Induction of granulysin in CD8 T cells by IL-21 and IL-15 is suppressed by human immunodeficiency virus-1

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1 Article Induction of granulysin in T cells by and is suppressed by human immunodeficiency virus-1 A. E. Hogg, G. C. Bowick,, N. K. Herzog, M. W. Cloyd,, and J. J. Endsley,,1 Departments of Microbiology and Immunology and Pathology, Center for Biodefense and Emerging Infectious Diseases, and Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas, USA RECEIVED APRIL 1, 29; REVISED JULY 4, 29; ACCEPTED JULY 13, 29. DOI: /jlb ABSTRACT Immunosuppression following infection with HIV-1 predisposes patients to a myriad of opportunistic pathogens, one of the most important of which is Mtb., expressed by NK cells and CTL, exhibits potent antimicrobial activity against Mtb and several other opportunistic pathogens associated with HIV-1 infection. The immune signals that promote granulysin expression in human CTL are not fully understood. Using primary human T cells, in this study, we identify as a strong inducer of granulysin, demonstrate that and activate granulysin expression within CD45RO T cells, and establish a role for Jak/STAT signaling in the regulation of granulysin within T cells. We show that infection of PBMC from healthy donors in vitro with HIV-1 suppresses granulysin expression by T cells, concomitant with reduced p-stat3 and p-stat5, following activation with and. Of note, simultaneous signaling through and could partially overcome the immunosuppressive effects of HIV-1 on granulysin expression by T cells. These results suggest that HIV-1 infection of PBMC may reduce the antimicrobial profile of activated T cells by disrupting signaling events that are critical for the induction of granulysin. Understanding the effects of HIV-1 on T cell activation is essential to understanding the physiological basis for inadequate cytotoxic lymphocyte activity in HIV patients and for informed guidance of cytokine-based therapy to restore T cell function. J. Leukoc. Biol. 86: ; 29. Introduction Chronic HIV-1 infection leads to a dysregulation of cell-mediated immunity through multifaceted effects on the immune system and increases host susceptibility to opportunistic infections [1, 2]. The loss and functional suppression of CD4 T Abbreviations: c -chain, C T comparative C T, APC allophycocyanin, C T comparative threshold, h human, MOI multiplicity of infection, Mtb Mycobacterium tuberculosis, p-stat phosporylated-stat, sirna small interfering RNA, t-stat total STAT cells following HIV-1 infection is well characterized [3], as is the suppression of the cytotoxic T cell response to viral antigens [4, 5]. T cells from HIV patients show impaired signaling through the TCR [6] and defective perforindependent CTL activity [7, 8]. The effects of HIV-1 infection on T cell expression of granulysin, however, have not been described yet. is a potent antimicrobial protein contained within the granules of CTL and NK cells [9 11]. Microbial targets of granulysin include Mtb, Plasmodium falciparum, Cryptococcus neoformans, Staphylococcus aureus, and many other important pathogens of immunocompetent and/or immunodeficient individuals [12 17]. Given this broad-spectrum antimicrobial activity, a greater understanding of how granulysin is regulated and affected by HIV-1 infection is important for efforts to promote protective immunity. The studies of granulysin regulation are limited compared with those of perforin and granzymes, due to the lack of a murine gene homologue. In primary human CTL, granulysin induction occurs following prolonged activation (5 7 days) with mitogen, specific antigen, CD4 ligation, or cytokine activation with IL-2 or [1, 14, 18 2]. IL-2 and IL-5 are members of the common c receptor family that includes IL-4, IL-7, IL-9, and. The members of this cytokine family that have been shown to promote cytotoxic activity of CTL are IL-2, IL- 15, and [21, 22]. IL-2 and have been demonstrated previously to regulate the granulysin-dependent killing of the fungus C. neoformans by human peripheral blood CD4 and T cells [14, 23]. Despite similarities in function with IL-2 and, the role of in the regulation of granulysin has not been described to date. is the most recently described member of the c cytokine family and is produced primarily by activated CD4 T cells as an autocrine growth factor [24 26]. Many of the reported effects of on T cells occur through synergy with other c cytokines. Together, exogenous and IL-7 or enhance proliferation and CTL activity of human and murine T cells toward tumor cells and virus-infected targets [27 29]. Recently, 1. Correspondence: University of Texas Medical Branch, 31 University Blvd., Galveston, TX , USA /9/ Society for Leukocyte Biology Volume 86, November 29 Journal of Leukocyte Biology 1191

2 was shown to induce perforin in T cells from HIV and not HIV patients [3]. Thus, is being incorporated currently in strategies to enhance immunogenicity of the vaccines against HIV and tuberculosis [31, 32] and as a therapeutic agent against melanoma tumors [33]. The signaling pathways that induce granulysin expression in T cells, via cytokines or other activation stimuli, are not described. The c cytokines share use of the MAPK, Jak/STAT, or the PI3K/AKT pathways for signal transduction and have individual and overlapping roles critical for the maintenance and function of T cell pools (for reviews, see refs. [34, 35]). In the one published report of granulysin signaling, induction in CD4 T cells by IL-2 is dependent on activation of the PI3K and Jak3 signaling pathways [36]. In peripheral blood, cytolytic CD4 T cells represent a minor population, and the predominant T cell source of granulysin and killing activity is T cells. Understanding the conserved and divergent pathways for regulation of granulysin in CD4 and T cells is critical to promoting an antimicrobial cell-mediated immune response. Disruption of the c cytokine network is an important mechanism that leads to reduced T cell function following HIV-1 infection. In HIV patients, CD4 T cells become defective in IL-2 secretion, even before loss of CD4 T cells is observed [37], and levels of are reduced subsequently [38]. Suppression of the high-affinity IL-2R -chain [39] and the common -chain (CD122) shared by IL-2R and R [4] is also evident in T lymphocytes from HIV patients. Recently, levels were also shown to be suppressed significantly in the serum of HIV patients and proposed as a biomarker of progression to AIDS [41]. The impact of reduced levels on cell-mediated immune function in HIV-1-infected individuals is not clear. Therapeutic intervention with exogenous c cytokines may augment T cell function following antiviral therapies. IL-2 has been used successfully to reconstitute CD4 T cell numbers following HAART therapy but has many sideeffects. Studies in nonhuman primates infected with SIV indicate that therapy is not beneficial and may even promote viral replication [42]. As an adjuvant for a SIV vaccine candidate, however, improves development of protective immunity [43, 44], and a combination of and shows promise to improve the efficacy of a HIV-1 vaccine construct [31]. Recent work indicates, as compared with, may induce perforin in T cells of HIV patients in the absence of a proliferative response that would promote viral replication [3]. Given the CTL-promoting properties of, we hypothesized that would strongly induce granulysin expression by cytotoxic CD4 and T cells. Further, we hypothesized that induction of granulysin would be suppressed by HIV-1 infection of the PBMC population via interference with c cytokine signaling. We show that induces granulysin expression in human peripheral blood T cells but not CD4 T cells. induction occurred in CD45RO T cells and via Jak/STAT-dependent signaling pathways following activation with or. Infection of PBMC in vitro with 4 HIV suppressed induction of granulysin in T cells by or and corresponded with decreased phosphorylation of STAT3 and STAT5 proteins. Identifying the mechanisms whereby HIV-1 dysregulates granulysin expression in T cells is critical to understanding suppression of cytotoxic lymphocyte activity and for implementation of prophylactic/therapeutic interventions against opportunistic pathogens. MATERIALS AND METHODS mab The following mab against human antigens were purchased from BD Biosciences (San Diego, CA, USA): granulysin-alexa Fluor 488, perforin-fitc, Ki67-FITC, perforin-pe, p-stat3 (Y72)-PE, CD45RO-PE, CD4-PerCPCy5.5, -PerCPCy5.5, IFN- -APC, CD45RO-APC, CD4-APC, -APCCy7, and p-stat5 (Y694)-Alexa Fluor 647. The following mab were purchased from ebioscience (San Diego, CA, USA): granulysin-pe and CD45RO-APC. The anti-human R-PE mab was purchased from R&D Systems (Minneapolis, MN, USA). Corresponding isotype controls purchased from BD Biosciences were: IgG2b-PE, IgG2b-FITC, IgG2a-APC, IgG1-PE, IgG1-APCCy7, IgG1-PerCPCy5.5, IgG1-Alexa Fluor 647, and IgG1-FITC. In addition, detection of HIV was performed using the KC57-FITC mab purchased from Beckman Coulter (Fullerton, CA, USA), which recognizes HIV-1 core proteins (p24). For Western blot analysis, the following antibodies were used: mouse anti-human -actin mab (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-granulysin (MBL International, Woburn, MA, USA), rabbit antihuman tstat1, tstat3, tstat5, and p-stat1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-stat3 and rabbit anti-human p-stat5 (Cell Signaling Technology, Beverly, MA, USA), and goat anti-rabbit IgG-HP and goat anti-mouse IgG-HP (Southern Biotechnology, Birmingham, AL, USA). Cell preparation and activation Peripheral blood was obtained from healthy human donors as approved by the University of Texas Medical Branch Institutional Review Board (Galveston, TX, USA). PBMC were isolated from heparinized peripheral blood using Accuprep (Accurate Chemicals, Westbury, NY, USA) and density centrifugation. T cells were purified from PBMC by negative selection using the AutoMACS system (Miltenyi Biotec, Auburn, CA, USA) following the manufacturer s instructions. Sample purity was assessed by FACS with anti-, anti-cd4, and anti- and was found to be 95% routinely. PBMC or purified T cells were cultured in complete culture media (RPMI 164; Invitrogen, Carlsbad, CA, USA), supplemented with 1% FBS, 2 mm L-glutamine, 1 mm sodium pyruvate,.1 mm nonessential amino acids, and penicillin-streptomycin (Invitrogen). For cytokine stimulations, PBMC or purified T cells were cultured in media alone (control) or with rhil-2 (5 ng/ml, R&D Systems), rh (15 ng/ml, R&D Systems), rh (15 ng/ml, Invitrogen) with plus (both 15 ng/ml), or microbead-conjugated antibodies to and CD28 (Dynabead /CD28 T cell expander reagents, Invitrogen) for 1, 3, and 5 days at 37 C and 5% CO 2. Cell viability was determined by trypan blue exclusion and was not altered significantly by any of the treatments. HIV-1 culture and infection CXCR4 virus HIV-1 213, which was isolated previously from PBMC of a HIVinfected individual and biologically cloned, was propagated in CEM cells [45]. Virus was collected following acute infection when cells were nearly 1% HIV-1 antigen-positive and concentrated by ultracentrifugation and resuspended in RPMI-164 cell culture medium (Gibco, Grand Island, NY, USA), supplemented with 1% human AB serum, penicillin-streptomycin, Hepes buffer, and L-glutamine, as described previously [45]. All HIV-1 frozen stock preparations were tested and found free of bacterial endotoxin. PBMC was exposed to HIV-1 at a MOI equal to 1 and cultured in complete culture media 5 h prior to addition of cytokines Journal of Leukocyte Biology Volume 86, November 29

3 Hogg et al. HIV-1 suppresses T cell expression of granulysin Pharmacological inhibitors The pathway inhibitor AG49, a pan tyrosine kinase inhibitor with enhanced activity for Jak 1/3 (Calbiochem, San Diego, CA, USA), was prepared in DMSO, stored at 2 C, and used at 1 M concentration, as described previously [46]. Purified T cells were obtained from PBMC and treated with AG49 or DMSO for 1hat37 C prior to addition of the cytokines. To determine T cell viability following culture with the pharmacological inhibitors, an aliquot of cells was stained with propidium iodide. T cell viability was similar among cells left untreated, treated with DMSO, or treated with AG49. Flow cytometry PBMC or purified T cells were harvested at the indicated times and labeled with antibodies against surface molecules:,, CD4, and CD45RO. Cells were washed and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences). Cells were then washed and labeled with antibodies to granulysin, perforin, Ki67, or HIV-1 core proteins. Lastly, cells were washed and resuspended in 4 l 2% ultrapure formaldehyde (Polysciences Inc., Warrington, PA, USA). A total of 5, or CD4 cells were collected within a live lymphocyte gate on a FACS- Canto (BD Biosciences), and compensation was performed using FACSDiva software (BD Biosciences). Analysis was performed using FCS Express Version 3 (De Novo Software, Los Angeles, CA, USA). Detection of cell viability Following 5 days of cytokine stimulation, mock- and HIV-1-infected PBMC were harvested to determine cell viability. An aliquot of PBMC was washed and incubated with LIVE/DEAD fixable far-red stain (Molecular Probes, Invitrogen Detection Technologies, Carlsbad, CA, USA) for 3 min on ice. Samples were washed and fixed in 2% ultrapure formaldehyde prior to flow cytometric analysis. Western blot Following cytokine stimulation for 3 days, T cells were negatively isolated from PBMC and washed in cold PBS. Cell suspensions were lysed in radioimmunoprecipitation assay buffer, supplemented with protease and phosphatase inhibitors (Calbiochem), and a bicinchoninic acid assay (Bio- Rad, Hercules, CA, USA) was performed to determine protein concentrations. Equal amounts of protein (2 g) were loaded in each lane of a 4 2% gradient precast Tris-glycine gel (Bio-Rad) and separated by electrophoresis. Proteins were then transferred to a nitrocellulose membrane (Bio-Rad) and blotted with primary antibodies against -actin, granulysin, and p- and tstat1, p- and tstat3, and p- and tstat5. Membranes were washed and incubated with HRP-conjugated secondary antibodies (1/2) for 1 h. Blots were developed with the enzyme chemiluminescence kit ECL (Amersham Biosciences, Piscataway, NJ, USA). Densitometry analysis of blots was performed using AlphaEase FC Software (Version 4., Alpha Innotech, San Leandro, CA, USA). Phosphorylation state-specific flow cytometry At indicated times, cultured PBMC or purified T cells were fixed with 2% ultrapure formaldehyde in PBS for 15 min on ice. Cells were pelleted and resuspended in ice-cold 8% methanol (Fisher Scientific, Hampton, NH, USA) for 3 min on ice. Cells were then washed and labeled with fluorochrome-conjugated antibodies to,, granulysin, and p- STAT3 or p-stat5 molecules for 1 h at room temperature. Samples were then washed and fixed in 2% ultrapure formaldehyde prior to flow cytometric analysis. Transwell assays PBMC were infected with HIV-1 or mock-infected overnight. Cells were then washed, counted, and plated out at 1 6 /ml in 24-well transwell plates containing a.4 m polyethylene terephthalate membrane insert (BD Biosciences). Total noninfected T cells were placed in the lower chamber of the well, and mock-infected or HIV-infected PBMC were placed in the upper chamber insert. After 5 days of activation with or, the inserts were removed carefully, and the T cells present in the lower chamber were analyzed by flow cytometry for expression of,, granulysin, and Ki67. Real-time PCR Total RNA was isolated from control (media) or cytokine-stimulated, purified T cells using RNeasy (Qiagen, Valencia, CA, USA). cdna was synthesized using Superscript RT (Invitrogen), according to the manufacturer s recommendations. Differences in gene expression were determined using specific primers and probes for: granulysin forward 5 -AGAAGTGTT- TCCAATGCTGCGACC-3, reverse 5 -TCACAGATCTGCTGGGCAGTTT-3, and FAM-labeled probe 5 -TGGCGCGACGTCTGCAGAAATTTCAT-3 ; perforin forward 5 -AGAAGAAGCACAAGATGACGGCCT-3, reverse 5 -ACAG- CAGGTCGTTAATGGAGGTGT-3, and FAM-labeled probe 5 -TTCCAC- CAAACCTACCGGGAGC-3 ; and IFN- forward 5 -CGGTAACTGACTT- GAATGTCCAACGC-3, reverse 5 -TCGACCTCGAAACAGCATCTGACT-3, and FAM-labeled probe 5 -ATGGCTGAACTGTCGCCAGCAGCTAAA-3 (Integrated DNA Technologies, Coralville, IA, USA). To standardize template, 18S ribosomal control primers and VIC-labeled probe (Applied Biosystems, Foster City, CA, USA) were included in each reaction. Relative gene expression was determined using an ABI PRISM 7 sequence detector. Duplicate C T values were analyzed using the C T method, as described by the manufacturer (Applied Biosystems). The amount of target (2 CT ) was obtained by normalizing to an endogenous reference (18S) and relative to control, untreated samples. sirnas Validated STAT-3- and STAT-5-targeting oligonucleotide duplexes (Stealth Select RNAi ) and a nonspecific control were purchased from Invitrogen. Purified T cells were transfected by electroporation with control sirna (medium guanine-cytosine content) or STAT-specific sirna using the Amaxa Nucleofector (Lonza Cologne AG, Germany) and transfection reagents and settings for unstimulated human T cells, as recommended by the manufacturer (Amaxa, Lonza Cologne AG). The efficiency of transfection was monitored for each donor and assay using a GFP control reagent (Amaxa, Lonza Cologne AG). Transfected T cells were cultured in media (control), (15 ng/ml), or (15 ng/ml) for 3 days and were then analyzed by flow cytometry for expression of granulysin, p-stat5, or p-stat3. Statistical analysis Data are shown as mean sem. One-way ANOVA followed by a Dunnett s multiple comparison test was used for group comparisons (GraphPad Software v4., San Diego, CA, USA). Statistically significant values are designated as follows:, P.5;, P.1;, P.1. RESULTS induces granulysin expression in human peripheral blood T cells Although IL-2 and have been shown previously to increase granulysin levels in human and CD4 T cells [14, 18], the role of in regulating granulysin has not been demonstrated. To determine whether induces granulysin in T cells, we cultured PBMC from healthy donors with or for 1, 3, and 5 days and examined expression of granulysin by flow cytometry (Fig. 1, A and B). We observed that the addition of resulted in a significant increase in the percentage of granulysin-expressing T cells (Fig. 1B). This Volume 86, November 29 Journal of Leukocyte Biology 1193

4 A Live cell gate B + T cells CD4 + T cells C + T cells CD4 + T cells FSC 1 SSC CD4 IgG1-PerCP IgG1-PerCP IgG2a-FITC IgG2a-FITC R D CD45RO / E % / 15 1 Figure 1. induces granulysin in human peripheral blood T cells. PBMC 5 from healthy donors were cultured in media () or in media with (15 ng/ml), (15 ng/ml), or plus for 5 days. Cells were labeled with antibodies to,, CD4, R, CD45RO, and granulysin, collected on a BD FACSCanto, and analyzed using FCS Express Version 3. (A) Gat- Total + CD45RO + CD45RO - ing of and CD4 T cells and isotype controls for intracellular staining are shown. FSC, Forward-scatter; SSC, side-scatter. (B) Representative dot plots of 16 healthy donors are shown, and granulysin expression is presented as the percent of and CD4 T cells. (C) Representative dot plots of 1 healthy donors, showing the percent of and CD4 T cells that express R. (D) Representative dot plots of the expression of CD45RO and granulysin by T cells. (E) Summary of the distribution of granulysin among T cells, including total, CD45RO, and CD45RO populations, from 16 healthy donors, shown as mean sem.,p.5;, P.1;, P.1, statistically significant differences between nonstimulated and cytokine-stimulated groups by one-way ANOVA and Dunnett s multiple comparison test. NS, Not significant. enhancing effect by on granulysin levels was restricted to T cells, as no detectable increase was observed in the CD4 T cells at 5 days (Fig. 1B) or even 7 days following stimulation (data not shown). The differential induction of granulysin among T cell populations was not a result of differences in expression of the R, as we found comparable levels of the receptor on CD4 and T cells (Fig. 1C). Similar to previous reports [14, 18], treatment promoted expression of granulysin in and CD4 T cells. Analysis of the kinetics of cytokine-induced granulysin expression showed that although increases in granulysin levels could be detected by Day 3 (data not shown), the highest levels were consistently detected at Day 5 of culture (Fig. 1B) following stimulation with or. Thus, similar to IL-2 and, induction of granulysin in T cells by occurs following prolonged stimulation. Using quantitative PCR analysis, we detected increased mrna levels of granulysin, perforin, and IFN- in purified T cells following 24 h of or stimulation, providing further evidence for the granulysin-promoting properties of (data not shown). To identify the populations of T cells, in which granulysin expression can be augmented by and, we next analyzed the phenotype of the responding T cells. As shown in Figure 1, D and E, the predominant population of T cells that up-regulate granulysin in response to or expresses CD45RO, a cell-surface antigen that largely defines activated T cells. We also detected a much smaller population of CD45RO T cells that constitutively expressed granulysin; however, this population did not increase significantly following cytokine stimulation and may represent a subset of effector T cells [47]. These results demonstrate that CD45RO T cells are poised to re Journal of Leukocyte Biology Volume 86, November 29

5 Hogg et al. HIV-1 suppresses T cell expression of granulysin spond to and in the cytokine microenvironment by up-regulating the expression of granulysin. Regulation of granulysin by and is dependent on Jak/STAT signaling The signaling pathways that regulate granulysin expression in T cells have not yet been characterized. The Jak/STAT pathway is known to regulate perforin expression and was shown recently to play a role in the induction of granulysin by IL-2 in CD4 T cells [36]. To investigate activation of the Jak/ STAT signaling pathway in our system, we cultured purified T cells with and for 1 and 3 days and analyzed t- and p-stat1, -STAT3, and -STAT5 and granulysin expression by Western blot. Consistent with our observations in PBMC cultures (Fig. 1B), we found that granulysin was increased by and activation of isolated T cells (Fig. 2A). Thus, granulysin can be induced in T cells by in the absence of CD4 T cells or accessory cells. As we observed previously in T cells within PBMCs, increased expression of granulysin by purified T cells is detectable by 3 days (Fig. 2A) and is greatest at 5 days poststimulation (data not shown). As shown in Figure 2A, the relative responsiveness of T cells to as compared with also varies with donor. We further observed increases in p-stat3 and p-stat5 within 24 h of cytokine stimulation (data not shown), and these differences increased after 3 days (Fig. 2, A C). At this time-point, we also detected an enhanced expression of granulysin in the cytokine-stimulated T cells, which correlated with an increase in p-stat3 and p-stat5 (Fig. 2A). The levels of p-stat1, however, did not correlate with cytokine induction of granulysin. Similar to previous reports [3], we found that preferentially induced p-stat3, and activated p-stat5 and p-stat3 in T cells (Fig. 2A), as confirmed by densitometry (Fig. 2, B and C). To determine whether Jak/STAT signaling is required for the induction of granulysin in T cells by and IL- 21, we pretreated purified T cells with AG49, a pharmacological inhibitor of Jak/STAT signaling. After 3 days of cytokine stimulation, we observed that augmentation of granulysin in T cells by or was abrogated in the A t-stat3 p-stat3 t-stat5 p-stat5 t-stat1 p-stat1 D % + G pstat3 β-actin sirna STAT3 sirna Donor A Donor B DMSO Ag B E pstat Densitometry units p-stat H pstat5 sirna STAT5 sirna C F DMSO Ag49 pstat Densitometry units p-stat DMSO Ag Figure 2. Induction of granulysin in human T cells by and requires Jak/STAT signaling. Protein extracts were prepared from purified T cells following 5 days of cytokine stimulation, and Western blots were performed for -actin, granulysin, t-stat1, p-stat1, tstat3, p-stat3, tstat5, and p-stat5. (A) Western blots representative of four healthy donors are shown. (B and C) Densitometry of p-stat3 and p-stat5 from four donors was calculated using AlphaEase FC and is presented as the mean sem expression relative to -actin. Purified T cells were incubated with DMSO or AG49 (pan tyrosine kinase inhibitor with specificity for Jak1/3) for 1 h prior to culture with media (), (15 ng/ml), or (15 ng/ml). After 3 days, cells were fixed in 2% formaldehyde, permeabilized in 8% methanol, labeled with antibodies to,, p-stat3, p-stat5, and granulysin, and analyzed using flow cytometry. (D) Percentage of T cells that express granulysin following cytokine activation in the presence of DMSO (shaded bars) or AG49 (open bars) is presented as the mean sem of five healthy donors. (E and F) Representative dot plots showing expression of granulysin and p-stat3 or p-stat5 within cytokine-activated T cells in the presence of DMSO or AG49. Purified T cells were transfected with control sirna, STAT3 sirna, or STAT5 sirna, cultured for 3 days in media (),, or, and analyzed by flow cytometry. (G and H) Representative dot plots showing granulysin expression and p-stat3 or p-stat5 in purified T cells., P.5;, P.1, indicate statistically significant difference between groups by one-way ANOVA and Dunnett s multiple comparison test. Volume 86, November 29 Journal of Leukocyte Biology 1195

6 presence of AG49 (Fig. 2, D F). Furthermore, coexpression of granulysin with p-stat5 or p-stat3 was also reduced in AG49-treated T cells (Fig. 2, E and F). To further confirm the specific requirement of STAT3 and STAT5 activation for the induction of granulysin by and in T cells, we performed sirna knockdown experiments to block de novo synthesis of STAT3 or STAT5B. We found that blockade of STAT3 protein synthesis by sirna resulted in abrogation of p-stat3 levels within T cells and that this inhibition strongly correlated with reduced expression of granulysin within -treated T cells (Fig. 2G). As a result of the large proportion of T cells that expresses p-stat5 following activation, we had difficulty detecting a sizable reduction in p-stat5 levels (Fig. 2H), although control reagents indicated transfection efficiency was 7%. We did, however, consistently detect a reduction in p-stat5 expression and a concomitant decrease in granulysin expression in T cells following STAT5B silencing (Fig. 2H). Induction of granulysin by was also reduced somewhat following STAT3 silencing and occurred within the p-stat3 granulysin population (Fig. 2G). Our data suggest that activation of Jak/STAT signaling is a critical event and that STAT3 and STAT5 may be important mediators in the regulation of granulysin by and in T cells. HIV-1 suppresses induction of granulysin by and in T cells Specific defects in T cell function occur during chonic HIV-1 infection, including suppressed proliferative capacity and reduced expression of perforin and IFN- [48, 49]. An important role for granulysin in the control of C. neoformans infection in HIV-1-infected individuals has been proposed [36]; however, the potential for HIV-1 to interfere with granulysin expression in T cells has not been described yet. To explore the impact of HIV-1 infection on the ability of T cells to up-regulate effector molecules in response to activating signals from c cytokines, we used an in vitro system in which PBMC from healthy donors were infected with HIV prior to stimulation with cytokines. We then compared the responsiveness of T cells to and in mock-infected and HIV-1- infected PBMC. As shown in Figure 3, A and B, we found that the percentage of granulysin-expressing T cells following stimulation with or was reduced in the cultures infected with HIV-1. Surprisingly, activation of granulysin by plus was refractive to the effects of HIV-1. We next examined whether HIV-1 altered the expression of other effector molecules by T cells in our system. We observed that HIV-1 infection of the PBMC population reduced induction of perforin significantly in response to (Fig. 3, C and D). In contrast to granulysin, HIV-1 did not alter perforin induction by significantly. However, expression of intracellular IFN- in response to was suppressed in the presence of HIV-1 (Fig. 3, E and F). In uninfected PBMC, and induced distinct effector profiles in T cells (Fig. 3, A F). Whereas preferentially induced expression of the cytotoxic molecules granulysin and perforin, induced a broader effector response characterized by increases in IFN-, perforin, and granulysin (Fig. 3, A F). These results from our in vitro system suggest the potential capacity of HIV-1 to dys- A Increase in % + cells 16 PBMC 14 PBMC / B / C Increase in % Perforin + cells / D / Perforin E Increase in % IFN-γ + cells /21 F / IFN-γ Figure 3. HIV-1 inhibits induction of effector molecules by and in T cells. PBMC from healthy donors were infected with 4 HIV for 5 h prior to addition of (15 ng/ml), (15 ng/ml), or plus for 5 days. Flow cytometry was used to analyze intracellular expression of granulysin, perforin, and IFN- within T cells. Data are shown as the mean ( sem) increase, relative to media control, in the percentage of cells expressing (A) granulysin, (C) perforin, and (E) IFN- from 1 healthy donors. (B, D, and F) Representative plots, including baseline levels., P.5;, P.1, indicate statistically significant differences between groups by one-way ANOVA and Dunnett s multiple comparison test Journal of Leukocyte Biology Volume 86, November 29

7 Hogg et al. HIV-1 suppresses T cell expression of granulysin regulate the activation of effector profiles by and in T cells. HIV-1 suppresses T cell activation by and in the absence of direct infection or altered cell viability To explore the mechanisms whereby HIV-1 infection of PBMC alters T cell expression of granulysin, we confirmed the cellular tropism of the virus strain and determined the effects of HIV-1 infection of PBMC on T cell viability. We found that CD4 T cells were consistently the predominant population infected with HIV-1, although infection rates in CD4 T cells varied slightly between donors and within treatment groups, ranging from 2% to 8% and were generally greater following activation (Fig. 4A). This largely mirrors the percentage of activated CD4 T cells present in PBMC, in which HIV-1 can replicate. Our system is most representative of a late-stage HIV-1 infection that is associated with detectable levels of infected CD4 T cells in peripheral blood and an increased frequency of the CXCR4 HIV-1 virus [5, 51]. After determining the infection rates and tropism, we evaluated whether the viability of T cells was affected by HIV-1 infection of CD4 T cells. Using the LIVE/ DEAD fixable far-red stain, we observed a low level of cell death attributable to cytokine stimulation; however, the frequency of T cell death was not altered significantly in HIV-1-infected PBMC (Fig. 4B). To determine whether HIV-1 can interact directly with T cells, we cultured purified T cells with virus for 5 h prior to cytokine addition and measured levels of granulysin after 5 days. We were unable to detect a suppressive effect of HIV-1 on induction of granulysin when purified T cells were stimulated with or (Fig. 4C). We next examined whether inhibition of T cell expression of granulysin by HIV-1 requires direct contact with HIV-infected cells or could be mediated though a transwell. When T cells were separated from the remaining HIV-1-infected PBMC by transwell, the suppression of - or -induced granulysin expression was similar to that observed when PBMC were cultured directly with HIV-1 (Fig. 4D). These results suggest that in our system, CD4 T cells are the predominant host of HIV-1 and that suppression of granulysin induction by or in T cells may be mediated by a soluble factor. HIV-1 inhibits granulysin induction by independent of cell-cycle progression It has been reported that an important difference between and in regulation of T cell function is the relative capacity to promote proliferation and that although has more moderate effects, is a strong inducer of T cell proliferation [3, 52]. The relationship between cell-cycle progression and induction of granulysin has not been described yet; therefore, we evaluated coexpression of the Ki67 protein and granulysin following cytokine stimulation A FSC CD4 C % + Live cell gate + CD4 + gate SSC CD4 CD4 HIV p24 IgG1-APC IgG1-FITC HIV p D % HIV B FSC SSC + + gate + HIV Cell death marker Contact Contact + HIV Transwell Transwell + HIV Figure 4. Suppression of T cell effector function by HIV-1 is indirect. PBMC were mock-infected () or infected with 4 HIV ( HIV) at a MOI of 1 for 5 h prior to addition of media (),, or. After 5 days, cells were labeled with antibodies to CD4,, and HIV-1p24 and analyzed by flow cytometry. (A) Representative dot plots of the frequency of HIV-1-infected CD4 T cells of three healthy donors are shown. (B) Cell viability of T cells, as determined using the LIVE/DEAD fixable far-red stain in mock-infected and 4 HIV infected PBMC, is shown as representative histograms of three healthy donors. (C) Expression of granulysin by purified T cells cultured for 5 days, with or without HIV, and media (),, or. (D) Fold increase in the percentage of granulysin-expressing T cells cultured for 5 days in a transwell or in direct contact with HIV-infected PBMC or mock-infected PBMC., P.5, indicates statistically significant differences between groups by one-way ANOVA and Dunnett s multiple comparison test. Volume 86, November 29 Journal of Leukocyte Biology 1197

8 with or and TCR activation using anti- and anti-cd28 microbeads (Fig. 5, A D). Ki67 is expressed only by cells that are in active stages of the cell cycle and is not expressed by resting (G ) cells [53]. Following TCR stimulation, HIV-1 infection of PBMC did not suppress induction of granulysin or cell-cycle progression in T cells (Fig. 5B), demonstrating the functional capacity of these cells. Similar to previous reports [3], we found that but not promoted the progression of T cells to enter the active phases of the cell cycle, indicative of cellular proliferation (Fig. 5, C and D). Interestingly, we observed that in response to, increases in granulysin occurred almost exclusively within cycling (Ki67 ) T cells (Fig. 5C). In contrast, activation led to a similar increase in granulysin levels in the absence of promoting cell-cycle progression. In HIV-1-exposed PBMC, we observed that the percentage of T cells that are cycling and expressing granulysin after stimulation is reduced greatly (Fig. 5, C and D). Moreover, we found that induction of granulysin in noncycling T cells was also reduced by HIV-1 (Fig. 5C). Our data suggest that HIV-1 can mediate suppression of granulysin within cycling and noncycling T cells following cytokine stimulation. HIV-1-mediated suppression of granulysin corresponds with the disruption of STAT signaling Having demonstrated an important role for Jak/STAT signaling in the regulation of granulysin in T cells by and (Fig. 2), we then examined whether the disruption of granulysin expression by HIV-1 in - and -activated T cells correlated with defective intracellular Jak/STAT signaling. Using multivariate flow cytometry, we examined the simultaneous expression of granulysin and p-stat3 or p-stat5 within gated T cells in response to and stimulation (Fig. 6). Following 3 days of stimulation, we observed markedly reduced p-stat3 following stimulation and a reduced p-stat5 following stimulation as a result of HIV-1 infection of the PBMC (Fig. 6, A D). Interestingly, we found that p-stat5 and p-stat3, within granulysinexpressing T cells, were abrogated in the presence of HIV-1 (Fig. 6, E and F). In response to stimulation, a significant number of granulysin-positive cells expressed low levels of p-stat3 (Fig. 6E), and this population was also diminished as a result of HIV-1. This difference may be a result of the prolonged activation time required for granulysin expression, as compared with the earlier p-stat3 signaling events required for induction of granulysin. Analysis of p-stat kinetics (15 min, 3 min, 1 h, 4 h) revealed that HIV-1 mediated a reduction of -induced p-stat3- and -induced p-stat5 by 4 h post-cytokine stimulation, although this reduction could not be associated with suppression of granulysin at this time-point (data not shown), as a result of the expression kinetics of granulysin. Alternatively, HIV-1 may suppress - induced granulysin through mechanisms that are independent of STAT3. Following stimulation, most T cells were positive for granulysin and p-stat5, and expression of both molecules was reduced by HIV-1 infection (Fig. 6F). Analysis of the PBMC indicated that -induced p- STAT5 was also decreased in populations by HIV-1 infection (data not shown) in agreement with other studies [36, 54]. Following stimulation, however, HIV did not suppress p-stat3 in the non- T cell population (data not shown). These observations were supported further by Western blot analysis of negatively sorted T cells from mock- and HIV-1-infected PBMC (Fig. 6G). In these experiments, we also measured levels of tstat proteins and did not observe any difference in expression levels of tstat3 or tstat5 (Fig. 6G). Our results suggest that the HIV-1-mediated suppression of T cell responsiveness to cytokines may occur at the level of activation of downstream Jak/STAT signaling. A Live cell gate + + gate Isotype controls D % + + Ki67 + FSC 8 6 SSC IgG2a IgG1 PBMC PBMC B.3.2 Ki /CD C Ki Figure 5. HIV-1 inhibits granulysin induction by independent of cell-cycle progression. PBMC from healthy donors were mock-infected or infected with 4 HIV for 5 h prior to addition of (15 ng/ml), (15 ng/ml), or anti-/cd28 microbeads for 5 days. Flow cytometry was 2 used to analyze intracellular expression of granulysin and Ki67 within T cells. (A) Gating of T cells and isotype controls for intracellular staining are shown. (B) Representative dot plots showing the expression of granulysin and Ki67 by T cells following TCR stimulation in the presence/absence of HIV. (C) Representative dot plots demonstrating the expression of granulysin and Ki67 by T cells following cytokine stimulation are shown. (D) Summary of the percentage of T cells expressing Ki67 from six healthy donors is shown as the mean sem., P.1, indicates statistically significant differences between groups by one-way ANOVA and Dunnett s multiple comparison test Journal of Leukocyte Biology Volume 86, November 29

9 Hogg et al. HIV-1 suppresses T cell expression of granulysin A B PBMC PBMC E C pstat3 pstat % p-stat5+ % p-stat3+ D F pstat pstat G t-stat3 p-stat3 t-stat5 p-stat5 β-actin + HIV Figure 6. Defective granulysin induction by and corresponds with reduced STAT activation in the presence of HIV-1. Mock-infected ( HIV) and 4 HIV infected ( HIV) PBMC were cultured in media () or in the presence of (15 ng/ml) or (15 ng/ml) for 3 days. Cells were then fixed in 2% formaldehyde, permeabilized in 8% methanol, and labeled with antibodies to,, p-stat5, p-stat3, and granulysin. (A F) cells were gated and analyzed for expression of p-stat3, p-stat5, and granulysin. Dot plots shown demonstrate levels of p-stat molecules in T cells (A, C, E, and F) and the coexpression of granulysin with p-stat3 or p-stat5 within T cells (E and F) and are representative of eight healthy donors. A summary of data from eight donors for levels of p-stat3 and p-stat5 in T cells (B and D) is presented as mean sem. (G) T cells were negatively sorted from uninfected or HIV infected PBMC after 3-day cytokine stimulation. Protein extracts were prepared, and Western blots were performed for -actin, t-stat3, p-stat3, t-stat5, and p-stat5. Western blots are representative of two healthy donors., P.5;, P.1, indicate statistically significant differences between groups by one-way ANOVA and Dunnett s multiple comparison test. DISCUSSION To gain insight into the mechanisms whereby HIV-1 infection promotes host susceptibility to opportunistic infection, it is important to understand how the virus alters antimicrobial protein expression by T cells. In this study, we used an in vitro system, first to define the mechanisms of granulysin regulation by and in primary human T cells and then to examine interference by HIV-1 in granulysin signaling through and. Our results provide the first evidence that granulysin is induced by and is expressed primarily by CD45RO T cells following activation by or. We show that Jak/STAT signaling is required for granulysin induction through R and R and that cytokine-specific p-stat3 and p-stat5 may regulate transcription. Furthermore, we demonstrate that HIV interferes with granulysin induction and suppresses p-stat5 and p-stat3 upon activation of T cells with and. We propose that the dysregulation of granulysin expression in T cells by HIV-1 is a contributing factor to the suppression of cell-mediated immune responses against opportunistic infections. is the most recently characterized member of the c cytokine family and has been implicated in infectious disease, tumor immunity, and autoimmunity (reviewed in ref. [21]). Of the c cytokines, IL-2,, and are known to promote cytotoxic activity of T cells [21, 22]. In our studies, exogenous induced granulysin expression by peripheral blood T cells, a function described previously for IL-2 and [14, 18]. In addition to sharing the c, IL-2 and share a common -chain receptor subunit (reviewed in ref. [22]). Significant homology between the R -chain and the IL-2R/R -chain has been reported and proposed to account for the shared biological effects of these cytokines [55]. The conserved signaling mechanisms activated by c cytokines may account for the similarities that we observe in the regulation of granulysin expression in T cells. These Volume 86, November 29 Journal of Leukocyte Biology 1199

10 results suggest that promotion of antimicrobial potential by T cells via granulysin induction is a conserved feature of this subset of c cytokines (IL-2,, and ). Our study shows that can influence cytotoxic granule protein expression in T cells from healthy donors. In addition to granulysin induction, we observed a lesser, but significant, increase in perforin expression by -activated T cells. Our data are in contrast to that of a previous report, where was shown to enhance perforin expression in T cells from HIV patients after HAART therapy but not from healthy donors [3]. In the same study, increases in perforin after stimulation are evident in T cells from healthy donors but did not reach statistical significance. One explanation for this difference may be the greater number of donors (n 16) that we examined compared with the previous report. Moreover, we also observed some donor variation in the magnitude of responsiveness of T cells to individual cytokines (Fig. 1) and would not have been able to observe significant differences in perforin using fewer than 1 donors. expression was less variable than perforin, and a substantial increase in granulysin levels in response to was evident for most donors. Also, in contrast to the report by White et al. [3], we observe that HIV infection suppresses induction of perforin in T cells by. It is likely that the differences observed in these studies are a result of the relative HIV load within the experimental systems. In the study by White et al. [3], PBMC were obtained from HIV patients after successful HAART therapy, in which viral load in the blood would be almost undetectable. In contrast, in our study, we infect PBMC with live virus and detect an infection rate of CD4 T cells of 2 8%. A differential induction of IFN- by and was also evident in our studies. Whereas induced poly-functional T cells expressing granulysin, perforin, and IFN-, induced granulysin and perforin in the absence of detectable IFN-. Similar studies in mice have shown that signaling, at the time of antigen presentation, promoted the unique differentiation of cytolytic perforin-expressing T cells that were defective in IFN- production [56]. Results from three clinical trials of patients with metastatic melanoma demonstrated that therapy could augment mrna levels of IFN-, perforin, and granzyme B in T cells [57 59]. In agreement with these studies, we also observed increases in mrna for perforin and IFN- in -treated T cells. In contrast to our results for perforin, however, we were unable to detect -induced increases in IFN- protein levels. This noted disparity may be a result of a differential effect of on IFN- and perforin translation or differences in the immune status of the subjects used in our study (healthy donors) and the clinical trials (patients with metastatic melanoma). In our study of healthy donors, activation promoted an effector molecule profile in T cells characterized by high cytotoxic (perforin) and antimicrobial (granulysin) potential in the absence of IFN-. Antigen-primed T cells can respond quickly following an infection and express the truncated form of the CD45 leukocyte antigen, CD45RO. In our studies, we observe that granulysin expression is restricted primarily to CD45RO T cell populations following activation with or. These studies are the first characterization of the distribution of granulysin among T cell memory subpopulations following cytokine activation. It is likely that our findings reflect the increased expression of R and R on activated and memory T cell populations [6, 61]. The high-affinity R is a marker of memory T cells, and expression of R is increased on T cells upon TCR activation [21]. A recent study by Stenger and colleagues [62] demonstrated that in the absence of stimulation, effector memory CD45RA CCR7 T cells expressed the highest constitutive levels of granulysin. This is similar to our observations that granulysin expression ex vivo or in the nonstimulated control can be detected in CD45RO and CD45RO T cells (Fig. 1, D and E). An unexpected finding of our studies was that in contrast to IL-2 (data not shown) and, -induced granulysin expression is in T cells with no significant effect on CD4 T cells. The R is expressed on all lymphocyte populations [63], and our studies demonstrate that the level of R on CD4 and T cells does not account for differential induction of granulysin by. Separate sources of these c cytokines control their presence in different microenvironments. Although there are other sources, IL-2 is expressed mostly by T cells, and is expressed by myeloid cells (monocytes, macrophages, dendritic cells) and epithelial cells. is expressed primarily by activated CD4 T cells and NKT cells [63, 64]. Our results demonstrate that the availability of c cytokines (IL-2,, and ) in the microenvironment and expression of their receptors during immune activation of T cells may direct the choice of antibacterial effector molecules. The c cytokines that promote cytotoxic activity (IL-2,, and ) have been shown to activate the Jak/STAT signaling pathway upon receptor binding and differentially induce p-stat1, p-stat3, or p-stat5 transcription factors. The IL- 15R signals predominantly through STAT5, and signals predominantly through STAT3. Past studies have identified a role for Jak/STAT signaling in the IL-2-mediated induction of perforin in T cells and granulysin in CD4 T cells [36, 65], and the intracellular signaling pathways that regulate granulysin in primary T cells had not been described previously. In this study, we identify a key role for the Jak/ STAT signaling pathway in induction of granulysin by and in T cells. Our gene-silencing studies further implicate an important role for STAT3 to regulate transcription of granulysin following stimulation of T cells. Although silencing of STAT5 was incomplete in our studies, a contribution of STAT5 to regulation of granulysin in response to was evident nonetheless. Taken together, our results support the proposition that Jak/STAT signaling represents an important pathway by which c cytokines, and, regulate granulysin expression in T cells. Chronic HIV-1 infection has multi-faceted effects on cellmediated immunity, which lead to defects in the function of CD4 and T cells. The mechanistic basis for the suppression of T cells during chronic HIV-1 infection is poorly understood. Although recent evidence suggests that in 12 Journal of Leukocyte Biology Volume 86, November 29

11 Hogg et al. HIV-1 suppresses T cell expression of granulysin vitro T cells can become infected with HIV-1 [66], these observations have not been confirmed yet in vivo. In these studies, we optimized an in vitro HIV-1 infection system using primary human PBMC to explore the basic mechanisms, whereby HIV-1 may reduce the antimicrobial potential of T cells. In our in vitro system, we detected the HIV-1 p24 antigen primarily in CD4 T cells and within a much smaller population of CD14 monocytes (data not shown) but did not detect HIV-1 within T cells. The relatively low infection rate of CD4 T cells that we observed is comparable with published data from untreated HIV patients showing that only a minor population of peripheral blood CD4 T cells harbor HIV-1 and that HIV-1 can replicate in a small fraction of these infected CD4 T cells [51, 67]. HIV-1 can also alter the function of resting CD4 T cells and monocytes by binding to CD4 on the cell surface and initiating immunosuppressive functions, including production of IL-1, in the absence of viral replication [68, 69]. In HIV patients with progressive disease, HIV-specific T cells show defective capacity to proliferate and express perforin in response to HIV-1 antigens and IL-2 stimulation. Using our system, we were able to reproduce these effects on T cell proliferation and expression of perforin. Further, we were able to determine that in vitro HIV-1 infection of CD4 T cells causes a suppression of granulysin in response to common c cytokines in T cells. Reduced expression of granulysin by CD4 T cells from HIV patients has been reported previously and was shown to result from defective IL-2 signaling and contribute to the poor control of C. neoformans [36]. Thus, HIV-1 infection is able to suppress the expression of granulysin in CD4 and T cells. Our studies demonstrate that these suppressive effects of HIV-1 on uninfected T cells likely occur via an accessory cell, are mediated by a contact-independent mechanism, and can be detected even following short-term in vitro infection of peripheral blood cells from healthy donors. Similar to the findings of Zheng et al. [36], demonstrating that Jak/STAT signaling is important for induction of granulysin by IL-2 in CD4 T cells, we observed that induction of granulysin in T cells by also requires activation of the Jak/STAT signaling pathway and correlates with p-stat5. The work of Zheng and colleagues [36] revealed an association between defective p-stat5 and poor induction of granulysin by IL-2 in CD4 T cells from HIV-infected subjects. Activation of granulysin expression by in T cells in our studies is also dependent on the Jak/STAT pathway but appears to correlate with STAT3 activation and occurs in the absence of significant p-stat5. Identification of STAT5-independent signaling pathways to promote CTL function is important for development of therapeutic interventions to promote/restore T cell activity in HIV patients, as a STAT5-binding site is present in the HIV-1 promoter and has been shown to drive viral replication [7]. CD4 T cells down-regulate STAT5 in vivo and in vitro following HIV-1 infection [71], an effect presumed to represent an antiviral response to slow viral replication. Perforin expression by T cells from HIV patients can be induced in vitro by exogenous in the absence of cellular proliferation by CD4 or T cells [3]. These effects shown by White and colleagues [3] have important implications for use of as cytokine therapy for HIV patients to promote antiviral T cell function. Our studies complement these observations further, demonstrating that granulysin, a broad-spectrum antimicrobial molecule, can also be induced in T cells by at levels that do not promote proliferation. However, we observed a HIV-1-dependent reduction of granulysin and signaling through STAT3 and STAT5 in T cells following activation with and, respectively. Our results suggest that despite the capacity of to augment granulysin levels in the absence of T cell proliferation or STAT5 activation, induction of granulysin by was still abrogated in the presence of HIV-1. The broad-spectrum antimicrobial activity of granulysin suggests an important effector role of this molecule in the activity of cytotoxic lymphocytes against many pathogens. To date, there is significant evidence to support a key role for granulysin in the immune response to Mtb and C. neoformans, two important pathogens of HIV patients [14, 2, 23, 36, 72]. A greater understanding of how HIV-1 may disrupt regulation of this molecule is imperative to characterizing and potentially restoring loss of T cell function following HIV-1 infection. We have shown that signaling through the R and R leads to an up-regulation of granulysin within CD45RO T cells. Furthermore, we demonstrate a requirement for Jak/STAT signaling in the induction of granulysin by and and implicate STAT3 and STAT5 as potential transcription factors involved in the regulation of granulysin within T cells. Using an in vitro system, we were able to determine that dysregulation of granulysin in T cells, through inhibition of and signaling, could be an important consequence of HIV-1 infection. These efforts to elucidate the mechanistic basis for suppression of T cell function by HIV-1 are critical for development of prophylactic and therapeutic interventions for opportunistic pathogens of immune-compromised populations. ACKNOWLEDGMENTS This work was supported by the Department of Microbiology and Immunology, the James W. McLaughlin Fellowship Fund, and the Sealy Center for Vaccine Development, University of Texas Medical Branch (Galveston, TX, USA). The authors thank Dr. Jiaxiang Ji for sharing his expertise and for preparation of HIV stocks and Professors Lynn Soong and Gary Klimpel for their critical review of this manuscript. REFERENCES 1. Lawn, S. D., Butera, S. T., Shinnick, T. M. (22) Tuberculosis unleashed: the impact of human immunodeficiency virus infection on the host granulomatous response to Mycobacterium tuberculosis. Microbes Infect. 4, Levy, J. A. (29) HIV pathogenesis: 25 years of progress and persistent challenges. AIDS 23, McMichael, A. J., Rowland-Jones, S. L. (21) Cellular immune responses to HIV. Nature 41, Mackewicz, C. E., Wang, B., Metkar, S., Richey, M., Froelich, C. J., Levy, J. A. (23) Lack of the cell anti factor in cell granules. Blood 12, Zhang, D., Shankar, P., Xu, Z., Harnisch, B., Chen, G., Lange, C., Lee, S. J., Valdez, H., Lederman, M. M., Lieberman, J. (23) Most antiviral Volume 86, November 29 Journal of Leukocyte Biology 121

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