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Journal of Virology, February 2008, p. 1155-1165, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01275-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Gamma/Delta T-Cell Functional Responses Differ after Pathogenic Human Immunodeficiency Virus and Nonpathogenic Simian Immunodeficiency Virus Infections{triangledown}

David A. Kosub,1 Ginger Lehrman,1 Jeffrey M. Milush,1,{dagger} Dejiang Zhou,1,{ddagger} Elizabeth Chacko,1 Amanda Leone,1,2 Shari Gordon,3,4 Guido Silvestri,3 James G. Else,4 Philip Keiser,1 Mamta K. Jain,1 and Donald L. Sodora1,2*

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas,1 Seattle Biomedical Research Institute, Seattle, Washington,2 Department of Pathology, University of Pennsylvania, Philadelphia, Pennsylvania,3 Emory Vaccine Center and Yerkes National Primate Research Center, Emory University, Atlanta, Georgia4

Received 12 June 2007/ Accepted 7 November 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to functionally assess gamma/delta ({gamma}{delta}) T cells following pathogenic human immunodeficiency virus (HIV) infection of humans and nonpathogenic simian immunodeficiency virus (SIV) infection of sooty mangabeys. {gamma}{delta} T cells were obtained from peripheral blood samples from patients and sooty mangabeys that exhibited either a CD4-healthy (>200 CD4+ T cells/µl blood) or CD4-low (<200 CD4 cells/µl blood) phenotype. Cytokine flow cytometry was utilized to assess production of Th1 cytokines tumor necrosis factor alpha and gamma interferon following ex vivo stimulation with either phorbol myristate acetate/ionomycin or the V{delta}2 {gamma}{delta} T-cell receptor agonist isopentenyl pyrophosphate. Sooty mangabeys were observed to have higher percentages of {gamma}{delta} T cells in their peripheral blood than humans did. Following stimulation, {gamma}{delta} T cells from SIV-positive (SIV+) mangabeys maintained or increased their ability to express the Th1 cytokines regardless of CD4+ T-cell levels. In contrast, HIV-positive (HIV+) patients exhibited a decreased percentage of {gamma}{delta} T cells expressing Th1 cytokines following stimulation. This dysfunction is primarily within the V{delta}2+ {gamma}{delta} T-cell subset which incurred both a decreased overall level in the blood and a reduced Th1 cytokine production. Patients treated with highly active antiretroviral therapy exhibited a partial restoration in their {gamma}{delta} T-cell Th1 cytokine response that was intermediate between the responses of the uninfected and HIV+ patients. The SIV+ sooty mangabey natural hosts, which do not proceed to clinical AIDS, provide evidence that {gamma}{delta} T-cell dysfunction occurs in HIV+ patients and may contribute to HIV disease progression.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Following human immunodeficiency virus (HIV) infection, progression to AIDS is typically associated with increased viral replication and generalized immune dysregulation that is manifested in a variety of malignancies or opportunistic infections. Immune dysfunction occurs in numerous immunologic cells, including CD4+ T cells (22, 30), CD8+ T cells (27, 29), B cells (16, 25, 45), macrophages (10), natural killer cells (50), and gamma/delta ({gamma}{delta}) T cells (13, 32, 39). In humans, {gamma}{delta} T cells comprise a minor subset (1 to 5% on average) of circulating T cells but may represent as much as 50% of the T cells present within the mucosa-associated lymphoid tissue (6). {gamma}{delta} T cells play an important role in the recognition of microbial pathogens and can influence adaptive immune responses by the production of both Th1 and Th2 cytokines (15). There are two main {gamma}{delta} T-cell subsets that express either the first variable region (V{delta}1) or the second variable region (V{delta}2) of the delta locus from the T-cell receptor (TCR) (19, 24). The V{delta}1+ {gamma}{delta} T cells are found predominately at mucosal sites and can respond to nonclassical major histocompatibility complex molecules expressed on stressed cells, while V{delta}2+ {gamma}{delta} T cells are predominately in the peripheral circulation and respond to nonpeptide phosphoantigens (19, 20). {gamma}{delta} T cells are influenced by HIV infection as evidenced by a phenotypic switch from predominately V{delta}2 before infection to predominately V{delta}1 within the peripheral blood of HIV-positive (HIV+) patients (2). In addition, a decrease in the number of effector (CD27– CD45RA–) {gamma}{delta} T cells was observed in immunocompromised patients (18) and in simian immunodeficiency virus (SIV)-infected rhesus macaques (53). Previous studies suggest this may be due to the induction of cellular anergy (32, 33, 39, 42) or the ability of {gamma}{delta} T cells to migrate in response to proinflammatory chemokines (43) and kill cellular targets (51). These data suggest that {gamma}{delta} T cells lose the ability to respond to the HIV/SIV or invading opportunistic pathogens potentially impacting the disease outcome in infected humans/monkeys.

In contrast to pathogenic HIV/SIV infections, natural host primate species in Africa, such as sooty mangabeys (Cercocebus atys), can be infected with SIV but generally remain free of clinical disease (17, 21, 23, 44, 47). The absence of clinical disease signs in SIV+ sooty mangabeys does not appear to be due to any differences inherent in virus replication, as mangabey viral loads are comparable to those of HIV-infected patients (105 to 107 copies per milliliter of plasma) and passage of virus to rhesus macaques results in simian AIDS (46). The presence of high levels of SIV replication in mangabeys suggests that protection from AIDS is not due to species-specific innate antiviral mechanisms or a more robust adaptive immune response (14, 37). To date, studies indicate that the ability of mangabeys to resist progression to AIDS may be due in part to reduced levels of immune activation and bystander apoptosis, as well as a preservation in the levels of peripheral CD4+ T cells (7, 37, 47). While investigating the nonpathogenic mechanisms associated with SIV+ mangabeys, we observed that some mangabeys undergo an extreme, persistent, and generalized depletion of CD4+ T cells to levels associated with disease during HIV infection yet do not exhibit clinical AIDS signs (37, 48). These findings suggested that low CD4+ T-cell levels are not sufficient to induce disease in these natural hosts of SIV. Furthermore, we observed that {gamma}{delta} T cells from both SIV+ CD4-healthy (>200 CD4+ T cells/µl blood) and CD4-low (<200 CD4 cells/µl blood) mangabeys maintained their ability to proliferate in response to the bacterial antigens isopentenyl pyrophosphate (IPP) and lipopolysaccharide (LPS) (37). Studies presented here expand upon the previous analyses through a comparative assessment of the levels and functions of {gamma}{delta} T cells during pathogenic HIV infection of humans and nonpathogenic SIV infection of mangabeys. Our findings indicate that {gamma}{delta} T cells from SIV+ mangabeys maintain their ability to express Th1 cytokines in response to both {gamma}{delta} T-cell-specific and nonspecific stimulation. These data provide a rationale for investigating the use of therapies aimed at increasing {gamma}{delta} T-cell functionality in HIV+ humans.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal and human subjects. HIV-negative blood samples utilized in this study were obtained voluntarily from donors in accordance with the University of Texas Southwestern Medical Center at Dallas Institutional Review Board guidelines. HIV+ patient samples were obtained from consenting donors from the University of Texas Southwestern Medical Center AIDS Clinic in accordance with Institutional Review Board approval. Patients enrolled in this study ranged between 24 and 55 years of age. HIV+ patients on highly active antiretroviral therapy (HAART) were taking at least three antiretroviral drugs for a minimum of 6 months. A summary of HIV viral loads, complete blood counts, and lymphocyte counts for the HIV+ patients are presented (Table 1).


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  		TABLE 1. Complete blood counts and viral loads of the HIV+ patientsa

 
Rhesus macaques (Macaca mulatta) and sooty mangabeys (Cercocebus atys) were colony bred at the Yerkes National Primate Research Center and cared for in accordance with NIH and local Institutional Animal Care and Use Committee guidelines. Animals ranged from 2 to 10 years of age when enrolled in the study and tested negative for SIV, simian T-cell leukemia virus, and simian retrovirus. The SIV+ sooty mangabeys utilized in this study were either naturally infected in the Yerkes colony or experimentally infected as previously described (40) (Table 2).


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  		TABLE 2. Complete blood counts of sooty mangabeysa

 
Real-time PCR to quantify V{delta}1 and V{delta}2 TCR. Mangabey V{delta}1 and V{delta}2 sequences (GenBank accession numbers 765393 and 765371, respectively) were utilized to design quantitative real-time PCR primers and probes to quantify {gamma}{delta} T-cell receptor expression. Total RNA was isolated from mangabey peripheral blood mononuclear cells (PBMCs) at 0, 12, and 100 weeks postinfection using the RNeasy kit (Qiagen, Valencia, CA) and cDNA synthesized (Invitrogen, Carlsbad, CA). Real-time PCR analysis was undertaken utilizing primers (Sigma Genosys, The Woodlands, TX) and probes (Applied Biosystems, Foster City, CA) as follows. For V{delta}1, the forward primer was 5'-TCGCCTTAACCATTTGAGCC-3', the reverse primer was 5'-AACGGATGGTTTGGTATGAGGT-3', and the probe was 5'- FAM-TACAGCTAGAAGACTCAGCAACATACTTCTGTGCTC-TAMRA-3' (where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine). For V{delta}2, the forward primer was 5'-GAGAACCAGGCTGTACTTAAGATCCTT-3', the reverse primer was 5'-TGACGAAAACGGATGGTTTG-3', and the probe was 5'-FAM-AGAGAGAGATGAAGGGTCTTACTACTGTGCCAGTG-TAMRA-3'. The quantitative real-time PCRs were performed and analyzed as previously described (36) with the following modifications: 1 µg of cDNA generated from total RNA was added to each reaction mixture, and unknowns were compared to plasmid standards containing both the V{delta}1 and V{delta}2 genes. Copy numbers were then normalized to represent the number of V{delta}1 or V{delta}2 cDNA copies per 1 x 106 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA copies, and the ratio was determined. Sequences for the GAPDH primer and probe set were obtained from published sequences (1).

Phenotypic analysis and cytokine flow cytometry of {gamma}{delta} T cells. Approximately 30 to 40 ml of whole blood was drawn from either humans or sooty mangabeys in acid-citrate-dextrose anticoagulant tubes. PBMCs were isolated utilizing a Ficoll-Hypaque gradient. To undertake a phenotypic assessment for the levels of {gamma}{delta} T cells within the PBMCs, the cells were first washed with flow cytometry wash buffer (phosphate-buffered saline [PBS] plus 4% fetal bovine serum) and stained with fluorescently labeled antibodies specific for cellular surface antigens for 30 min. The antibodies were conjugated to fluorescein isothiocyanate (Pan-negative [Pan–] {gamma}{delta} TCR] clone 5A6.E9]), phycoerythrin (PE) (V{delta}2 {gamma}{delta} TCR [clone B6]), or PerCP-Cy5.5 (CD3 [SP34-2]). Following antibody staining, the PBMCs were washed twice with PBS and then fixed in 1% paraformaldehyde.

To perform cytokine flow cytometry, PBMCs were aliquoted into a 96-well plate at a concentration of 2 x 106 cells/200 µl medium and stimulated overnight at 37°C with medium alone (negative control), the bacterial antigens isopentenyl pyrophosphate (IPP; 30 µg/ml) (Sigma, St. Louis, MO), or LPS (1 µg/ml) (Sigma, St. Louis, MO). The mitogen phorbol myristate acetate (PMA) (25 ng/ml) (Sigma, St. Louis, MO) in combination with the calcium ionophore, ionomycin (1 µg/ml) (Sigma, St. Louis, MO) was added to a separate well for 1 h at 37°C to represent a generalized stimulant. Following overnight stimulation (medium alone, IPP, or LPS) (or 1 h after PMA/ionomycin stimulation) 1 µg/ml brefeldin A (Sigma, St. Louis, MO) was added to each sample, and the cells were incubated an additional 5 h to allow cytokines to accumulate within the cells. The cells were washed with fluorescence-activated cell sorting (FACS) wash buffer and then permeabilized by the addition of 200 µl of FACSJuice (2x FACSLyse [BD Pharmingen, San Diego, CA] with 0.05% Tween 20 [Sigma, St. Louis, MO]) and incubated for 3 min at room temperature. The cells were washed twice with FACS wash buffer, centrifuged, and stained with fluorescent antibodies specific for cellular surface antigens and cytokines for 30 min. The antibodies were directly conjugated to fluorescein isothiocyanate (Pan– {gamma}{delta} TCR [clone 5A6.E9]), PE (V{delta}2 {gamma}{delta} TCR [clone B6]), PerCP-Cy5.5 (CD3 [SP34-2]), PE-Cy7 (interleukin 4 [IL-4] [clone 8D4.8]), allophycocyanin (tumor necrosis factor alpha [TNF-{alpha}]) (clone MAb11)], or allophycocyanin-Cy7 (gamma interferon [IFN-{gamma} [[4SB3]). Following antibody staining, the PBMCs were washed twice with PBS and then fixed in 1% paraformaldehyde. Flow cytometric analysis was performed on a Cyan flow cytometer (Dako-Cytomation; Fort Collins, CO) and analyzed utilizing FlowJo software (Flowjo, Ashland, OR).

Assessment of {gamma}{delta} T cells from lymph node biopsy, rectal biopsy, and BAL samples. Lymph node biopsies (axillary or inguinal) and rectal mucosal biopsies and bronchoalveolar lavages (BALs) were performed on two SIV+ CD4-low (SM1 and SM2) and two SIV+ CD4-healthy (SM3 and SM4) mangabeys (37). Rectal mucosal biopsies were obtained using a sigmoidoscope with forceps, and mononuclear cells were isolated by collagenase digestion (two sequential 30-min incubations at 37°C in RPMI containing 0.75 mg/ml collagenase). The digested suspension was passed through 70-µm cell strainers and then enriched for lymphocytes by Percoll density gradient. Mononuclear cells were collected (present at the interface between the 35 and 60% Percoll layers), washed, and resuspended in complete RPMI. For the BALs, a fiber-optic bronchoscope was placed into the trachea after local anesthetic was applied to the larynx. Four 35-ml aliquots of warmed saline were injected into the right primary bronchus and collected by aspiration before a new aliquot was instilled. Following aspiration and collection of the BAL cells, PBMCs were isolated utilizing Ficoll density gradient centrifugation as described earlier. After the cells were counted, they were stained with antibodies recognizing the Pan– {gamma}{delta} TCR, and CD3 was then assessed utilizing flow cytometric analysis. Utilizing FlowJo software, a lymphocyte gate was determined utilizing forward and side scatter, followed by gating on CD3+ cells, and then gating on {gamma}{delta} TCR+ cells. The percentage of {gamma}{delta} TCR+ T cells within the total CD3+ population was determined and graphed using GraphPad software.

Statistical analysis. Statistical analyses were performed using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). A Mann-Whitney test (nonparametric, two-tailed, and unpaired) was performed to determine the differences between the values for uninfected and infected groups; comparison of uninfected humans to HIV+ patients and uninfected mangabeys to SIV+ mangabeys was also done. Statistical significance is identified as P values less than 0.05 (95% confidence interval).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparative phenotypic assessment of peripheral {gamma}{delta} T-cell levels in humans, macaques, and mangabeys. Rhesus macaques and sooty mangabeys are nonhuman primates used to study pathogenic and nonpathogenic models of HIV infection, respectively (36, 47, 49, 53). PBMCs from these monkeys and humans were assessed to identify changes within the {gamma}{delta} T cells following SIV/HIV infection. A representative gating strategy to assess the levels of {gamma}{delta} T cells in peripheral blood is depicted (Fig. 1A to C). The frequency of {gamma}{delta} T cells is presented as the percentage of CD3+ T cells expressing the {gamma}{delta} T-cell receptor within the different species and with regard to infection status (Fig. 1D and E). A significantly higher frequency of {gamma}{delta} T cells was observed in uninfected mangabeys (12.5%) than in macaques (6.1%) and humans (3.8%) (Fig. 1D). A cross-sectional analysis of HIV+ patients enrolled at the University of Texas Southwestern Medical Center AIDS Clinic (Table 1) was performed to examine the relationship between peripheral blood {gamma}{delta} T-cell frequencies and disease progression. HIV+ patients were divided into three groups as follows: (i) HIV+ CD4-healthy (>200 CD4+ T cells/µl blood), (ii) HIV+ CD4-low (<200 CD4+ T cells/µl blood), and (iii) HIV+ on HAART (350 to 2,100 CD4+ T cells/µl blood). The percentage of {gamma}{delta} T cells was elevated two- to threefold in both the CD4-healthy and CD4-low HIV+ patients, representing a significantly higher level than in the uninfected donors (Fig. 1E). The increased percentage of {gamma}{delta} T cells in the HIV+ patients may be due to a decrease in the overall percentage of CD4+ cells within the CD3+ T-cell population. HAART treatment resulted in {gamma}{delta} T-cell frequencies that were intermediate between HIV+ and uninfected donors (Fig. 1E). In addition, an analysis of peripheral {gamma}{delta} T-cell frequencies was also undertaken in both CD4-healthy and CD4-low SIV+ mangabeys (Table 2). In contrast to HIV-infected patients, the frequency of {gamma}{delta} T cells significantly decreased in the CD4-healthy SIV+ mangabeys in comparison to their uninfected counterparts (Fig. 1E). Some of the SIV+ CD4-low mangabeys exhibited very low {gamma}{delta} T-cell frequencies, although others exhibited frequencies comparable to those of the uninfected mangabeys (Fig. 1E). This decrease in the levels of {gamma}{delta} T cells in some mangabeys is likely due to redistribution of the cells to other tissue sites (such as mucosa).


Figure 1
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  		FIG. 1. Peripheral blood mononuclear cells from humans and sooty mangabeys were stained with fluorescently labeled cross-reactive antibodies against the Pan– {gamma}{delta} TCR and CD3 and assessed using flow cytometric analysis. (A to C) A representative gating strategy from an uninfected donor is shown, indicating the forward and side scatter to gate on lymphocytes (A), pulse width to gate on single cells (B), and CD3 and Pan– {gamma}{delta} TCR to gate on {gamma}{delta} T cells (C). (D) Graphical representation of the percentage of CD3+ T cells expressing the Pan– {gamma}{delta} TCR from uninfected humans ({blacksquare}), macaques ({blacktriangleup}), and mangabeys (•). The percentage of {gamma}{delta} T cells in uninfected mangabeys was significantly higher than the percentages in both humans and macaques. (E) Assessment of the levels of {gamma}{delta} T cells was performed in the CD4-healthy (dark blue triangles [{blacktriangleup}]) and CD4-low (light blue triangles [{blacktriangledown}]) HIV+ patients and SIV+ mangabeys, as well as HIV+ patients on HAART (green circles) and compared to uninfected donors (red squares). There were increased percentages of {gamma}{delta} T cells in the peripheral circulation of HIV+ patients compared to uninfected donors. The SIV+ CD4-healthy mangabeys had decreased percentages of peripheral blood {gamma}{delta} T cells compared to their uninfected counterparts, whereas on average, the SIV+ CD4-low mangabeys demonstrated similar levels as uninfected mangabeys. The short horizontal lines in panels D to F indicate the means for each group.

 
Assessment of {gamma}{delta} T-cell subtypes in humans and mangabeys. The ability of the {gamma}{delta} TCR to recognize antigens is determined predominantly by the particular delta variable region expressed (6, 19, 24). {gamma}{delta} T cells in the peripheral blood primarily express the V{delta}2 {gamma}{delta} TCR and, to a lesser extent, the V{delta}1 {gamma}{delta} TCR. Indeed, in this study, {gamma}{delta} T cells from uninfected individuals demonstrated a predominance of V{delta}2+ cells in the peripheral circulation as indicated by a negative V{delta}2–/V{delta}2+ ratio (Fig. 2A and B). However, the HIV+ patient cohort had a predominately V{delta}2– {gamma}{delta} T-cell population in the peripheral blood (Fig. 2B, flow cytometric analysis) in agreement with previous studies (2, 11, 43). In order to assess the {gamma}{delta} TCR usage in mangabeys, a quantitative real-time PCR approach was utilized due to the lack of a cross-reacting anti-human V{delta}2 antibody. Prior to infection, the predominate {gamma}{delta} TCR transcripts in the mangabey's peripheral blood were V{delta}1, rather than V{delta}2 observed in humans (Fig. 2C, real-time PCR). Following SIV infection, the V{delta}1 TCR transcripts continued to be the predominate receptor expressed at 12 weeks and 100 weeks postinfection (Fig. 2C). The declining V{delta}1/V{delta}2 ratio observed throughout infection was principally due to a decrease in the absolute number of V{delta}1 TCR transcripts (Fig. 2D) rather than an increase in V{delta}2 transcripts which remained either stable or declined slightly (data not shown). In summary, the V{delta}2 to V{delta}1 phenotypic switch observed in the peripheral blood {gamma}{delta} T cells of HIV+ patients did not occur in the SIV+ mangabeys, as they maintained preferentially elevated levels of V{delta}1 {gamma}{delta} T cells both before and after SIV infection.


Figure 2
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  		FIG. 2. {gamma}{delta} T cells from HIV+ patients and SIV+ sooty mangabeys predominately express the V{delta}1 {gamma}{delta} TCR. PBMCs from humans were stained with fluorescently labeled cross-reactive antibodies recognizing the Pan– {gamma}{delta} TCR, V{delta}2 {gamma}{delta} TCR, and CD3, and the ratio of V{delta}2-negative (V{delta}2neg) {gamma}{delta} T cells to V{delta}2+ {gamma}{delta} T cells is reported. (A) A representative histogram shows the V{delta}2neg/V{delta}2+ T-cell ratio from an uninfected donor. The ratio is determined by dividing the percentage of V{delta}2– cells by the V{delta}2+ cells. (B) Graphical representation of the V{delta}2neg/V{delta}2+ {gamma}{delta} T-cell ratio from uninfected, HIV+ CD4-healthy, and HIV+ CD4-low patients and HIV+ patients on HAART. HIV+ patients had an increased V{delta}2neg/V{delta}2+ T-cell ratio compared to their uninfected counterparts. (C) To assess {gamma}{delta} T-cell ratios in sooty mangabeys, a real-time PCR approach was utilized to quantify the levels of V{delta}1 and V{delta}2 mRNA. An assessment of the V{delta}1/V{delta}2 T-cell ratio was determined in six sooty mangabeys (SM1 to SM6) before infection (0 week postinfection [WPI]) and 12 weeks and 100 weeks postinfection (12 and 100 WPI, respectively). The V{delta}1 TCR was predominately expressed by {gamma}{delta} T cells both before and after SIV infection in mangabey PBMCs. (D) Determination of the levels of V{delta}1 in 1 x 106 equivalents of GAPDH mRNA is depicted for six sooty mangabeys before infection and 12 weeks and 100 weeks postinfection. These data indicate that the decrease in the V{delta}1/V{delta}2 ratio in SIV+ mangabeys in panel C is principally due to a decrease in V{delta}1 {gamma}{delta} TCR expression levels.

 
Th1 cytokine responses are maintained or increased by {gamma}{delta} T cells from SIV+ mangabeys but not HIV+ patients. {gamma}{delta} T cells demonstrate a variety of functions which include the production of cytokines to augment the adaptive immune response at the sites of infection or tumors (15, 31, 38). Here, the cytokine expression of {gamma}{delta} T cells was assessed utilizing cytokine flow cytometry on peripheral blood {gamma}{delta} T cells from both HIV+ patients and SIV+ mangabeys. To induce cytokine production ex vivo, the peripheral blood cells were incubated with medium alone (negative control), PMA/ionomycin (PI) (global stimulation), the {gamma}{delta} T-cell-specific bacterial ligand isopentenyl pyrophosphate (specific for V{delta}2 {gamma}{delta} TCR), or LPS (TLR-4 ligand). To determine the impact of nonpathogenic SIV infection and CD4+ T-cell levels, the percentage of {gamma}{delta} T cells expressing IFN-{gamma} (Fig. 3) or TNF-{alpha} (Fig. 4) from both CD4-healthy or CD4-low SIV+ mangabeys was compared to uninfected mangabeys. In general, neither medium alone nor LPS induced Th1 cytokine secretion by peripheral {gamma}{delta} T cells from either uninfected or SIV+ mangabeys (Fig. 3A and 4A). However, {gamma}{delta} T cells from both CD4-healthy and CD4-low SIV+ mangabeys stimulated with PI or IPP maintained or slightly increased their ability to express IFN-{gamma} (Fig. 3A) and TNF-{alpha} (Fig. 4A). Unlike SIV+ mangabeys, an assessment of {gamma}{delta} T cells from HIV+ patients revealed significantly decreased percentages of {gamma}{delta} T cells expressing these Th1 cytokines (IFN-{gamma} and TNF-{alpha}) following stimulation with PI and IPP (Fig. 3B and 4B). {gamma}{delta} T cells from HIV+ patients on HAART had a partially restored ability to express these Th1 cytokines following stimulation, though not to the extent that were produced by {gamma}{delta} T cells from uninfected patients (Fig. 3B and 4B). Within all {gamma}{delta} T cells, the changes in cytokine expression of the V{delta}2+ T-cell subset was our primary interest due to the observations of alterations/dysfunction of this subset (32, 33, 39, 42) as well as the high levels of this subset within the peripheral blood of uninfected people. When exposed to PI, the majority of V{delta}2+ {gamma}{delta} T cells from uninfected human donors expressed IFN-{gamma} or TNF-{alpha} (Fig. 3C and 4C). However, the percentages of V{delta}2+ {gamma}{delta} T cells expressing these Th1 cytokines following PI stimulation significantly declined in both the CD4-healthy and CD4-low HIV+ patients (Fig. 3C and 4C). Similarly, the specific V{delta}2 TCR agonist IPP stimulated fewer {gamma}{delta} T cells to produce Th1 cytokines in the HIV+ patients than in uninfected patients (Fig. 3C and 4C). HAART treatment partially restored the ability of V{delta}2+ {gamma}{delta} T cells to express Th1 cytokines following PI and IPP treatment although not to the extent observed in uninfected patients (Fig. 3C and 4C). The V{delta}2– population is predominately composed of V{delta}1+ {gamma}{delta} T cells, and these cells were responsive to PI stimulation but did not produce Th1 cytokines when stimulated with IPP or LPS (Fig. 4D). Therefore, the majority of the decline in responsiveness in the HIV+ patients could be attributed to decreased cytokine production by the V{delta}2+ {gamma}{delta} T cells.


Figure 3
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  		FIG. 3. IFN-{gamma} responses were maintained by {gamma}{delta} T cells from SIV+ sooty mangabeys but not HIV+ patients. (A and B) PBMCs were stimulated ex vivo with medium alone (M) (negative control), PI, IPP, or LPS, and the expression of IFN-{gamma} by total {gamma}{delta} T cells from SIV+ mangabeys (A) and HIV+ patients (B) was assessed utilizing cytokine flow cytometry. The percentage of {gamma}{delta} T cells from SIV+ mangabeys expressing IFN-{gamma} (IFN-{gamma}+) was maintained following ex vivo stimulation with PI and IPP. Unlike mangabeys, significantly decreased percentages of {gamma}{delta} T cells from HIV+ patients expressed IFN-{gamma} following stimulation. (C and D) Utilizing an anti-human V{delta}2 {gamma}{delta} TCR antibody, the V{delta}2+ (C) and V{delta}2-negative (V{delta}2neg) (D) {gamma}{delta} T-cell subsets from patients was also determined to permit a comparison between the uninfected and HIV+ humans. A decreased percentage of IFN-{gamma} expressing V{delta}2+ {gamma}{delta} T cells was observed in all HIV+ patient groups. The short horizontal lines indicate the means for each group.

 

Figure 4
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  		FIG. 4. TNF-{alpha} expression was maintained or increased by {gamma}{delta} T cells from SIV+ sooty mangabeys but not HIV+ patients. (A and B) PBMCs were stimulated ex vivo with medium alone (M) (negative control), PI, IPP, or LPS, and the expression of TNF-{alpha} by total {gamma}{delta} T cells from SIV+ mangabeys (A) and HIV+ patients (B) was assessed utilizing cytokine flow cytometry. The percentage of {gamma}{delta} T cells from SIV+ mangabeys expressing TNF-{alpha} (TNF-{alpha}+) was maintained or increased following ex vivo stimulation with PI and IPP. Unlike mangabeys, significantly decreased percentages of {gamma}{delta} T cells from HIV+ patients expressed TNF-{alpha} following stimulation. (C and D) Utilizing an anti-human V{delta}2 {gamma}{delta} TCR antibody, the V{delta}2+ (C) and V{delta}2-negative (V{delta}2neg) (D) {gamma}{delta} T-cell subsets from patients was also determined to permit a comparison between uninfected and HIV+ humans. A decreased percentage of TNF-{alpha}-expressing V{delta}2+ {gamma}{delta} T cells was observed in all HIV+ patient groups. The short horizontal lines indicate the means for each group.

 
Although stimulation of the {gamma}{delta} T cells tended to produce both IFN-{gamma} and TNF-{alpha} in most situations, the levels of the production of the two cytokines did vary. Indeed, a higher percentage of {gamma}{delta} T cells expressed TNF-{alpha}, compared to IFN-{gamma}, after PI and IPP stimulation in the majority of conditions assessed. For example, when V{delta}2+ {gamma}{delta} T cells from uninfected donors were stimulated with PI, nearly 90% of the cells expressed TNF-{alpha} compared to 60% expressing IFN-{gamma} (Fig. 3B and 4B). Moreover, TNF-{alpha} was almost exclusively expressed by human V{delta}2+ {gamma}{delta} T cells following stimulation with IPP (Fig. 3 and 4). The increased expression of TNF-{alpha} suggests that {gamma}{delta} T cells may preferentially express this cytokine for the potential killing of HIV/SIV-infected cells or modulating the immune system in response to opportunistic pathogens (3, 15, 20, 31, 51).

To more easily compare the HIV+ humans and SIV+ mangabeys, the {gamma}{delta} T-cell Th1 cytokine responses following either PI or IPP stimulation is depicted in Fig. 5. SIV infection of mangabeys resulted in {gamma}{delta} T cells with maintained IFN-{gamma} and increased TNF-{alpha} response following ex vivo stimulation with PI and IPP (Fig. 5). The level of CD4+ T cells did not appear to impact Th1 cytokine production, as the maintained or increased levels were observed in both CD4-healthy and CD4-low SIV+ mangabeys. In contrast, the ability of {gamma}{delta} T cells from HIV+ patients, both CD4-healthy and CD4-low, to produce IFN-{gamma} and TNF-{alpha} decreased significantly (Fig. 5). The increased percentages of {gamma}{delta} T cells expressing Th1 cytokines in the HIV+ HAART-treated patients indicated some recovery of IFN-{gamma} and TNF-{alpha} production, but not to the levels observed in the uninfected patients (Fig. 5). These results indicate that {gamma}{delta} T cells maintain their functionality in SIV+ mangabeys; however, {gamma}{delta} T cells from HIV+ patients have an impaired functional response that is only partially restored with HAART treatment.


Figure 5
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  		FIG. 5. Graphical representation of the percentage of total {gamma}{delta} T cells from uninfected, CD4-healthy, or CD4-low sooty mangabeys and humans and HIV+ patients on HAART therapy is presented. The ability of the {gamma}{delta} T cells to express the Th1 cytokines IFN-{gamma} and TNF-{alpha} in response to PMA/ionomycin (PI) (A) or isopentenyl pyrophosphate (IPP) (B) is presented such that comparisons can be easily made between species and infection status. {gamma}{delta} T cells from CD4-healthy and CD4-low SIV+ mangabeys had the same IFN-{gamma} responses and increased TNF-{alpha} responses to ex vivo PI and IPP stimulation compared to their uninfected counterparts. In contrast, HIV+ patients showed decreased percentages of {gamma}{delta} T cells expressing these Th1 cytokines compared to healthy donors. {gamma}{delta} T cells from HIV+ patients on HAART had a partially restored ability to express these Th1 cytokines following stimulation, though not to the extent that was produced by {gamma}{delta} T cells from uninfected patients. Asterisks indicate statistically significant differences from the results for uninfected mangabeys or humans.

 
{gamma}{delta} T cells are present at the mucosal sites in SIV-infected mangabeys. Assessment of {gamma}{delta} T cells at two mucosal sites (rectal and pulmonary) and lymphoid sites was undertaken in both CD4-healthy and CD4-low SIV+ mangabeys. Overall, the rectal mucosa contained the highest percentage of {gamma}{delta} T cells, ranging from 3.0 to 9.8% of the CD3+ cells, with no discernible impact on these frequencies in the mangabeys with the CD4-low phenotype (Fig. 6). In comparison to rectal mucosa, lower percentages of {gamma}{delta} T cells were present in the BAL and lymph nodes, ranging from 0.5 to 3.5% of CD3+ cells. These data indicate that {gamma}{delta} T cells are present at mucosal and lymphoid sites during chronic SIV infections of mangabeys irrespective of CD4+ T-cell levels which likely represent major reservoirs for this T-cell subset.


Figure 6
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  		FIG. 6. Presence of {gamma}{delta} T cells at mucosal and lymphoid sites in SIV+ CD4-low and CD4-healthy sooty mangabeys. Cells were obtained from lymph node (LN), rectal mucosal (RM) biopsy, and bronchoalveolar lavage (BAL) samples from four sooty mangabeys (SM1 to SM4) (37) and analyzed for the expression of CD3 and the Pan– {gamma}{delta} TCR. The percentage of CD3+ T cells expressing the Pan– {gamma}{delta} TCR from the RM, BAL, and LN samples are depicted. The RM biopsy samples exhibited the highest percentage of {gamma}{delta} T cells within the CD3+ T-cell population; lower percentages of {gamma}{delta} T cells were observed in BAL and LN samples.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The finding that dramatic and sustained CD4+ T-cell depletion in SIV+ mangabeys was not sufficient to induce clinical signs of simian AIDS indicated that along with low levels of immune activation, other immune cells, such as {gamma}{delta} T cells, may be important in preventing AIDS disease progression in this species (37). {gamma}{delta} T cells have important roles in bridging the innate and adaptive immune responses (31, 38) primarily by responding to bacterial antigens, such as isopentenyl pyrophosphate, or the recognition of stress-induced nonclassical major histocompatibility complex molecules expressed on virally infected cells (6, 19, 20). The role of {gamma}{delta} T cells during HIV/SIV infection is not clear, although there is evidence that they may participate in defense against acute SIV infection of vaccinated macaques following oral (49) or rectal (26) challenge and are activated (express cytokines) following in vitro stimulation with HIV-infected cells (20, 41). However, the functional responses of {gamma}{delta} T cells are impaired following chronic pathogenic HIV/SIV infections as evident by a decreased ability to proliferate in response to the opportunistic pathogen mycobacteria (42, 53) and a decreased capacity to express IFN-{gamma} following in vitro stimulation (13, 32, 33). The data presented in this study demonstrated a switch in the predominate V{delta}2+ peripheral {gamma}{delta} T-cell population to a V{delta}2– subset following HIV infection (Fig. 2) in agreement with other reports (2, 11, 33). In addition, the {gamma}{delta} T cells from CD4-low and CD4-healthy HIV+ patients showed an impaired ability to express IFN-{gamma} and TNF-{alpha} following bacterial antigen stimulation (Fig. 3, 4, and 5). The studies of HIV+ patients described here therefore support previous reports whereby alterations in both the phenotypic and functional responses of {gamma}{delta} T cells occur following chronic HIV infection (2, 11, 13, 32, 33).

Prior to HIV infection, CD4+ T cells express predominately Th1 cytokines in response to ex vivo stimulation with phorbol esters, but this response is switched to predominantly Th2 cytokines during chronic HIV infection (8, 9, 22, 35). The mechanism for the observed Th1 to Th2 cytokine skewing remains unknown. In agreement with the majority of published reports in the {alpha}β T-cell literature (8, 9, 22, 35), decreased Th1 cytokine expression by {gamma}{delta} T cells in HIV+ patients was observed (following ex vivo IPP and PI stimulation) (Fig. 3, 4, and 5). The fact that Th1 responses are impaired in both {alpha}β and {gamma}{delta} T-cell subsets suggests that this phenomenon may be due in part to immune dysfunction as opposed to direct viral cytopathogenicity. Through an analysis of the V{delta}2+ and V{delta}2– {gamma}{delta} T-cell subsets these findings indicate that the dysfunction is primarily localized to the V{delta}2+ subset. Therefore, not only is the percentage of this subset declining in the blood throughout infection but the ability of V{delta}2+ {gamma}{delta} T cells to produce Th1 cytokines is also compromised. Although {gamma}{delta} T cells can express Th2 prohumoral/anti-inflammatory cytokines, such as IL-4, in response to antigenic stimulation (15, 38), there was no evidence for a Th1 to Th2 shift in any subset of {gamma}{delta} T cells, as IL-4 expression generally remained low in HIV+ patients and SIV+ mangabeys (data not shown). The preserved functionality of the {gamma}{delta} T cells in the SIV+ mangabeys provides a foundation for future studies to assess the role of {gamma}{delta} T cells in preventing opportunistic infections and SIV disease progression in this species.

Dysfunctional responses by {gamma}{delta} T cells were historically attributed to the loss of CD4+ T cells during pathogenic HIV/SIV infections. The studies presented here addressed the dependence of {gamma}{delta} T cells on CD4+ T-cell help for proper function by comparing the {gamma}{delta} T-cell responses of CD4-healthy and CD4-low cohorts (Fig. 3, 4, and 5). In sooty mangabeys, {gamma}{delta} T cells maintain the ability to proliferate (37) and express Th1 cytokines when stimulated with bacterial antigens despite depletion of CD4+ T cells in the CD4-low cohort (Fig. 3, 4, and 5). Furthermore, during HIV infection of humans, decreased levels of Th1 cytokine responses were observed in the {gamma}{delta} T cells, and depletion of CD4+ T cells in the CD4-low patients did not further abrogate Th1 cytokine levels (Fig. 3, 4, and 5). These data suggest that the impairment of Th1 cytokine expression by {gamma}{delta} T cells in the HIV+ humans may not be due solely to the loss of CD4+ T cells, but rather other indirect HIV-induced immunologic changes (Fig. 5). We hypothesize that the persistent immune activation observed in pathogenic HIV infection may be a key factor in the alteration of {gamma}{delta} T-cell function. Therefore, low levels of immune activation in chronically SIV+ mangabeys, as opposed to CD4+ T-cell levels, may contribute to the preservation of Th1 cytokine expression by {gamma}{delta} T cells.

The presence of {gamma}{delta} T cells at mucosal sites (Fig. 6) suggests that these cells may contribute to the protection against pathogenic mucosal microorganisms. During the CD4+ T-cell depletion in the mucosa of SIV and HIV infections (5, 28, 34), the ability of {gamma}{delta} T cells to respond to microbes at mucosal surfaces may be important to prevent disease progression following infection. We propose that mangabey {gamma}{delta} T cells may prevent opportunistic bacterial pathogens from establishing infections which otherwise might contribute to persistent immune activation due to the fact that {gamma}{delta} T cells from SIV+ mangabeys retain their ability to express Th1 cytokines (Fig. 3 to 5) and are present at mucosal sites (Fig. 6). Alternatively, {gamma}{delta} T cells may be important in maintaining an intact mucosal epithelium that prevents commensal bacteria from entering into the systemic circulation (4). We hypothesize that augmenting Th1 responsiveness by human {gamma}{delta} T cells may enhance innate cellular immune defenses against opportunistic infections in HIV+ patients. Clinical augmentation of {gamma}{delta} T-cell functions has been demonstrated previously whereby the antitumor properties of {gamma}{delta} T cells can be effectively increased in melanoma patients through the administration of bisphosphates (12, 52), which are a class of compounds related to IPP. However, the timing, administrative route, and dosages of any drugs designed to increase {gamma}{delta} T-cell function would require careful assessment during pathogenic SIV-macaque infections prior to administration in HIV+ patients. In this regard, the findings depicted here assessing the nonpathogenic SIV-mangabey infections might be useful in understanding the importance of effective {gamma}{delta} T-cell immune responses during a nonpathogenic infection.


   ACKNOWLEDGMENTS
 
This study was supported by NIH grants R01-AI035522 (D.L.S.), R21-AI060451 (D.L.S.), and RR00165 (Yerkes). D.A.K. was supported by the Integrative Immunology Training Program NIAID 5T32 AI005284.

We acknowledge the excellent staff, particularly Michael Lowery, at the University of Texas Southwestern HIV Research Unit for enrolling HIV+ patients into the study. We thank Christopher Cano for excellent technical assistance. We also thank Harold McClure, Stephanie Ehnert, and the animal care and veterinary staff at the Yerkes National Primate Research Center where the mangabeys and macaques were housed.


   FOOTNOTES
 
* Corresponding author. Mailing address: Seattle Biomedical Research Institute, 307 Westlake Ave. N., Seattle, WA 98109. Phone: (206) 256-7413. Fax: (206) 256-7229. E-mail: Don.Sodora{at}SBRI.org Back

{triangledown} Published ahead of print on 28 November 2007. Back

{dagger} Present address: Department of Experimental Medicine, University of California San Francisco, San Francisco, CA. Back

{ddagger} Present address: University of California San Diego, San Diego, CA. Back


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