Detection of Polyfunctional Mycobacterium tuberculosis Specific T Cells and Association with Viral Load in HIV-1 Infected Persons

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1 MAJOR ARTICLE Detection of Polyfunctional Mycobacterium tuberculosis Specific T Cells and Association with Viral Load in HIV-1 Infected Persons Cheryl L. Day, 1,3,4,a Nompumelelo Mkhwanazi, 1 Sharon Reddy, 1 Zenele Mncube, 1 Mary van der Stok, 1 Paul Klenerman, 2 and Bruce D. Walker 1,3,4,5 1 HIV Pathogenesis Programme, Doris Duke Medical Research Institute, University of KwaZulu Natal, Durban, South Africa; 2 Nuffield Department of Medicine, The Peter Medawar Building for Pathogen Research, Oxford University, Oxford, United Kingdom; 3 Partners AIDS Research Center, Massachusetts General Hospital, and 4 Division of AIDS, Harvard Medical School, Boston, Massachusetts; and 5 Howard Hughes Medical Institute, Chevy Chase, Maryland (See the editorial commentary by Lalvani and Millington, on pages ) Background. The human immunodeficiency virus type 1 (HIV-1) epidemic is associated with a significant increase in the incidence of tuberculosis (TB); however, little is known about the quality of Mycobacterium tuberculosis (MTB) specific cellular immune responses in coinfected individuals. Methods. A total of 137 HIV-1 positive individuals in Durban, South Africa, were screened with the use of overlapping peptides spanning Ag85A, culture filtrate protein 10 (CFP-10), early secretory antigen target 6 (ESAT-6), and TB10.4, in an interferon (IFN) enzyme-linked immunospot (ELISPOT) assay. Intracellular cytokine staining for MTB-specific production of IFN-, tumor necrosis factor (TNF), and interleukin (IL) 2 was performed, as was ex vivo phenotyping of memory markers on MTB-specific T cells. Results. A total of 41% of subjects responded to ESAT-6 and/or CFP-10, indicating the presence of latent MTB infection. The proportion of MTB-specific IFN- /TNF- CD4 cells was significantly higher than the proportion of IFN- /IL-2 CD4 cells (P.0220), and the proportion of MTB-specific IL-2 secreting CD4 cells was inversely correlated with the HIV-1 load (P.0098). MTB-specific CD8 T cells were predominately IFN- /TNF- /IL-2. Ex vivo memory phenotyping of MTB-specific CD4 and CD8 T cells indicated an early to intermediate differentiated phenotype for the population of effector memory cells. Conclusions. Polyfunctional MTB-specific CD4 and CD8 T cell responses are maintained in the peripheral blood of HIV-1 positive individuals, in the absence of active disease, and the functional capacity of these responses is affected by HIV-1 disease status. Approximately 90% of HIV-1 negative individuals infected with Mycobacterium tuberculosis (MTB) do not Received 24 September 2007; accepted 29 October 2007; electronically published 4 March Potential conflicts of interest: none reported. Financial support: National Institutes of Health (grant R01 AI067073); Mark and Lisa Schwartz Foundation. Presented in part: 3rd South African AIDS Conference, Durban, South Africa, June 2007 (poster A61). a Present affiliation: South African Tuberculosis Vaccine Initiative, Institute for Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa. Reprints or correspondence: Dr. Cheryl L. Day, South African Tuberculosis Vaccine Initiative, Institute for Infectious Diseases and Molecular Medicine, University of Cape Town, S1.02 Wernher and Beit Bldg., Anzio Rd., Observatory 7925, Cape Town, South Africa (cheryl.day@uct.ac.za). The Journal of Infectious Diseases 2008; 197: by the Infectious Diseases Society of America. All rights reserved /2008/ $15.00 DOI: / develop active disease after initial MTB infection; this observation provides compelling evidence that the host immune response is usually successful in containing the infection [1, 2]. Coinfection with HIV-1, which impairs cellular immune responses, greatly increases the risk of development of active tuberculosis (TB) [3, 4], and the HIV-1 epidemic has contributed significantly to the rapid spread of TB, as well as to subsequent increases in the number of cases of active disease in such areas as sub-saharan Africa [5]. Previous studies in interferon (IFN) deficient mice and in humans with IFN- receptor abnormalities have indicated an essential role of IFN- producing T cells in the containment of MTB infection [6 9]. IFN- secreting T cells specific for multiple immunogenic antigens of MTB have been identified, including the Ag85 complex, culture filtrate protein 10 (CFP-10), 990 JID 2008:197 (1 April) Day et al.

2 early secretory antigenic target 6 (ESAT-6), heat shock protein 65, and TB10.4 [10 16]. Recently, tests have been developed for immunodiagnosis of MTB infection on the basis of the presence of IFN- secreting cells in response to the following antigens in the region of difference 1 (RD1): ESAT-6 and CFP-10; these tests have been shown to be more specific and sensitive than tuberculin skin tests [17]. The RD1 antigens have been deleted from all Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccines [18] and are, therefore, useful in differentiating individuals who have been exposed to MTB within a BCG-vaccinated population. Although the majority of studies of the performance of IFN- release assays for the detection of MTB infection have been performed in HIV-1 negative cohorts, the performance of these assays has not been found to be significantly affected by HIV-1 infection in studies involving HIV-1 positive subjects [19 24]. Despite the high prevalence of coinfection with HIV-1 and MTB, very little information is currently known about the functional capacity of MTB-specific cellular immune responses in HIV-1 positive individuals. A high sensitivity for the detection of MTB infection has been noted with the use of an RD1-based IFN- enzyme-linked immunospot (ELISPOT) assay in the screening of HIV-1 positive patients with TB, including both adults and children [19, 21, 25]. However, the degree to which HIV-1 coinfection influences the magnitude of responding MTB-specific T cells in patients with TB varies in different study cohorts [19, 26, 27]. Moreover, detailed analyses of the effector functions of MTB-specific T cell responses in HIV-1 positive individuals with latent and active TB, as well as the extent to which HIV-1 disease progression influences the functional capacity of these responses, have not been well described. In the present study, we characterized the MTB-specific T cell response in HIV-1 positive individuals with latent TB from KwaZulu Natal, South Africa, where 80% of active TB cases are associated with HIV-1 infection [28]. Our aims were to determine the magnitude and breadth of responses to mycobacterial antigens, to determine the relationship between MTB-specific T cell responses and parameters of HIV-1 disease progression, and to assess the ex vivo phenotype of MTB-specific T cells and their capacity to secrete multiple cytokines in individuals with chronic HIV-1 infection. MATERIALS AND METHODS Subjects. One hundred thirty-seven subjects were recruited from the Sinikithemba Care Center at McCord Hospital in Durban, South Africa. All subjects were HIV-1 positive and antiretroviral therapy naive, and they had no previous history of TB symptoms, diagnosis, or treatment, as well as no current symptoms of active TB. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by means of density centrifugation with ficoll-hypaque (Sigma). The HIV-1 load was measured using the Roche Amplicor assay (version 1.5). The median viral load of the cohort was 20,500 HIV-1 RNA copies/ml of plasma, and the median absolute CD4 cell count was 359 cells/ L of whole blood. Five HIV-1 negative subjects from the United States who were not vaccinated with BCG and who had no previous history of TB were included as negative controls. All study participants provided written, informed consent for the study, which was approved by institutional review boards. Synthetic peptides. Peptides (16- to 18-mers overlapping by 10 aa) corresponding to the sequence of the MTB antigens Ag85A, CFP-10, ESAT-6, and TB10.4 were synthesized on an automated peptide synthesizer. For initial screening purposes, peptides were arranged into pools of 9 peptides each in a matrix fashion, such that each peptide was uniquely represented in 2 pools [29]. IFN- ELISPOT assays. Ninety-six well polyvinylidene plates (Millipore) were precoated with 0.5 g/ml anti human IFN- monoclonal antibody (1-D1K; Mabtech). A total of 100,000 freshly isolated PBMCs were added per well in a volume of 100 L of R10 medium (RPMI 1640 supplemented with 10% fetal calf serum, 10 mmol/l HEPES buffer, 2 mmol/l L-glutamine, and 50 U/mL penicillin-streptomycin). Peptides were added at a final concentration of 2 g/ml. Negative controls consisted of wells containing PBMCs incubated with medium alone. Positive controls consisted of wells containing PBMCs and 12.5 g/ml phytohemagglutinin (PHA; Murex Biotech). Plates were incubated overnight at 37 C in a 5% CO 2 incubator and were washed the next morning; 0.5 g/ml biotinylated anti IFN- antibody (7-B6-1; Mabtech) was then added for 1.5 h, and the plates were washed and incubated with streptavidin-alkaline phosphatase (Mabtech) for 45 min. The plates were developed by the addition of the alkaline phosphatase color reagents 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium chloride in Tris buffer (ph 9.5). The reaction was stopped by washing with tap water after the appearance of intense blue spots in the positive control wells. Spots were counted on an ELISPOT reader (AID), and responses were considered to be positive if the number of spots per well minus the background value was 50 spot-forming cells (SFCs)/10 6 PBMCs (i.e., if the value was greater than the mean value plus 3 times greater than the standard deviation of the responses in the negative control subjects). Intracellular cytokine staining and immunophenotyping. A total of PBMCs were incubated with 1 g of peptide in 1 ml of R10 medium overnight (12 14 h) at 37 C in a 5% CO 2 incubator. After the first hour of incubation, 10 g of brefeldin A (Sigma) was added. The cells were washed and stained with the following surface antibodies at room temperature for 15 min: peridinin-chlorophyll-protein complex (PerCP) conjugated anti-cd4, allophycocyanin (APC) conjugated anti-cd8, fluorescein isothiocyanate (FITC) conjugated anti-cd27, APCconjugated anti-cd28, APC-conjugated anti-cd45ra, FITC- MTB-Specific T Cells in HIV-1 Infection JID 2008:197 (1 April) 991

3 conjugated anti-ccr7 (R&D Systems), and APC-conjugated CD62L. The cells were washed, fixed, and permeabilized (Caltag reagents A and B; Caltag Laboratories). The following intracellular antibodies were then added: FITC-conjugated anti-ifn-, phycoerythrin (PE) conjugated TNF-, PE-conjugated IL-2, and APC-conjugated Granzyme B (GrB; Caltag Laboratories). All antibodies were from Becton Dickinson, unless otherwise noted. The cells were incubated for 15 min and then were washed and acquired on a FACSCalibur flow cytometer with the use of CellQuest software (Becton Dickinson). Negative controls consisting of PBMCs incubated with medium alone were included in each assay. Responses 0.03% above the background value were considered to be positive. For phenotypic analysis of CD4 cells, peptide-stimulated cells were gated on CD4 TNF- cells and analyzed for expression of CD27, CD28, CD45RA, CCR7, and CD62L. For phenotypic analysis of CD8 cells, peptidestimulated cells were gated on CD8 TNF- cells and analyzed for expression of CD27, CD28, CD45RA, CCR7, and GrB. Statistical analysis. The Mann-Whitney U test and Spearman rank correlation were performed using GraphPad Prism software (version 4.0; GraphPad Prism). P.05 was considered to be statistically significant. RESULTS Identification of MTB-specific T cell responses in HIV-1 positive individuals with latent MTB infection. To characterize the MTB-specific cellular immune response in HIV-1 positive individuals in an area of endemicity for TB who are at high risk for reactivation and development of active disease, we screened a large cohort of individuals with chronic HIV-1 infection for the presence of IFN- producing T cells. Screening was performed using an ELISPOT assay for the detection of MTB RD1 antigens CFP-10 and ESAT-6, as well as the immunogenic secreted antigens Ag85A [11] and TB10.4 [30, 31]. Fifty-six (41%) of 137 subjects had positive responses to ESAT-6 and/or CFP-10, according to the ELISPOT assay (data not shown), and they were thus identified as subjects with latent MTB infection. No significant differences in either absolute CD4 cell count or viral load were observed between individuals with detectable and undetectable MTB-specific responses in the IFN- ELISPOT assay (data not shown). Forty-one of the 56 subjects with positive responses in the initial screening performed with the peptide pools had sufficient numbers of cells available with which to perform a second ELISPOT assay the next day to confirm the individual peptides eliciting the positive response. All of the subjects responded to at least one peptide in either CFP-10 and/or ESAT-6, thus confirming all initial positive responses at an individual peptide level; 44% of subjects responded to peptides from both CFP-10 and ESAT-6, whereas 34% responded to ESAT-6 only and 22% responded to CFP-10 only (figure 1A). All subjects responding to Figure 1. Magnitude and breadth of Mycobacterium tuberculosis (MTB) specific T cell responses in HIV-positive individuals with latent infection. 18-mer overlapping peptides spanning the Ag85A, culture filtrate protein 10 (CFP-10), early secretory antigen target 6 (ESAT-6), and TB10.4 proteins were arranged in pools of 9 peptides each in a matrix fashion such that each peptide was represented in 2 pools. Freshly isolated peripheral blood mononuclear cells (PBMCs) were screened for positive responses to the peptide pools in an interferon (IFN)- enzymelinked immunospot (ELISPOT) assay; responses to individual peptides were reconfirmed in a second ELISPOT assay. Positive responses to individual 18-mer peptides from the confirmation assay are shown; for 41 individuals, a second confirmatory ELISPOT assay with individual peptides was performed. A, Frequency (percentage) of subjects with a positive IFN- ELISPOT assay response who demonstrated a response to each of the 4 antigens tested. Individuals were considered to be responders if they targeted at least one peptide within the antigen indicated. B, Magnitude of T cell responses to individual peptides (Ag85A, CFP-10, ESAT-6, and TB10.4 peptides) in an IFN- ELISPOT assay. Horizontal lines denote the median number of spot-forming cells (SFCs) per million PBMCs. C, No. of peptides targeted per individual within each antigen and across all antigens tested. Horizontal lines denote median values. 992 JID 2008:197 (1 April) Day et al.

4 peptides in Ag85A or TB10.4 also responded to at least one other peptide in ESAT-6 and/or CFP-10 (data not shown). The magnitudes of the positive responses to individual peptides within the 4 antigens tested ranged from 50 to 2000 SFCs/ million PBMCs. The median magnitude was highest for TB10.4- specific cells, although this finding was not significantly different from responses directed against CFP-10 (median magnitude, 219 SFCs/million PBMCs) or ESAT-6 (median magnitude, 171 SFCs/million PBMCs) (figure 1B). The number of epitopes targeted (per individual) within each antigen tested ranged from 1 to 6 (figure 1C). Overall, summing up the responses to epitopes in all 4 antigens tested, the breadth of responses ranged from 1 to 16 epitopes simultaneously targeted in a single subject, with a median of 2 epitopes targeted (figure 1C). Identification of immunodominant epitopes in CFP-10 and ESAT-6. We next analyzed the frequency of recognition of individual overlapping peptides in CFP-10 and ESAT-6, to further map immunodominant epitopes targeted in this population. Ten of 12 overlapping peptides for CFP-10 were recognized by 1 subject, with 2 dominant epitopes (CFP and CFP ) each targeted by 37% of subjects (figure 2A). Eleven of 12 ESAT-6 peptides were recognized by 1 subject. The most frequently targeted peptides (ESAT and ESAT ) were each recognized by 27% of subjects. Peptides overlapping with the sequences of the 4 most dominant peptides identified in the present study have previously been reported to be frequently recognized by HIV-1 negative subjects in Indian, European, Cambodian, and Middle Eastern cohorts with both latent and active TB [14, 21, 32 35]. Furthermore, ESAT and CFP have been described to be immunodominant in HIV-1 positive patients with TB and asymptomatic adults in Zambia [19]. Taken together, these data indicate that the pattern of epitope recognition in these antigens may be similar among ethnically diverse populations of HIV-1 positive and HIV-1 negative individuals. Analysis of cytokine production by MTB-specific CD4 and CD8 T cells at the single-cell level. The aforementioned experiments using the IFN- ELISPOT assay were designed to identify HIV-1 positive subjects with evidence of latent TB. Because IFN- is but one of the cytokines produced by antigenspecific T cells, we next determined the ability of MTB-specific T cells in these subjects to secrete additional cytokines by use of an intracellular cytokine staining assay to detect IFN-, TNF-, and IL-2 after short-term stimulation with individual ESAT-6 or CFP-10 peptides. Representative data are shown in figure 3A, and composite data (n 40) are shown in figure 3B. There was no statistical difference in the median frequency of IFN-, TNF-, and IL-2 secreting CD4 cells (0.065%, 0.095%, and 0.075% of CD4 cells, respectively). The majority of MTB-specific T cell responses identified in this cohort were CD4 mediated; however, we were able to identify subjects with detectable MTB-specific CD8 T cell responses. Figure 2. Frequency of recognition of individual culture filtrate protein 10 (CFP-10) and early secretory antigen target 6 (ESAT-6) peptides. The percentage of individuals responding to each individual overlapping 18-mer peptide spanning the length of the secreted antigens culture filtrate protein 10 (CFP-10) (A) or ESAT-6 (B) in an ex vivo interferon- enzyme-linked immunospot (ELISPOT) assay is shown. Subjects were screened for responses to overlapping 18-mer CFP-10 and ESAT-6 peptides, as described in the figure 1 legend. Forty-one subjects had positive ELISPOT assay responses to either CFP-10 and/or ESAT-6. Representative data are shown in figure 3C, with composite data of CD8 responses in 14 subjects shown in figure 3D. The median frequency of MTB-specific cells secreting IFN- and TNF- to an individual ESAT-6 or CFP-10 peptide was 0.060% and 0.055%, respectively, of CD8 cells. The frequency of IFN- and TNF- secreting CD8 cells was significantly higher than the median frequency of MTB-specific IL-2 secreting CD8 cells (P.0001 and P.0009, respectively) (figure 3D). Assessment of production of multiple cytokines by antigenspecific T cells is thought to be an indication of functionality, MTB-Specific T Cells in HIV-1 Infection JID 2008:197 (1 April) 993

5 Figure 3. Cytokine production by Mycobacterium tuberculosis (MTB) specific CD4 and CD8 T cells. A, Representative intracellular cytokine staining (ICS) data for a subject whose cells were stimulated with peptide culture filtrate protein 10 (CFP-10; in this case, CFP ) and stained intracellulary for interferon (IFN), tumor necrosis factor (TNF), and interleukin (IL) 2. The percentages in the upper right quadrant denote the percentage of CD4 cytokine-positive cells, with background cytokine production values subtracted. Top, no antigen stimulation; Bottom, peripheral blood mononuclear cells (PBMCs) stimulated for 12 h with CFP peptide. B, Composite data on IFN-, TNF-, and IL-2 production by CD4 T cells in response to individual TB peptides, as tested by ICS (n 40). For the ICS assay, each subject was tested with individual early secretory antigen testing 6 and CFP-10 peptides that elicited a positive response from that subject in the initial IFN- enzyme-linked immunospot (ELISPOT) screening assay. Horizontal bars denote the median percentage of CD4 cytokine-positive cells. C, Representative ICS data for a subject whose cells were stimulated with peptide CFP and stained intracellularly with IFN-, TNF-, and IL-2. The percentage in the upper right quadrant denotes the percentage of CD8 cytokine-positive cells, with background cytokine production values subtracted. Top, no antigen stimulation; Bottom, PBMCs stimulated for 12 h with CFP peptide. D, Summary data of IFN- (n 14), TNF- (n 12), and IL-2 (n 11) production by CD8 T cells in response to individual MTB peptides tested by ICS. Horizontal lines denote median percentages of CD8 cytokine-positive cells. with loss of cytokine production indicative of impairment of function [36]. To address whether there was evidence of impaired function of MTB-specific T cells in HIV-1 positive individuals, we assessed the frequency of MTB-specific CD4 and CD8 T cells that were able to secrete multiple cytokines simultaneously. Because only a 4-color flow cytometer was available on site in Durban, we performed costaining for IFN- /IL-2 and IFN- /TNF- separately. Representative data shown in figure 4A indicated that the majority ( 75%) of MTB-specific CD4 T cells are polyfunctional: IFN- /IL-2 and IFN- /TNF-. The proportion of INF- /TNF- cells was significantly greater than the proportion of IFN- /IL-2 cells (P.0220). Although there were virtually no IFN- /TNF- cells detectable, a small but significant proportion of MTB-specific CD4 cells that were IFN- /IL-2 was detected (P.0001). We similarly addressed the polyfunctional cytokine-secreting capacity of MTB-specific CD8 cells by costaining with IFN- / IL-2 and IFN- /TNF- (figure 4C), and we found a dominance of IFN- /IL-2 cells and IFN- /TNF- cells (figure 4D). The proportion of TB-specific IFN- /IL-2 CD8 cells was significantly lower than the proportion of IFN- /TNF- cells (P.0001) (figure 4D). Relationship between functionality of MTB-specific T cell responses and parameters of HIV-1 disease progression. To determine the impact of HIV-1 coinfection on the functionality of MTB-specific T cell responses, we next analyzed the relationship between the breadth and magnitude of MTB-specific T cell responses, as determined by the IFN- ELISPOT assay, in relation to HIV-1 disease progression, including absolute CD4 cell count and HIV-1 load, and we found no correlations (data not shown). However, analysis of the cytokine costaining data indicated an inverse correlation between the proportion of TBspecific IL-2 secreting (IFN- /IL-2 and IFN- /IL-2 ) CD4 cells and the viral load, and a corresponding positive correlation 994 JID 2008:197 (1 April) Day et al.

6 Figure 4. Polyfunctional cytokine analysis of Mycobacterium tuberculosis (MTB) specific CD4 and CD8 T cells. A, Representative intracellular cytokine staining (ICS) data from peripheral blood mononuclear cells (PBMCs) stimulated with culture filtrate protein 10 (CFP-10; in this case, CFP ) peptide and costained with interferon (IFN) /interleukin (IL) 2 and IFN- /tumor necrosis factor (TNF). Cells were gated on CD4 events, and the percentages denote the percentage of CD4 cytokine-positive cells in each quadrant. The background cytokine production value has been subtracted from the percentages shown in the lower 2 plots. B, Analysis of the proportion of MTB-specific CD4 cells that are IFN- /IL-2, IFN- /IL-2, IFN- /IL-2, IFN- /TNF-, IFN- /TNF-, and IFN- /TNF-. PBMCs were stimulated with peptide overnight and were costained with either IFN- and IL-2 or IFN- and TNF-, as indicated in panel A. The median value and interquartile range for each population are shown (40 subjects had IFN- /IL-2 costaining done, and 26 had IFN- /TNF- costaining done). Statistical comparisons were made using the Mann-Whitney test. C, Representative ICS data for MTB-specific CD8 cells stimulated with CFP peptide and costained with IFN- /IL-2 and IFN- /TNF-. Cells were gated on CD8 events, and the percentage of CD8 cytokine cells is shown in the quadrants; the background cytokine production value has been subtracted in the lower 2 plots. D, The proportion of MTB-specific CD8 cells that are IFN- /IL-2, IFN- /IL-2, IFN- /IL-2, IFN- /TNF-, IFN- /TNF-, and IFN- /TNF-. The median value and interquartile range are shown for each population (10 subjects had IFN- /IL-2 costaining done, and 9 had IFN- /TNF- costaining done). Statistical comparisons were made using the Mann-Whitney test. E, Inverse correlation between the proportion of MTB-specific IL-2 secreting CD4 cells and HIV-1 load (P.0098, by Spearman correlation; n 40). Results are presented as the percentage of IL-2 positive cells (IFN- /IL-2 and IFN- /IL-2 ) contributing to the total MTB-specific response to individual CFP-10 or early secretory antigen target 6 peptides.

7 Figure 5. Memory phenotypic analysis of Mycobacterium tuberculosis (MTB) specific CD4 and CD8 T cells. A, Representative MTB-specific CD4 memory phenotyping data for a subject with cells stimulated with peptide culture filtrate protein 10 (CFP-10; in this case, CFP ). Peripheral blood mononuclear cells (PBMCs) were gated on CD4 TNF- cells and analyzed for expression of memory markers CD27, CD28, CD45RA, CCR7, and CD62L, as indicated. The percentages in the flow plots denote the percentage of CD4 TNF- cells that express the markers indicated. B, Summary data of the memory phenotype of MTB-specific CD4 T cells. PBMCs were stimulated with individual peptides and gated on CD4 TNF- cells, as shown in panel A (n 14 subjects). Horizontal lines denote median expression of the indicated marker on MTB-specific CD4 T cells. C, Representative MTB-specific CD8 memory phenotyping data for a subject with cells stimulated with peptide CFP PBMCs were gated on CD8 TNF- cells and were analyzed for expression of the memory and effector markers CD27, CD28, CD45RA, CCR7, and Granzyme B (GrB; Caltag Laboratories), as indicated. The percentages in the quadrants denote the percentage of CD8 TNF- cells that express the markers indicated. D, Summary of phenotyping data of MTB-specific CD8 T cells (n 6 subjects). PBMCs were stimulated with individual MTB peptides and gated on CD8 TNF- cells, as indicated in panel C. Horizontal lines denote median expression of the indicated marker on MTB-specific CD8 T cells. between IFN- /IL-2 CD4 cells and the viral load (P.0098) (figure 4E). These data indicate that IL-2 secreting MTBspecific CD4 T cells are diminished or have impaired function under conditions of high levels of HIV-1 viremia. Phenotypic analysis of MTB-specific CD4 and CD8 T cells. Antigen-specific T cells can be classified into different populations of memory and effector cells on the basis of the expression of phenotypic markers associated with maturation status. To determine the phenotypic profile of MTB-specific CD4 T cells associated with latent TB, we used the intracellular cytokine staining assay to stimulate PBMCs with individual peptides, gating on CD4 TNF- cells and analyzing expression of the memory markers CD27, CD28, CCR7, CD45RA, and CD62L (figure 5A). MTB-specific CD4 T cells were found to be CCR7 lo CD45RA lo CD62L lo, indicating an effector memory phenotype (n 14) (figure 5B). The high level of expression of CD28 and the intermediate level of CD27 expression denote an early to intermediate differentiated phenotype. Phenotypic analysis of MTB-specific CD8 T cells was performed by stimulating PBMCs with individual peptides and gating on CD8 TNF- cells for analysis of CD27, CD28, CCR7, CD45RA, and GrB expression (figure 5C). MTB-specific CD8 T cells were found to be CCR7 lo CD45RA lo GrB hi, indicating an effector phenotype (n 6) (figure 5D). Similar to MTB-specific CD4 cells, MTB-specific CD8 cells demonstrated high levels of expression of CD28 and intermediate levels of expression of CD27, suggesting an early to intermediate differentiated phenotype. DISCUSSION This is the first detailed study to characterize MTB-specific CD4 and CD8 T cell responses at the single-cell level in HIV-1 positive adults with latent TB. We used an IFN- ELISPOT-based assay using overlapping peptides spanning the RD1 antigens ESAT-6 and CFP-10 to identify HIV-1 positive subjects with 996 JID 2008:197 (1 April) Day et al.

8 latent TB in KwaZulu Natal Province, South Africa, where extensively drug-resistant TB recently has been detected [28]. Our rate of detection of latent TB by use of this RD1-based IFN- ELISPOT assay in this cohort was 41%, a rate similar to that reported in a previous study of HIV-1 positive asymptomatic adults in Zambia, in which 9 (43%) of 21 subjects responded to RD1 peptides in an IFN- ELISPOT assay [19]. The frequency of latent TB in our study population in South Africa is likely to be even higher than that identified here, because some individuals, particularly those with extremely low CD4 cell counts ( 100 cells/ L), may have responses below the level of detection of the ELISPOT assay [20, 24], or they may respond to antigens other than CFP-10 or ESAT-6. Tests for the identification of individuals with latent TB may be useful in predicting who will develop active disease [37], although longitudinal studies are still required to determine the prognostic value of positive responses to CFP-10/ESAT-6 in the development active disease. Analysis of production of multiple cytokines by antigenspecific T cells is an important measurement of their functionality, and such production is thought to be associated with antigen load [38, 39]. Because of the limitations associated with the use of a 4-color flow cytometer in KwaZulu Natal, we costained cells with either IFN- and IL-2 or IFN- and TNF-, and, because a median of 75% of MTB-specific CD4 cells were IFN- /TNF- and IFN- /IL-2, we can infer that the majority of MTB-specific CD4 cells are polyfunctional and secrete all 3 cytokines simultaneously. However, we found a significantly higher proportion of MTB-specific CD4 cells that were IFN- / TNF-, compared with IFN- /IL-2, suggesting that IL-2 secretion capacity may be lost first (before the loss of TNF- or IFN- ). This finding is consistent with murine models of lymphocyte choriomeningitis virus that indicate a hierarchal loss of function of antigen-specific T cells under conditions of persistent antigen stimulation, with loss of IL-2 production preceded by loss of TNF- synthesis and then, finally, by loss of IFN- production [40]. Previous studies involving HIV-1 positive individuals have also indicated loss of IL-2 function by HIV-1 specific CD4 T cells in subjects with progressive disease [41]. The inverse correlation found between the proportion of MTB-specific IL-2 secreting CD4 cells and viral load suggests that MTB-specific CD4 T cells in HIV-1 positive subjects may gradually lose IL-2 secretion capacity as HIV-1 disease progresses. This finding has important implications for MTB pathogenesis, because these responses may already be impaired by the time of reactivation, thus potentially contributing to the rapid course of TB disease often seen in HIV-1 positive individuals. The time of MTB infection is not known in this cohort, and longitudinal studies would need to be performed to determine how the duration of MTB and HIV-1 coinfection affects the IL-2 secretion capacity of MTB-specific CD4 cells. It is important to note that there was no correlation between the total frequency of MTB-specific IFN-, IL-2, or TNF- CD4 cells and viral load or CD4 count, thus underscoring the need to analyze multiple cytokine functions simultaneously to determine the impact of HIV-1 infection on the functional capacity of MTB-specific T cells. A previous study indicated reduced frequencies of purified protein derivative specific IFN- /IL-2 CD4 T cells in BCGvaccinated HIV-positive subjects, compared with BCGvaccinated HIV-negative subjects [42]. In the present study, we were unable to recruit HIV-1 negative individuals with latent TB to directly compare the functional profile of MTB-specific T cell responses in HIV-1 positive and negative individuals and to determine the relative contributions of these 2 pathogens in the impairment of MTB-specific T cell function. However, a recent study assessing the capacity for IFN- and IL-2 secretion by MTB-specific T cells in HIV-1 negative subjects with active TB indicated codominance of IFN- /IL-2 and IFN- /IL-2 cells, followed by a shift to dominance of IFN- /IL-2 cells after treatment [39]. Taken together, these data imply that establishment of active disease may further impair MTB-specific T cell function, as evidenced by lack of IL-2 secretion capacity. Phenotypic analysis of antigen-specific T cells provides important information regarding effector function and maturation status, and it has been well described in human viral infections [43 47] but less so in such bacterial infections as MTB infection. We found the phenotype of MTB-specific CD4 and CD8 cells to be intermediate differentiated effector memory cells, consistent with findings of previous studies of chronically infected mice [48, 49]. Our phenotypic analysis of MTB-specific T cells provides new insights into the development of MTB-specific memory cells in humans. MTB-specific CD8 T cells displayed an unusual phenotype for antigen-specific CD8 T cells in that they exhibited high CD28 expression and intermediate CD27 expression, while they also maintained high GrB expression, which may represent a dysregulated phenotype associated with MTB infection. Antigen-specific CD8 T cells in viral infections are thought to start off as CD27 /CD28 (early differentiated phenotype) and then lose CD28 expression (CD27 /CD28 ; intermediate differentiated phenotype) before losing CD27 expression (CD27 /CD28 ; late differentiated phenotype) [44]. However, we found MTB-specific CD8 T cells to be high in CD28 expression and lower in CD27 expression, suggesting that they lose CD27 expression first (before they lose CD28 expression), consistent with the model that has been proposed for antigen-specific CD4 cell differentiation rather than antigenspecific CD8 T cells. There is no accurate test for antigen levels associated with MTB infection in humans, and it is not known whether there is some degree of bacterial replication ongoing in these HIV-1 positive subjects (despite the absence of symptoms of active TB) that may drive these cells to maintain an effector memory phenotype. Furthermore, we did not have access to chest radiographs of these subjects, and it is possible that a pro- MTB-Specific T Cells in HIV-1 Infection JID 2008:197 (1 April) 997

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