD8+ T cells and CD73CD8+ T cells and their functions compared. (B) FACS was used to determine the expression level of CD107a/b on tetramer gated CD8+ T cells specific to VP11/12220–228 epitope, as described in the Materials and Methods section. VP11/12220–228 epitope-specific CD73+CD8+ T cells express high levels of CD107a/b cytotoxic degranulation compared to CD73CD8+ T cells. The numbers on the top of each histogram represent the mean fluorescent intensity (MFI) depicting the expression level of CD107a/b molecules. Numbers in bold represent mean fluorescent intensity (MFI) on CD73+CD8+ T cells from ASYMP individual. Representative FACS data (C) and average percentage (D) of VP11/12220–228 epitope-specific CD107a/b+CD73+CD8+ T cells versus CD107a/b+CD73CD8+ T cells. (E) Level of CD107a/b molecules expression on VP11/12220–228 epitope-specific CD8+ T cells following co-stimulation with (i) mAbs anti-CD3 alone, (ii) mAbs anti-CD3 + mAbs anti-CD28 (iii) anti-CD3 + mAbs anti-CD73. Representative FACS data (F) and average percentage (G) of VP11/12220–228 epitope-specific IFN-γ+CD73+CD8+ T cells versus IFN-γ+CD73CD8+ T cells are shown. (H) IFN-γ expression level on VP11/12220–228 epitope-specific CD8+ T cells following co-stimulation on with (i) mAbs anti-CD3 alone, (ii) mAbs anti-CD3 + mAbs anti-CD28 (iii) anti-CD3 + mAbs anti-CD73. Representative FACS data (I) and average percentages (J) of VP11/12220–228 epitope-specific TNF-α+CD73+CD8+ T cells versus TNF-α+CD73CD8+ T cells are shown. (K) TNF-α expression levels on VP11/12220–228 epitope-specific CD8+ T cells following co-stimulation on with (i) mAbs anti-CD3 alone, (ii) mAbs anti-CD3 + mAbs anti-CD28 (iii) anti-CD3 + mAbs anti-CD73. The average frequencies of CD8+ T cells from 10 HLA-A*02:01-positive ASYMP and 10 SYMP individuals in response to stimulation with the VP11/12220–228 peptide are shown. The results are representative of two independent experiments in each individual. The indicated P values, calculated using one-way ANOVA Test, show statistical significance between SYMP and ASYMP individuals.
Figure 4.
Figure 4.. HSV-1 VP11/12220–228 epitope-specific CD73+CD8+ T cells are more functional:
Correlation of VP11/12220–228 epitope-specific CD73+CD8+ T cells percentage in PBMC with (A) expression of CD107a/b cytotoxic degranulation molecules, and production of (B) IFN-γ and (C) TNF-α. Correlation of VP11/12220–228 epitope-specific CD73CD8+ T cells percentage in PBMC with (D) expression of CD107a/b cytotoxic degranulation molecules, and production of (E) IFN-γ and (F) TNF-α. The nominal P values show statistical significance between the percentage of VP11/12220–228 epitope-specific CD8+ T cells and production of IFN-γ function.
Figure 5.
Figure 5.. HSV-1 VP11/12220–228 epitope-specific CD73CD8+ T cells are dysfunctional (exhausted):
VP11/12220–228 epitope-specific CD8+ T cells from ASYMP and SYMP individuals were divided into CD73+CD8+ T cells and CD73CD8+ T cells and their function compared. VP11/12220–228 epitope-specific CD73CD8+ T cells express high level of PD-1 marker of exhaustion compared to CD73+CD8+ T cells. FACS was used to determine the expression level of PD-1 on tetramer gated CD8+ T cells specific to VP11/12220–228 epitope, as described in the Materials & Methods section. The numbers on the top of each histogram represent mean fluorescent intensity (MFI) depicting the expression level of PD-1 molecule. (A) Representative FACS data (B) average MFI (C) and average percentage of VP11/12220–228 epitope-specific PD-1highCD73+CD8+ T cells versus PD-1highCD73CD8+ T cells are shown. (D) An inverse correlation of VP11/12220–228 epitope-specific CD73+CD8+ T cells percentage in PBMC with PD-1 expression in SYMP and ASYMP individuals. The results are representative of two independent experiments in each individual. The indicated P values, calculated using unpaired t test, show statistical significance between SYMP and ASYMP individuals.
Figure 6.
Figure 6.. Frequent functional gB498–505 epitope-specific CD73+CD8+ T cells detected in the cornea and trigeminal ganglia of HSV-1 infected ASYMP B6 mice:
(A and B) Kinetics of CD73 molecule expression in cornea- and trigeminal ganglia-derived CD8+ T cells. Flow cytometry of the frequency of gB498–505 epitope specific CD73+CD8+ T cells in the cornea and trigeminal ganglia (TG) of B6 mice (n =10) following ocular infection with HSV-1 (McKrae, 2 × 105 pfu/eye). (C) Representative FACS data (left panel) and average frequency (right panel) of the gB498–505 epitope-specific CD73+CD8+ T cells in the cornea and trigeminal ganglia (TG) of ASYMP (n = 5) versus SYMP (n = 5) mice. (D) Representative FACS data (left panel) and average frequency (right panel) of the gB498–505 epitope-specific IFN-γ+CD8+ T cells (E) and CD107+CD8+ T cells in the cornea and trigeminal ganglia (TG) of ASYMP (n = 5) versus SYMP (n = 5) mice. Data are representative of two independent experiments. The indicated P values, calculated using unpaired t test, show statistical significance between SYMP and ASYMP individuals.
Figure 7.
Figure 7.. CD73−/− deficient mice developed more corneal infection and severe herpetic disease associated with less functional HSV-specific CD8+ T cells in cornea and trigeminal ganglia compared to WT B6 mice:
(A) Schematic representation of the timeline of HSV-1 infection and UV-B induced recurrent disease in WT B6 mice and CD73−/− deficient mice. A group of CD73−/− deficient mice and WT mice (6–8 weeks old) were ocularly infected on day 0 with 2 × 105 pfu of HSV-1 (strain McKrae) following scarification. After establishment of latency (35 days post-infection), reactivation of latent virus was induced following irradiation with UV-B. Tears were collected daily for six days post-UV-B and recurrent corneal disease was observed daily for eye disease for 25 days post UV-B exposure. (B) Presence of infectious virus in the tears of WT and CD73−/− mice post UV-B treatment. Viral titer estimation detected in tear samples six days post UV-B irradiation expressed as mean of virus load. The data are expressed as mean of virus load (plaque forming units (pfu)/ml). (C) Recurrent corneal herpetic disease detected for up to 25 days following UV-B irradiation and scored on a scale of 0 to 4. (D) Representative slit lamp images of WT and CD73−/− mice corneas. Mice were euthanized on day 60 post UV-B exposure and single cell suspensions from cornea and TG was obtained after collagenase treatment and stained for markers of CD8+ T cells, IFN-γ and CD107 and analyzed by immunostaining and FACS. Sections of the cornea (E) and TG (I) from WT and CD73−/− mice were co-stained using Blue - DAPI (DNA stain), mAb specific to CD103-Red - Green - CD8 Infiltration. Single cell suspension from the cornea was obtained after collagenase treatment at 370C for an hour and stained for markers of CD8+ TRM cells, IFN-γ and CD107a/b and analyzed by FACS. Representative FACS plot of the frequency of HSV-specific CD103+CD8+ T cells (F and J), IFN-γ+CD8+ T cells (G and K) and CD107a/b+CD8+ T cells (H and L) detected in cornea and TG of WT and CD73−/− deficient mice. The results are representative of two independent experiments. The indicated P values, calculated using an unpaired t test, show statistical significance between WT and CD73−/− deficient mice.

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