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. 2014 Feb 10;9(2):e88884.
doi: 10.1371/journal.pone.0088884. eCollection 2014.

Levels of CD56+TIM-3- effector CD8 T cells distinguish HIV natural virus suppressors from patients receiving antiretroviral therapy

Bhawna Poonia  1 , C David Pauza  1
Affiliations

Levels of CD56+TIM-3- effector CD8 T cells distinguish HIV natural virus suppressors from patients receiving antiretroviral therapy

Bhawna Poonia et al. PLoS One. .

Abstract

Prolonged antiretroviral therapy (ART) with effective HIV suppression and reconstitution of CD4 T cells, fails to restore CD8 T cell lytic effector function that is needed to eradicate the viral reservoir. Better understanding of the phenotype and function of circulating CD8 cells in HIV patients will contribute to new targeted therapies directed at increasing CD8 T cell lytic effector function and destruction of the viral reservoir. We show that CD8 T cells from ART treated patients had sharply reduced expression of CD56 (neural cell adhesion molecule-1), a marker associated with cytolytic function whereas elite patients who control HIV in the absence of ART had CD56+ CD8 T cell levels similar to uninfected controls. The CD56+ CD8 T cells had higher perforin upregulation as well as degranulation following stimulation with HIV gag peptides compared with CD56 negative CD8 T cells. Elite patients had the highest frequencies of perforin producing CD56+ CD8 T cells among all HIV+ groups. In patients receiving ART we noted high levels of the exhaustion marker TIM-3 on CD56+ CD8 T cells, implying that defective effector function was related to immune exhaustion. CD56+ CD8 T cells from elite or treated HIV patients responded to PMA plus ionomycin stimulation, and expressed transcription factors T-bet and EOMES at levels similar to uninfected controls. Consequently, the lytic effector defect in chronic HIV disease is due to immune exhaustion and quantitative loss of CD56+ CD8 T cells and this defect is not repaired in patients where viremia is suppressed and CD4 T cells are recovered after ART. Reconstituting the cytotoxic CD56+ subset of CD8+ T cells through new interventions might improve the lytic effector capacity and contribute to reducing the viral reservoir. Our initial studies indicate that IL-15 treatment partly reverses the CD56 defect, implying that myeloid cell defects could be targeted for immune therapy during chronic HIV disease.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Preserved CD56+ CD8 T lymphocytes in elite controllers.
(A, B) Higher expression of CD56 on CD8dim T compared with CD8bright T lymphocytes. (C) Representative samples shows CD56+ CD8 T lymphocytes are mostly effector memory type whereas (D) CD56- CD8 T lymphocytes have a significant subset of naïve and central memory and type cells. (E) Both CD56+ and CD56- subset of CD8 T cells have similar frequency of HIV tetramer binding cells. Cells from an HLAB5701 HIV infected individual were stained with TW10 tetramer. Cells are gated on CD8+ CD3+ lymphocytes. (F) Significantly elevated CD8 T cell frequencies in both ART treated and low viremia (VIR) groups but not in elite controllers (NVS). (G) The frequency of CD56+ CD8+ T lymphocytes is significantly reduced in HIV+ ART treated group compared with uninfected controls. NVS (elite controllers) did not show this defect and have normal levels of CD56 expression on CD8 T cells. (H) Analysis of CD8bri (H) or CD8dim (I) populations of T cells show that NVS group has superior levels of CD56 subset among both populations whereas ART group has lowest levels of CD8bri cells expressing CD56. VIR group has high frequency of CD56 expressing CD8bri but not CD8dim cells when compared with ART patients. For F-I, Kruksal-Wallis test followed by Dunn's test for multiple correction was used to analyze the four groups.
Figure 2
Figure 2. Elite controllers have higher levels of perforin/GranzymeB+CD56+ CD8 T lymphocytes.
Intracellular expression of preformed perforin (A) and Granzyme B (B) is significantly higher on gated CD8 T lymphocytes from elite controllers and low viremia groups compared to uninfected controls or HIV+ART subjects. The level of CD56 expression on perforin (C) or Granzyme B (D) expressing CD8 T lymphocytes was significantly reduced in HIV+ ART treated and low viremia groups but not in elite controllers. Results in panels E-G show perforin upregulation in response to stimulation with HIV gag peptides measured with DG48 clone of anti-perforin antibody. (E) Both elite controller and low viremia group have significantly higher production of perforin upregulation in response to stimulation compared with ART treated individuals. (F). Representative sample from each infected group showing majority of CD56+ CD8 T cells upregulated perforin in response to stimulation while a minority of CD56- CD8 T cells upregulate perforin. Cells are gated on CD8 T lymphocytes. (G) Combined results from all infected groups show proportion of CD56 fraction of CD8 T cells upregulating perforin in response to stimulation with HIV peptides is significantly higher than proportion of CD56- CD8 T cell that upregulate perforin. Significantly greater proportions of CD56+ CD8 T cells degranulate (H) as well as produce IFNγ (I) in response to stimulation with HIV gag peptides. Results in G, H and I show show comparison of CD56+ and CD56- subsets irrespective of the patient's disease status.
Figure 3
Figure 3. Transcription factors and MAPK signaling in CD56 subset.
EOMES and T-bet control cytotoxic function of effector T cells. (A) Majority of CD56+ CD8 T cells expressed T-bet and/or EOMES and were double positive for these transcription factors, whereas a significant proportion of CD56- CD8 T cells were double negative for EOMES and T-bet. The representative plots show the trends presend in CD56+ or CD56- subsets from all patient groups irrespective of disease status (B) NVS group had highest frequency of T-bet expressing CD8 T cells whereas there was no significant difference in EOMES positive CD8 T cells among different cohorts of HIV infected patients. (C) Overall, the CD56+ fraction of CD8 T lymphocytes had higher levels of Erk phosphoryation upon PMA+inomycin stimulation compared with the CD56- CD8 T lymphocytes when samples from all groups were combined for analysis. CD56+ or CD56- CD8 T lymphocytes showed similar levels of Erk phosphorylation in all infected and control groups.
Figure 4
Figure 4. Exhausted CD56+ CD8 T lymphocytes in treated HIV infection.
The expression of Tim-3 on T cells identified exhausted T lymphocytes. (A) A higher proportion of CD56+ CD8 T cells express Tim-3 compared to the CD56- subset. (B) Cumulative data from all individuals irrespective of disease status shows significantly higher Tim-3 expression by CD56+ CD8 T cells (C) The proportion of CD56+CD8+ T cells that are exhausted as judged by their Tim-3 expression were significantly higher in HIV+ART but not in elite controllers when compared to uninfected group. (D) The levels of Tim-3 on CD56- CD8 T cells is comparable in all HIV- and HIV+ groups.
Figure 5
Figure 5. IL-15 restores CD56 on CD8 T cells from HIV infected patients.
(A) Most CD8 T cells and almost all CD56 + CD8 T cells express the common gamma chain (γc) (or CD132). Representative plots (from left ND, PD, NVS, VIR) show there was no significant downregulation of this receptor on CD8 T lymphocytes from any infected group (B) Culture of PMBC in IL-15 resulted in a significant upregulation of CD56 on CD8 T lymphocytes at day 12 relative to start of culture in infected and uninfected samples.
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This work was supported by Institute of Human Virology Institutional Grant 2010-01 (BP) and Public Health Services grant AI42458 (CDP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.