Association between Peripheral γδ T-cell Profile and Disease Progression in Individuals Infected with HIV-1 or HIV-2 in West Africa

Abstract

Background:

Human gammadelta (γδ) T cells play an important role in protective immunity in HIV-1 and SIV infection; their role in HIV-2 infection is unknown.

Objective:

To determine the role of γδ T cells in control of plasma viral load and CD4⁺ T-cell count in HIV-1 and HIV-2 infections in West Africa.

Methods:

Thirty HIV-1 and 25 HIV-2 treatment-naïve chronically infected individuals, as well as 20 HIV-seronegative individuals from Senegal were studied using multi-parametric flow cytometry to investigate the frequencies and phenotypes of peripheral γδ T-cells. γδ T-cells parameters and correlates of HIV disease progression were assessed.

Results:

We observed an expansion of Vδ1⁺ T-cell populations in both HIV-1 and HIV-2 infection. However, unlike HIV-1 infection, no significant contraction of the frequency of total Vδ2⁺ T cells was observed in HIV-2 infection. Significantly lower frequencies of CD4⁺Vδ2⁺ T cells were observed in HIV-2 infected individuals. Furthermore, frequencies of CD28⁻CD45RO⁺ and CD27⁻CD28⁻CD45RO⁻ Vδ2⁺ T-cell were low in HIV-1 infected individuals. Vδ2⁺ T-cell activation levels were elevated in both HIV-1 and HIV-2 infected individuals. The frequency of HLA-DR⁻CD38⁺ activated Vδ1⁺ and Vδ2⁺ T-cells was associated with a decline in CD4⁺ T-cell counts and increased viral load in both HIV-1 and HIV-2 infection.

Conclusion:

While maintaining the normal frequency of total Vδ2⁺ T cells, HIV-2 infection reduces the frequency of CD4⁺Vδ2⁺ T cells and alters the frequencies of subsets of Vδ1⁺ T cells. Both HIV-1 and HIV-2 infection induce γδ T cell activation, and this activation is associated with the disease progression.

INTRODUCTION

Two types of human immunodeficiency virus, HIV-1 and HIV-2, co-circulate in West Africa. Compared to HIV-1, infection with HIV-2 is associated with a reduced rate of progression to AIDS, slower decline in CD4⁺ T-cell count and significantly lower levels of plasma viral RNA, despite having similar levels of proviral DNA^1–4.

Gammadelta (γδ) T cells play an important role in protective immunity through cytokine secretion, cytotoxic activity and induction of adaptive immune responses^5–8. Two subsets of γδ T cells, Vδ1⁺ and Vδ2⁺, are commonly found in humans. Vδ2⁺ T cells, which express higher levels of IL-12 receptor and CCR5, are the more prevalent type in peripheral blood, whereas Vδ1⁺ T cells, which express higher levels of homing receptors such as L-selectin and CCR7, are more prevalent among intestinal intraepithelial lymphocytes and lymph nodes in epidermis^9–14. Vδ1⁺ and Vδ2⁺ T cells also appear to have different functions: when analyzed separately by microarrays following non-specific stimulation, Vδ2⁺ T cells expressed more genes involved in promoting inflammation, including TNF-α, IFN-γ, macrophage-colony-stimulation factor, IL-17 and IL-21; Vδ1⁺ T cells expressed higher levels of regulatory cytokine genes, including IL-10 and IL-11¹⁵.

In HIV-1 infection, these subsets change dramatically in frequency in peripheral blood and mucosa, with significant expansion of Vδ1⁺ and contraction of Vδ2⁺ cell populations^{9, 11, 16}. This poorly understood change in frequencies is first observed during acute HIV-1 infection and persists throughout the chronic phase without apparent reversion, even after highly active anti-retroviral therapy¹¹. The change in frequencies is not caused by either an antigen-driven clonal expansion of Vδ1⁺ T cells or a clonal deletion of Vδ2⁺ T cells^17–18; instead, a recent study on uninfected and SIV-infected rhesus macaques suggested that peripheral Vδ1⁺ T cell expansion is caused by the microbial translocation during SIV infection¹⁹. Expansion of Vδ1⁺ T cells may also contribute to the lysing of bystander CD4⁺ T lymphocytes and other cells, leading eventually to exacerbation of immunopathology and AIDS in HIV-1 infected individuals²⁰. Conversely, Vδ2⁺ T cells lyse HIV-1 infected cells and can block entry of HIV-1 in to CD4⁺ T cells via β-chemokine secretion²¹. However, their decreased numbers might be due to their own infection by HIV, because they express high levels of CCR5¹³. CD4⁺ γδ T cells expressing CCR5 and CXCR4 can be infected with HIV-1 in vitro, and can contribute between 3% and 45% of proviral DNA load in HIV-1 infected individuals²². Furthermore, after HIV-1 infection Vδ2⁺ T cells are frequently dysfunctional and anergic²³. Therefore, decreased Vδ2⁺ T-cell frequency and function may contribute not only to the loss of immunity against HIV-1 but also to diminished immune protection in general. Monitoring the frequency of γδ T-cell effectors has been suggested to be clinically relevant, and boosting them could be useful for vaccine strategies aimed at improving the immune responses in immunocompromised hosts and during active tuberculosis²⁴.

In contrast to HIV-1 infection, quantitative analysis regarding the frequency and function of γδ T cells in HIV-2 infection is lacking. In this study, we investigated the frequencies, activation levels and memory phenotypes of peripheral Vδ1⁺ and Vδ2⁺ T cells from HIV-2 as compared to HIV-1 infected individuals and HIV-seronegative individuals from Senegal. We show that HIV-2 infection disturbs the distributions of γδ T cells in peripheral. γδ T-cell activation is associated with HIV-2 disease progression and γδ T cells play a role in control of HIV-2 infection.

METHODS

Study Population

Study subjects were enrolled into a longitudinal study of HIV-1 and HIV-2 immunology at the University of Dakar Infectious Disease Clinic (Fann Hospital, Dakar, Senegal) and the sexually transmitted diseases clinic in Dakar (Institut d’Hygiene Sociale) since 2000. The details of the cohort have been previously described^25–26. All subjects were 16 years of age or older, provided written informed consent, and participated in protocols approved by the Senegalese AIDS National Committee and the University of Washington Human Subjects Institutional Review Boards. All untreated HIV-2 infected patients, as well as untreated HIV-1 infected patients with CD4⁺ T-cell counts of 350 cells/μl or greater at their screening visit, were invited to enroll in the study. Plasma HIV-1 and HIV-2 RNA levels were determined by a quantitative real-time RT-PCR assay as previously described^26–27.

Multiparameter Flow Cytometry

Peripheral blood mononuclear cells (PBMC) were isolated from anti-coagulated blood, cryopreserved in Senegal and shipped on liquid nitrogen vapor to Seattle. After thawing, 2×10⁶ viable cells from each individual were stained with previously-titrated LIVE/DEAD Violet Fixable Dead Cell Stain (ViViD; Molecular Probes/Invitrogen, Carlsbad, CA), washed, and surface stained at room temperature with anti-CD3-Qdot 655, −CD4-PerCP-Cy5.5, −CD8-APC-Cy7, −CD27-Qdot 605, −CD28-PE-Cy5, −CD57-Qdot 565, −CD45RO-PE-Texas Red, −HLA-DR-APC, −CD38-APC-Cy5.5, −Vδ1-FITC and −Vδ2-PE antibodies. All directly conjugated antibodies were obtained from BD Biosciences, except anti-CD45RO (Beckman Coulter, Fullerton, CA), anti-CD38 (Caltag/Invitrogen, Carlsbad, CA), and anti-Vδ1 (Pierce/Endogen, Rockford, IL). Qdot antibodies were conjugated in our laboratory per manufacturers’ protocols, using purified unconjugated monoclonal antibodies from BD Biosciences and Quantum Dots from Molecular Probes/Invitrogen. Following staining, cells were washed, fixed with 2% paraformaldehyde, and assayed within eight hours on an LSRII flow cytometer (BD Biosciences). Data analysis was conducted using FlowJo version 8.1 (TreeStar, Ashland, OR). The gating scheme is shown in Supplemental Figure 1.

Statistical Analysis

HIV-1 and HIV-2 plasma RNA viral load values were log10 transformed to normalize their distribution for statistical analyses. Pearson χ² or Fisher exact tests were performed to assess univariate associations between HIV infection status (HIV-2 vs. HIV-1 vs. HIV seronegative) and categorical factors of interest, ANOVA and t-tests were performed to compare groups with respect to continuous factors of interest. Additionally, continuous factors of interest that were not normally distributed were analyzed by Kruskal-Wallis and Wilcoxon rank sum tests. Spearman’s correlation coefficients were used to assess the correlation of T-cell subsets with both CD4⁺ T-cell counts and viral loads. Statistical analyses were conducted using SAS version 9.1 (SAS Institute, Cary, NC) and Prism version 5.0 (GraphPad Software, La Jolla, CA).

RESULTS

Study Population

The Senegalese study population consisted of 30 HIV-1 and 25 HIV-2 treatment-naïve chronically infected individuals, as well as 20 HIV-seronegative individuals who served as a comparison group (Table 1). The three groups were similar with respect to age and gender. As expected, HIV-1 and HIV-2 infected individuals had significantly lower CD4⁺ T-cell counts, (p<0.0001 and p=0.007, respectively), significantly higher CD8⁺ T-cell counts (p<0.0001 and p=0.01, respectively), and similar CD3⁺ T-cell counts compared to seronegative individuals.

Table 1.

Demographic, clinical and virological profiles of participants at time of sample draws.

	HIV Seronegative	HIV-1 Infected	HIV-2 Infected	p-valuea
Subjects (n)	20	30	25
Femaleb	14 (78%)	26 (87%)	23 (92%)	0.4
Age in years (mean years)c	34	36	40	0.07
Cell counts (mean, IQR cells/μl)
CD3+ T-cells	1688 (1152–2149)	1725 (1226–2243)	1614 (971–2213)	0.84
CD4+ T-cells	1071 (754–1333)	531 (237–834)	734 (362–1025)	<0.0001
CD8+ T-cells	527 (340–702)	1089 (684–1363)	781 (551–932)	<0.0001
HIV-1 plasma RNAd
Mean (IQR) RNA (log10 copies/ml)	-	4.3 (3.2–5.5)	-
Undetectable	-	4 (13%)	-
50–1000	-	3 (10%)	-
1000–10,000	-	5 (17%)	-
10,000–100,000	-	7 (23%)	-
≥100,000	-	11 (37%)	-
HIV-2 plasma RNAd
Undetectable	-	-	14 (56%)
Positive, unquantifiable	-	-	5 (20%)
25–100	-	-	2 (8%)
100–1,000	-	-	1 (4%)
1,000–10,000	-	-	3 (12%)

-, not applied

^aKruskal-Wallis test for all three groups.

^bmissing gender for two HIV seronegatives.

^cmissing two ages among HIV seronegatives and one among HIV-1 infected individuals.

^dThe limit of reliable detection and quantification was 50 copies/ml for HIV-1 and 25 copies/ml for HIV-2. For statistical analyses, samples that were below the threshold for reliable quantification were assigned a value equal to the limit of detection of each assay.

In order to evaluate comparable, relatively healthy HIV-1 and HIV-2 infected subjects prior to the onset of AIDS, we restricted enrollment of HIV-1 infected subjects to individuals with CD4⁺ T-cell counts above 350 cells/μl at their initial screening visit, although there was some decrease prior to study sample draws. As a result, mean CD4⁺ T-cell counts were not significantly different between HIV-1 and HIV-2 infected individuals (p=0.09). However, HIV-1 infected subjects had significantly higher CD8⁺ T-cell counts than HIV-2 infected subjects (p=0.02). HIV-1 infected individuals had a mean of 4.3 log₁₀ copies/ml plasma RNA load, with 24 (87%) having detectable plasma RNA load. As expected, HIV-2 infected individuals had less viremia, with only 11 (44%) having detectable plasma RNA load.

Alteration of γδ T-cell Subsets in HIV Infections

The total percentage of γδ T cells among all CD3⁺ cells was similar in HIV-2 infected, HIV-1 infected and seronegative individuals (data not shown). However, in agreement with previous reports, the individual frequencies of Vδ1⁺ and Vδ2⁺ T cells in HIV-1 infected individuals were significantly different from those in seronegative individuals (p=0.05 and p=0.01, respectively; Figure 1A), causing the median Vδ2⁺/Vδ1⁺ T-cell ratio to be significantly lower in HIV-1 infected subjects compared to HIV seronegatives (p=0.0009, Figure 1B). Although we also observed a trend toward expansion of Vδ1⁺ T-cell population in HIV-2 infected individuals, the frequencies of Vδ1⁺ and Vδ2⁺ T cells were not significantly different than those in seronegative individuals (Figure 1A). Similarly, while the median Vδ2⁺/Vδ1⁺ T-cell ratio in HIV-2 infected individuals was somewhat lower than that observed in seronegatives, the difference was not statistically significant (p=0.08). Furthermore, differences in the percentages of Vδ1⁺ and Vδ2⁺ T cells in HIV-2 compared to HIV-1 infected individuals did not achieve statistical significance (p=0.44 and p=0.09, respectively). However the median Vδ2⁺/Vδ1⁺ T-cell ratio was significantly higher in HIV-2 infected subjects compared to HIV-1 infected subjects (p=0.02; Figure 1B).

Figure 1:

Frequencies of peripheral Vδ1⁺ and Vδ2⁺ T cells from HIV-1 or HIV-2 infected and HIV seronegative individuals. All significant differences between populations are shown. Cells were gated as described in Supplemental Figure 1. A) Overall frequencies of Vδ1⁺ and Vδ2⁺ T cells. B) Ratio of Vδ2⁺/Vδ1⁺ T cells.

Frequency of CD4⁺ and CD8⁺ γδ T cells in HIV Infection

Consistent with previous reports^{22, 28}, CD4 was expressed on a subset of γδ T cells, and HIV-1 infection was associated with an increase in the frequency of CD8-expressing γδ T cells (Figure 2A). In our study sample, a median of 27% of Vδ1⁺ T cells expressed CD4 in HIV seronegatives, while HIV-1 and HIV-2 infections were each similarly associated with moderately decreased frequency of CD4⁺Vδ1⁺ T cells (HIV-1: median 15%, p=0.02; HIV-2: median 15%, p=0.10). A median of 26% of Vδ1⁺ T cells expressed CD8 in HIV seronegatives, while HIV-1 and HIV-2 infections were each associated with a significantly increased frequency of CD8⁺Vδ1⁺ T cells (HIV-1: median 40%, p=0.0006; HIV-2: median 38%, p=0.01).

Figure 2:

Frequencies of Vδ1⁺ and Vδ2⁺ T cells expressing (A) CD4 or CD8 and (B) HLA-DR and/or CD38. The boxes show the interquartile range, the horizontal line within each box shows the median, and the vertical lines extending from each box mark the minimum and maximum observed values.

A median of 38% of Vδ2⁺ T cells expressed CD4 in seronegatives, which was similar to the percentage observed in HIV-1 infected individuals (median 41%). HIV-2 infection was associated with a significantly decreased frequency of CD4⁺Vδ2⁺ T cells (median 29%) compared both to seronegatives (p=0.03) and to HIV-1 infected individuals (p=0.003). A median of 10% of Vδ2⁺ T cells expressed CD8 in seronegatives, which was significantly lower than the percentage observed in HIV-1 infection (median 13%, p=0.02). However, the percentage of CD8⁺Vδ2⁺ T cells in HIV-2 infected individuals (median 11%) was similar compared both to seronegatives and to HIV-1 infected individuals.

γδ T-cell Activation

Previous studies have shown that HIV-1 infection activates γδ T cells as indicated by increased expression levels of activation markers HLA-DR and CD38^29–30. We used multicolor flow cytometry to study the frequency of HLA-DR and CD38 expression on γδ T cells in HIV-1 or HIV-2 infected individuals (Figure 2B). Most HLA-DR-expressing Vδ1⁺ and Vδ2⁺ T cells also co-expressed CD38. The percentage of HLA-DR⁺CD38⁻Vδ1⁺ T cells in HIV-infected individuals (HIV-1: median 1.9%, HIV-2: median 1.3%) was comparable to HIV seronegatives (median 0.9%). Similarly, no significant difference was seen in the frequency of HLA-DR⁻CD38⁺Vδ1⁺ T cells in HIV infected individuals (HIV-1: median 53%; HIV-2: median 52%) compared to HIV seronegatives (median 48%). However, the percentage of HLA-DR⁺CD38⁺Vδ1⁺ T cells in HIV-1 infected individuals (median 10%) was significantly larger compared to HIV seronegative individuals (median 5%, p=0.01) and was modestly larger compared to that in HIV-2 infected individuals (median 8%, p=0.08). No significant difference was seen in the frequency of HLA-DR⁺CD38⁺Vδ1⁺ T cells in HIV-2 infected individuals compared to HIV seronegatives.

The percentage of HLA-DR⁺CD38⁻Vδ2⁺ T cells in HIV-infected individuals (HIV-1: median 0.8%; HIV-2: median 0.9%) was comparable to HIV seronegatives (median 0.7%). Similarly, no significant difference was seen in the frequency of HLA-DR⁻CD38⁺Vδ2⁺ T cells in HIV infected individuals (HIV-1: median 17%; HIV-2: median 16%) compared to HIV seronegatives (median 14%). However, the percentage of HLA-DR⁺CD38⁺Vδ2⁺ T cells (albeit small) in HIV-1 infected individuals (median 1.0%) and HIV-2 infected individuals (median 1.0%) was significantly larger compared to that in HIV seronegative individuals (median 0.6%; p=0.01 vs. HIV-1; p=0.01 vs. HIV-2).

Memory Phenotype of γδ T cells

To evaluate the frequency of naïve, effector and memory Vδ T-cell populations, we used multi-parameter flow cytometry to look at the surface expression of CD27, CD28 and CD45RO on those cells (Table 2). In agreement with previous reports³¹, in all three study groups a majority of the Vδ1⁺ T cells expressed an effector phenotype (median, 60% in seronegatives, 63% in HIV-1 and 55% in HIV-2 infected individuals), while a majority of the Vδ2⁺ T cells expressed a memory-like phenotype (median, 69% in seronegatives, 70% in HIV-1 and 64% in HIV-2 infected individuals), with a majority of them expressing CD28. Compared to seronegatives, both HIV-1 and HIV-2 infections were associated with a significantly smaller percentage of naïve Vδ1⁺ T cells and significantly larger percentages of intermediate (CD27⁺CD28⁻) and effector memory-like (CD27⁻CD28⁻) subsets of Vδ1⁺ T cells. The percentage of effector Vδ1⁺ T cells with the CD27⁺CD28⁻ phenotype was significantly smaller in HIV-2 infected individuals compared to HIV-1 infected and seronegative individuals. A trend was also seen toward a larger percentage of cytolytic-like CD57⁺Vδ1⁺ T cells in HIV-1 and HIV-2 infected individuals compared to seronegatives, but the differences were not statistically significant.

Table 2.

Frequencies of naïve and memory Vδ T-cells in seronegative, HIV-1 and HIV-2 infected West Africans.

				p-value
	HIV Seronegative (n=15)	HIV-1 Infected (n=30)	HIV-2 Infected (n=25)	Overalla	HIV-1 vs. HIV-2	HIV-1 vs. Neg.	HIV-2 vs. Neg.
Median percentage of Vδ1 T-cells (IQR)
Naïve cells
CD27+CD28+CD45RO−	21.2 (14.5–31.9)	9.2 (4.8–18.3)	7.8 (4.0–14.8)	0.001	0.6	0.0007	0.001
Memory-like cells (CD45RO+)
CD27+CD28+	12.8 (4.2–19.0)	7.4 (4.1–10.7)	8.1 (5.8–14.7)	0.4	0.3	0.3	0.9
CD27+CD28−	1.9 (1.2–3.2)	9.3 (4.7–17.7)	12.2 (4.8–23.2)	<0.0001	0.3	<0.0001	0.0004
CD27−CD28+	1.5 (0.6–2.7)	1.5 (0.9–2.1)	1.4 (0.8–1.6)	0.6	0.4	0.7	0.6
CD27−CD28−	0.3 (0.2–0.8)	2.4 (1.2–4.1)	1.1 (0.7–1.6)	<0.0001	0.004	<0.0001	0.01
Effector cells (CD45RO-)
CD27+CD28−	30.0 (19.4–47.7)	28.5 (23.6–36.1)	18.9 (14.3–30.0)	0.008	0.005	0.7	0.02
CD27−CD28+	1.6 (0.9–2.9)	2.1 (1.4–2.9)	1.4 (0.8–4.1)	0.5	0.4	0.3	1.0
CD27−CD28−	19.6 (14.1–36.0)	29.1 (14.6–39.7)	28.1 (14.7–40.0)	0.4	0.7	0.2	0.3
Cytolytic-like cells
CD57+	19.8 (13.0–35.5)	29.3 (19.9–39.2)	37.0 (25.2–48.0)	0.1	0.3	0.1	0.07
Median percentage of Vδ2 T-cells (IQR)
Naïve cells
CD27+CD28+CD45RO−	5.8 (2.9–6.1)	3.9 (3.0–7.3)	8.3 (3.2–13.0)	0.2	0.09	0.9	0.2
Memory-like cells (CD45RO+)
CD27+CD28+	28.3 (23.8–38.9)	22.4 (12.2–30.3)	30.6 (20.2–37.5)	0.02	0.02	0.02	0.8
CD27+CD28−	4.8 (3.2–12.5)	10.2 (5.9–15.1)	3.2 (2.2–8.1)	0.0008	0.0003	0.04	0.1
CD27−CD28+	22.2 (17.2–32.7)	19.1 (13.3–25.6)	20.7 (10.0–34.1)	0.4	0.6	0.1	0.7
CD27−CD28−	9.7 (4.0–14.1)	9.4 (7.4–14.1)	2.3 (1.2–5.9)	<0.0001	<0.0001	0.5	0.002
Effector cells (CD45RO−)
CD27+CD28−	7.3 (3.0–9.6)	8.4 (5.1–11.1)	6.7 (1.4–9.5)	0.2	0.1	0.1	0.5
CD27−CD28+	5.3 (2.8–7.2)	4.0 (2.0–7.7)	5.8 (2.7–16.5)	0.3	0.1	0.8	0.3
CD27−CD28−	9.7 (4.6–16.9)	8.8 (6.7–17.7)	4.5 (2.4–13.3)	0.04	0.01	0.6	0.1
Cytolytic-like cells
CD57+	11.7 (4.5–27.7)	21.2 (12.4–42.1)	17.7 (11.8–43.2)	0.1	0.8	0.04	0.09

^aKruskal-Wallis test for all three groups

The percentage of naïve Vδ2⁺ T cells was comparable between seronegatives and HIV-1 or HIV-2 infected individuals. HIV-1 infection was associated with a significantly smaller percentage of early memory-like CD27⁺CD28⁺Vδ2⁺ T cells compared to HIV-2 infected and seronegative individuals, while the percentage of intermediate memory-like CD27⁺CD28⁻Vδ2⁺ T cells was significantly larger than that in HIV-2 infected and seronegative individuals. Interestingly, the percentage of effector memory-like CD27⁻CD28⁻Vδ2⁺ T cells was significantly smaller only in HIV-2 infected individuals. Furthermore, the percentage of effector Vδ2⁺ T cells with the CD27⁻CD28⁻ phenotype was significantly smaller in HIV-2 infected individuals compared to HIV-1 infected and seronegative individuals. A trend was also seen toward a larger percentage of cytolytic-like CD57⁺Vδ2⁺ T cells in HIV-1 and HIV-2 infected individuals compared to seronegatives, but the differences were not statistically significant.

Correlation between the Frequency of γδ T-cell Subsets and Markers of HIV Disease Progression

To evaluate the role of γδ T cells in control of HIV infection, we correlated the frequency of γδ T-cell subsets and CD4⁺ T-cell count and plasma viral load (Tables 3).

Table 3.

Association between the frequency of Vδ T-cell count and CD4⁺ T-cell count or viral load in HIV-1 and HIV-2 infections.

	HIV-1				HIV-2
	CD4+ T-cell count		Viral loadb		CD4+ T-cell count		Undetectable viral loadb	Detectable viral loadb
	p	r	p	r	p	r	Median (IQR)	Median (IQR)	pa
Vδ1+ T-cell frequency	0.90	0.03	0.86	−0.03	0.39	0.18	3.58 (2.19–12.4)	2.49 (1.27–5.66)	0.29
Vδ1+ T-cell population
CD4+	0.008	0.47	0.27	−0.21	0.34	−0.20	11.20 (5.74–19.40)	32.00 (8.44–35.60)	0.08
CD8+	0.43	−0.15	0.88	0.03	0.60	0.11	34.75 (26.38–46.48)	40.90 (33.50–49.60)	0.26
Activated cells
HLADR+CD38-	0.004	0.51	0.006	−0.47	0.15	0.29	1.50 (0.81–2.35)	0.68 (0.13–1.47)	0.07
HLADR−CD38+	0.0008	−0.58	0.05	0.36	0.04	−0.41	37.95 (27.63–65.85)	55.9 (0.81–2.35)	0.03
HLADR+CD38+	0.38	0.17	0.07	−0.33	0.99	0.002	7.34 (4.61–9.77)	8.39 (3.71–15.40)	0.89
Naïve cells
CD27+CD28+CD45RO−	0.001	0.57	0.02	−0.43	0.40	0.18	10.22 (4.90–10.97)	7.10 (2.99–10.30)	0.28
Memory-like cells (CD45RO+)
CD27+CD28+	0.0003	0.62	0.02	−0.43	0.11	0.32	10.43 (6.65–22.54)	8.01 (2.57–14.68)	0.24
CD27+CD28−	0.8	0.05	0.62	−0.09	0.44	0.16	16.58 (7.75–29.98)	11.58 (1.57–20.49)	0.20
CD27−CD28+	0.03	0.39	0.85	−0.04	0.60	0.11	1.43 (0.86–2.11)	1.14 (0.74–1.60)	0.53
CD27−CD28−	0.40	−0.16	0.04	0.38	0.34	−0.20	0.87 (0.33–1.25)	1.49 (0.96–1.94)	0.05
Effector cells (CD45RO-)
CD27+CD28−	0.66	−0.08	0.74	0.06	0.43	0.16	20.63 (16.37–30.28)	16.49 (11.50–32.00)	0.29
CD27−CD28+	0.30	−0.19	0.33	0.18	0.41	−0.17	1.20 (0.84–2.88)	3.74 (0.75–10.20)	0.24
CD27−CD28−	0.08	−0.33	0.15	0.27	0.79	−0.06	22.84 (11.06–38.75)	29.46 (17.52–51.60)	0.24
Cytolytic-like cells
CD57+	0.44	−0.14	0.43	0.15	0.96	−0.01	34.72 (20.58–49.75)	38.88 (28.08–47.95)	0.43
Vδ2+ T-cell frequency	0.55	0.11	0.60	−0.10	0.48	0.15	1.60 (1.16–2.91)	0.92 (0.42–1.46)	0.05
Vδ2+ T-cell population
CD4+	0.15	0.27	0.64	−0.09	0.17	−0.28	1.02 (0.57–38.98)	40.00 (29.00–41.80)	0.08
CD8+	0.64	0.09	0.59	−0.10	0.88	−0.03	9.26 (6.08–17.03)	11.10 (9.27–21.30)	0.37
Activated cells
HLADR+CD38−	0.29	0.20	0.39	−0.16	0.84	−0.04	1.01 (0.45–2.25)	0.93 (0.55–1.60)	0.85
HLADR−CD38+	0.0005	−0.30	0.03	0.39	0.005	−0.54	7.04 (3.91–16.75)	26.9 (15.6–55.6)	0.002
HLADR+CD38+	0.01	−0.45	0.02	0.42	0.06	−0.38	0.67 (0.44–1.07)	2.79 (1.02–4.16)	0.02
Naïve cells
CD27+CD28+CD45RO−	0.16	0.26	0.008	−0.47	0.09	0.35	12.24 (7.51–13.21)	3.18 (1.71–9.18)	0.01
Memory-like cells (CD45RO+)
CD27+CD28+	0.75	0.06	0.42	0.15	0.38	0.19	34.38 (24.54–41.45)	26.14 (9.92–34.07)	0.13
CD27+CD28−	0.78	−0.05	0.58	0.10	0.33	−0.20	2.57 (1.10–7.96)	4.70 (2.93–8.98)	0.08
CD27−CD28+	0.05	−0.37	0.008	0.48	0.82	−0.05	19.82 (7.52–33.08)	20.72 (9.97–44.47)	0.57
CD27−CD28−	0.44	−0.15	0.63	0.09	0.06	−0.38	1.30 (0.78–2.50)	5.64 (2.97–11.85)	0.001
Effector cells (CD45RO-)
CD27+CD28−	0.005	0.50	0.02	−0.41	0.98	−0.005	7.50 (1.36–13.50)	3.44 (1.43–9.00)	0.68
CD27−CD28+	0.49	0.13	0.60	−0.10	0.59	0.11	6.62 (1.77–20.13)	5.11 (2.68–11.01)	0.89
CD27−CD28−	0.23	0.23	0.19	−0.25	0.86	−0.04	3.96 (2.28–8.54)	6.64 (2.08–23.98)	0.29
Cytolytic-like cells
CD57+	0.58	−0.11	0.62	−0.09	0.44	−0.16	16.17 (8.91–36.65)	25.72 (14.77–62.78)	0.34

^at-test between individuals with undetectable and detectable HIV viral load;

^bThe limit of reliable detection and quantification was 50 copies/ml for HIV-1 and 25 copies/ml for HIV-2. For statistical analyses, samples that were below the threshold for reliable quantification were assigned a value equal to the limit of detection of each assay.

Interestingly, in HIV-1 infection the frequency of CD4⁺-expressing Vδ1⁺ T cells was significantly positively correlated with CD4⁺ T-cell count. Furthermore, the frequencies of HLADR⁺CD38⁻, naïve, CD27⁺CD28⁺, CD27⁻CD28⁺ memory Vδ1⁺ T cells and CD27⁺CD28⁻ effector Vδ2⁺ T cells were significantly positively correlated with CD4+ T-cell count. On the other hand, CD4⁺ T-cell count was significantly negatively correlated with the frequencies of HLADR⁻CD38⁺ Vδ1⁺ T cells and HLADR⁻CD38⁺ and HLADR⁻CD38⁺ Vδ2⁺ T cells.

Similar to HIV-1 infection, CD4⁺ T-cell count was significantly negatively correlated with the frequencies of HLADR⁻CD38⁺ Vδ1⁺ and Vδ2⁺ T cells in HIV-2 infection). However, no association was observed between CD4⁺ T-cell count and the frequencies of other subsets of Vδ1⁺ or Vδ2⁺ T cells.

In HIV-1 infection, the frequencies of HLADR⁺CD38⁻, naïve, CD27⁺CD28⁺ memory like Vδ1⁺ T cells and naïve, CD27⁺CD28⁻ effector like Vδ2⁺ T cells were significantly negatively correlated with plasma viral load, while the frequencies of activated HLADR⁻CD38⁺ Vδ1⁺ T cells and HLADR⁻CD38⁺, HLADR⁺CD38⁺, CD27⁻CD28⁺ memory like Vδ2⁺ T cells were significantly positively correlated with plasma viral load.

In HIV-2 infection, the frequency of the Vδ2⁺ T cells was significantly positively associated with undetectable HIV-2 plasma viral load. Furthermore, as in HIV-1 infection, the frequencies of activated HLA-DR⁻CD38⁺ Vδ1⁺ and Vδ2⁺ T cells were both positively associated with detectable viral load in HIV-2 infection. The frequency of naïve Vδ2⁺ T cells was positively associated with an undetectable HIV-2 viral load, while the frequency of CD27⁻CD28⁻ memory-like Vδ2⁺ T cells was negatively associated with an undetectable HIV-2 viral load.

DISCUSSION

Human γδ T cells have been shown to play an important role in protective innate and adaptive immunity through cytokine secretion, cytotoxic activity and ability to present antigens. The results reported are from our in-depth phenotypic analysis of γδ T cells comparing West African HIV seronegative individuals with those infected with either HIV-1 or HIV-2. Similar to what has been reported in HIV-1 infection, HIV-2 infection also alters the subsets of γδ T cells in the blood. A trend toward expansion of Vδ1⁺ T-cell populations was seen in peripheral blood in HIV-2 infected individuals. More importantly, we observed the expansion of CD8⁺, CD28⁻CD45RO⁺ memory like and activated (CD38⁺) Vδ1⁺ T cells in blood of both HIV-1 and HIV-2 infected individuals.

A recent study of SIV infection in rhesus macaques and African green monkeys showed that peripheral expansion of Vδ1⁺ T-cell subset in SIV-infected macaques was associated with SIV-related microbial translocation in those animals¹⁹. In vitro studies have shown that HIV-1 can directly breach the integrity of mucosal epithelial barrier and allow translocation of virus and bacteria³². Furthermore, studies on chronically HIV-1-infected individuals have shown that disease progression is associated with increased circulating lipopolysaccharide (LPS) levels, an indicator of microbial translocation^33–34. This suggests that as in SIV and HIV-1 infection, HIV-2 infection is likely to be associated with damage of mucosal epithelial barrier, microbial translocation and T-cell activation, although no comparable studies of gut mucosa in HIV-2 infected individuals have been reported. However, elevation of plasma LPS concentrations has been recently observed in HIV-2 infected individuals who have progressed to AIDS³⁵. We are currently assessing mucosal integrity and activation in these individuals.

Interestingly, although we observed a significant reduction in the frequency of naïve Vδ1⁺ T cells in HIV-1 as well as HIV-2 infection, no association between the frequency of naïve Vδ1⁺ T cells and CD4⁺ T-cell count or the ability to detect viral load was found in HIV-2 infection, unlike in HIV-1 infection. This is likely due to the relatively less replicative fitness of the HIV-2 as reported in some ex vivo replicative viral fitness tests³⁶ and more importantly, the relatively preserved immune functions of adaptive and innate cells in HIV-2 as compared to HIV-1 infected individuals^{25, 37–41}. Furthermore, we observed a significant positive correlation between the frequency of CD4-expressing Vδ1⁺ T cells and CD4⁺ T-cell counts in only HIV-1 but not HIV-2 infection. This suggests that CD4⁺ Vδ1⁺ T cells might be preferentially depleted by HIV-1 infection. Further studies on the role or function of CD4⁺ Vδ1⁺ T cells will provide insights into why HIV-2 infection is much better controlled than HIV-1.

We showed that Vδ1⁺ as well as Vδ2⁺ T-cell activation levels were associated with decreased CD4⁺ T-cell counts and increased HIV viral load in both HIV-1 and HIV-2 infection. This further indicates that increased activation of γδ T cells is associated with disease progression in not just HIV-1 but also HIV-2 infection. Further studies on longitudinal samples from these individuals will be needed to confirm this hypothesis.

Furthermore, unlike as observed in HIV-1 infected individuals, individuals infected with HIV-2 were not only able to maintain their total number of peripheral Vδ2⁺ T cells, but also were able to maintain a relatively normal frequency of naïve Vδ2⁺ and CD28⁻CD45RO⁺ Vδ2⁺ T cells, as compared to that in HIV-1individuals. Interestingly, maintaining such cell frequencies was associated with undetectable viral load in those infected with HIV-2. Our data implies that individuals with HIV-2 infection maintain enough functional (or cytolytic-like) Vδ2⁺ T cells to control HIV-2 replication, although we observed no significant difference in the frequency of cytolytic-like (CD57⁺) Vδ2⁺ T cells between the two types of HIV infection. Further functional studies on individual subsets of Vδ2⁺ T cells from HIV-2 infected individuals will be important to clarify this hypothesis. Our data also implies that interventions to control HIV infection could potentially benefit from boosting Vδ2⁺ T cells. Furthermore, we observed that the frequency of CD4⁺Vδ2⁺ T cells in HIV-2 infected individuals was significantly reduced compared with seronegative and HIV-1 infected individuals. This reduction in the frequency of CD4⁺Vδ2⁺ T cells might prevent the further depletion of the Vδ2⁺ T cells that control HIV-2 replication in those infected individuals. Interestingly, a recent study by Beaumier and colleagues demonstrated that during coevolution with SIV_agm, African green monkeys have developed the ability to downregulate CD4 on memory CD4⁺ T cells to evade progressive SIV infection⁴². Whether this phenomenon occurs in HIV-2 infected individuals is worthy of further investigation.

Lastly, we observed a slight expansion of the Vδ1⁺ T-cell population in our HIV seronegative cohort as compared to that reported in HIV seronegative healthy Caucasians^{31, 43}. This is not surprising as similar observations have been reported in other studies conducted in Africans^44–45, although the reason for this increased frequency of circulating Vδ1⁺ T cells is still unknown⁴⁴. Infections other than HIV, such as malaria and Human herpesvirus 8 (HHV-8), have been associated with the peripheral expansion of the Vδ1⁺ T-cell population^{43, 45}. None of our samples were collected during the malaria season; however the history of malaria and HHV-8 infection were not recorded in our study.

In summary, HIV-2 infection alters the subsets of γδ T cells in the blood, particularly of Vδ1⁺ T cells, and the increased activation of γδ T cells is associated with unfavorable clinical outcome in both HIV-1 and HIV-2 infection. In this study we have shown that the maintaining the frequencies of naïve γδ T cells, especially within the Vδ2⁺ subset, and of memory-like Vδ2⁺ T cells are important in control of HIV-2 replication. Developing interventions to reduce γδ T-cell activation T cells will be important to prevent transmission and to combat HIV infection.