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The Journal of Immunology, 2004, 173: 641-648.
Copyright © 2004 by The American Association of Immunologists

Abundant Expression of Granzyme A, but Not Perforin, in Granules of CD8+ T Cells in GALT: Implications for Immune Control of HIV-1 Infection1

Barbara L. Shacklett2,*, Catherine A. Cox*, Máire F. Quigley§, Christophe Kreis*, Neil H. Stollman{ddagger}, Mark A. Jacobson{dagger},{ddagger}, Jan Andersson§, Johan K. Sandberg*,§ and Douglas F. Nixon*,{ddagger}

* Gladstone Institute of Virology and Immunology (GIVI), {dagger} University of California San Francisco (UCSF)-GIVI and Center for AIDS Research (CFAR), San Francisco General Hospital (SFGH), and {ddagger} Department of Medicine, University of California, San Francisco, CA 94141; and § Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Because GALT is a major portal of entry for HIV-1 and reservoir for viral replication, we hypothesized that an ineffective cellular immune response in intestinal mucosa might partially explain the failure of immune control in AIDS. In this study, we demonstrate that the vast majority of CD8+ T cells in rectal tissue, including HIV-1-specific cells, fail to express the cytolytic protein, perforin. However, rectal CD8+ T cells do express granzyme A, and are also capable of releasing IFN-{gamma} upon stimulation with cognate peptide. Confocal microscopy showed that granzyme A was located in intracellular granules in the absence of perforin. The majority of rectal CD8+ T cells exhibit an effector memory phenotype, expressing CD45RO but not CCR7. Quantitative real-time PCR analysis demonstrated that perforin RNA is expressed in rectal CD8+ T cells from healthy and HIV-1-positive individuals. In HIV-1-positive individuals, similar amounts of perforin RNA were detected in CD8+ T cells from rectal tissue and PBMC, despite a relative absence of perforin protein in rectal tissue. These findings demonstrate an important difference in perforin expression between CD8+ T cells in blood and mucosa. Furthermore, the relative absence of armed effector cells may serve to protect the integrity of rectal mucosa under normal conditions, but might also provide an early advantage to HIV-1 and other sexually transmitted viruses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell-mediated immune responses to HIV-1 can be vigorous in peripheral blood (1, 2). However, few studies have evaluated HIV-1-specific T cell responses in lymphoid tissues of the gastrointestinal and genitourinary tracts (3, 4). GALT is the largest lymphoid tissue in the human body and harbors a majority of the body’s lymphocytes (5). GALT consists of organized aggregates (Peyer’s patches and lymphoid follicles), as well as scattered lymphoid cells disseminated throughout the intestinal epithelium and lamina propria (5). The gastrointestinal tract is a frequent site of AIDS-associated opportunistic infections and malignancies, and serves as a major portal of entry for HIV-1.

HIV-1 infection leads to depletion of intestinal lamina propria CD4+ T cells (6); in animal model systems this occurs as early as 7–14 days after infection (7). This depletion is accompanied by a strikingly increased frequency of CD8+ T cells in the lamina propria (8). HIV-1 infection is characterized by significant intestinal inflammation, as evidenced by increased production of MIP-1{alpha}, MIP-{beta}, and RANTES (8).

HIV-1-specific CD8+ T cells have been isolated from rectal and duodenal mucosa (3, 9). When expanded in vitro, these cells were capable of lysing MHC class I-matched target cells expressing HIV-1 Ags (3). However, little is known of the effector functions exercised by human intestinal T cells in situ. Murine intestinal T cells have been demonstrated to differ from blood T cells in activation status (10, 11), yet are capable of a range of cytotoxic effector functions (12, 13).

CD8+ T cells use several mechanisms to induce target cell death, including granule exocytosis and apoptosis mediated by Fas, TNF, or TRAIL (14). The majority of HIV-1-specific CD8+ T cells are believed to kill via perforin-dependent granule exocytosis (15), and this was recently demonstrated for HIV-1-specific CD8+ T cells in mucosal tissues (9). This pathway is believed to require expression of both perforin and granzymes (14, 16, 17, 18).

Several recent studies (reviewed in Ref.19) suggest that HIV-1-specific CD8+ T cells in blood are impaired in maturation to end-stage effector cells (20), secretion of perforin and/or IFN-{gamma} (20, 21, 22, 23), MHC class I-restricted killing (20, 21, 22), signal transduction, and trafficking to lymphoid tissues (24, 25). We hypothesized that an ineffective CD8+ T cell response in mucosal tissues might partially explain the high level of viral replication and CD4+ T cell depletion observed in GALT. To test this hypothesis, we assessed rectal T cells from healthy controls and HIV-1-positive individuals for their ability to produce cytokines and cytoxic effector molecules. Our results suggest that rectal CD8+ T cells express granzyme A and secrete cytokines in response to antigenic stimulation, yet rarely express perforin. These findings demonstrate an important difference between CD8+ T cells in blood and mucosa, and suggest that intestinal lymphocytes have developed regulatory mechanisms limiting perforin expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Flexible sigmoidoscopy and rectal biopsy

Rectal tissue was obtained from 12 HIV-1-positive individuals and 6 healthy controls by fiberoptically guided flexible sigmoidoscopy and biopsy at ~10 cm from the anal verge (26, 27). A flexible sigmoidoscope with a biopsy channel (EC3831L; Pentax Precision Instruments, Orangeburg, NY) was used with single-use biopsy forceps (Radial Jaw 3; Boston Scientific, Miami, FL) for rectal biopsies. At each biopsy procedure, no more than 20–25 pinch biopsies were obtained and the tissue samples were pooled for lymphocyte extraction and analysis. Informed consent was obtained from all volunteers, and the study was approved by the Committee on Human Research of the University of California (San Francisco, CA). All biopsies were taken from noninflamed rectal tissue that was normal in appearance. It was not possible to distinguish lymphoid follicles in rectal tissue using this approach. Healthy controls were HIV-1-negative volunteers without known gastrointestinal diseases such as inflammatory bowel disease, ulcerative colitis, Crohn’s disease, colon cancer, or diverticulitis.

Patient screening and inclusion criteria

Twelve individuals were selected from among clinically stable HIV-1-positive volunteers (Table I), including nine men and three women. Six of the men reported homosexual contact; two of the men and one of the women reported a history of i.v. drug use, and all had been infected for more than 1 year (range 1.25–15 years, mean 10.2 years). Most patients had CD4+ T cell counts above 250/µl and were not currently on antiretroviral therapy.


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  		Table I. Patient characteristics

 
Mononuclear cell preparation

Rectal lymphocytes were prepared from biopsy tissue as previously described (26, 27). Briefly, tissues were disrupted by incubating for 3 x 30 min at 37°C in medium containing 0.5 mg/ml collagenase type II (Sigma-Aldrich, St. Louis, MO). Lymphocytes were then isolated by centrifuging on a discontinuous 35%/60% Percoll gradient (Pharmacia Biotech, Uppsala, Sweden). Yield and variability were assessed by manual cell counts with trypan blue staining. Typical yields ranged from 3 to 8 x 106 viable cells for 20–25 pinch biopsies. Because of the limited yield of rectal mononuclear cells, it was not possible to perform all experiments on all samples. The number of samples evaluated for each set of experiments is specified in the text and figure legends.

Phenotypic analysis of rectal T cells

Phenotypic analysis of fresh rectal lymphocytes and PBMC was performed using fluorescent Abs to CD3, CD4, CD8, CD45RO, CCR7 (BD Biosciences/BD Pharmingen, San Diego, CA). Samples were analyzed on a FACSCalibur (BD Immunocytometry Systems, San Jose, CA). Appropriate isotype controls were used to set quadrant markers. For multicolor analysis, electronic compensation for spectral overlap was set using PBMC stained with single color reagents.

MHC class I tetramer staining

PBMC were screened for the HLA-A2 allele by surface staining using an A2-specific mAb (ExAlpha, Watertown, MA), and analyzed by flow cytometry. Cells from HLA-A*0201-positive subjects were then stained with MHC class I tetramer complexes containing peptides from HIV-1 Gag (SL9, Gag aa 77–85, SLYNTVATL) (Beckman Coulter, Hialeah, FL). Responses were considered positive if a distinct population consisting of ≥0.03% of CD8+ T cells bound tetramer. This cutoff was similar to that used in previous studies (1, 28). To increase the confidence level in low frequency populations, apparent tetramer-binding populations representing <0.1% of CD8+ T cells were considered equivocal unless observed in at least two independent experiments. Specificity of tetramer staining was confirmed using HIV-1-negative and HLA-mismatched controls.

To determine the percentage of Ag-specific CD8+ T cells expressing perforin and granzyme A, cells were first stained with surface Abs and tetramer for 30 min at 4°C, then washed, permeabilized (FACSPerm; BD Biosciences), and subsequently stained with Abs to detect intracellular perforin (clone {delta}G9; BD Biosciences), granzyme A (clone CB9; BD Biosciences), or appropriate isotype controls.

Cytokine flow cytometry

Cytokine production in response to antigenic stimulation was assessed using standard methods (29, 30, 31), as described in detail by Maecker et al. (32). To assess production of IFN-{gamma} by Ag-specific T cells, mononuclear cells from rectal tissue or blood were incubated with peptide (10 µg/ml) and anti-CD28 costimulatory Ab (clone L293; BD Biosciences) or medium alone. Staphylococcal enterotoxin B (SEB)3 (5 µg/ml; Sigma-Aldrich) was used as a positive control. After 1 h, brefeldin A (10 µg/ml; Sigma-Aldrich) was added to block cytokine release. After an additional 5–6 h, cells were washed, fixed in 4% formaldehyde (Sigma-Aldrich) and stored overnight at 4°C. The following day, cells were permeabilized (FACSPerm; BD Biosciences), stained with Abs specific for CD3, CD8, CD69 and IFN-{gamma} (BD Biosciences), then analyzed by flow cytometry.

Immunohistochemistry

Rectal biopsies were frozen in OCT (Tissue-TEK, Elkhart, IN). Sections (8 µm) were prepared, fixed, and stained as described (33) with Abs to granzyme A (clone CB9, provided by Dr. J. Lieberman, Harvard University, Boston, MA), perforin (clone {delta}G9; Endogen, Woburn, MA), and CD8 (clone MCA351; Serotec, Oslo, Norway). Photomicrographs were obtained using a Leica DMR-X microscope at x275 magnification. For triple stainings, secondary Abs were Alexa 546-goat anti-rat IgG and Alexa 488-goat anti-mouse IgG (Molecular Probes, Eugene, OR). Nucleic acid was stained with Toto3 (Molecular Probes). Images were obtained by sequential scanning using a Leica TCS-SP2 confocal microscope equipped with lasers exciting at 488, 543, and 633 nm.

Quantitative real-time PCR

CD8+ T cells were purified from PBMC and rectal cell suspensions using magnetic beads (Miltenyi Biotec, Auburn, CA). Total cellular RNA was extracted using commercial reagents and treated with DNase (RNeasy reagents; Qiagen, Valencia, CA). In some cases, mRNA was isolated from total RNA using an Oligotex kit (Qiagen). Total RNA preparations were checked for DNA contamination by performing real-time PCR in the presence and absence of reverse transcriptase. Only samples with <0.1% DNA contamination were used in subsequent experiments.

Primers and probes for perforin and granzyme A (GenBank NM_005041 and BC015739, respectively) were designed with Primer Express software, version 1.5 (Applied Biosystems, Foster City, CA), and checked by a basic local alignment search tool search of GenBank (34). Probes were synthesized at Applied Biosystems and carried a 5' fluorescent reporter dye (6-CR 6G, also known as "VIC"; Applied Biosystems) and a 3' minor groove binding nonfluorescent quencher (MGB-NFQ; Applied Biosystems). The sequences of perforin primers and probes were as follows: primers, 5'-CTGGCAGGGACGATGACCT-3' (nt 1460–1478); 5'-GGGAACCAGACTTGGGAGC-3'(1516–1498); probe: 5'-VIC-TTGGCACCTGTGATCA-MGB-NFQ-3' (nt 1481–1496). The sequences of granzyme A primers and probes were as follows: primers, 5'-GAGACTCGTGCAATGGAGATTCT-3' (nt 622–644); 5'-AAGTGACCCCTCGGAAAACA-3' (nt 687–668); probe: 5'-VIC-AGCCCTTTGTTGTGCGMGBNFQ-3' (nt 648–663). Primer and probe concentrations were optimized for each target mRNA by constructing primer matrices with increments at different molarities, and choosing conditions that yielded the lowest threshold cycle (CT) and the highest slope. Primers and probes for the 18s rRNA gene were obtained commercially (Applied Biosystems) and used according to the manufacturer’s instructions.

Reverse transcription and real-time quantitative PCR were performed using total RNA or mRNA with the TaqMan Gold RT-PCR kit (Applied Biosystems) in a one-tube reaction. The ingredients of a 50-µl RT-PCR were as recommended by the manufacturer except that target RNA was present at a concentration of 200 ng/50 µl and linear polyacrylamide (Ambion, Austin, TX) was added at 100 ng/50 µl. The same gene-specific primers were used for reverse transcription and PCR. Amplifications were performed using the ABI-PRISM 7700 Sequence Detection System (Applied Biosystems). The program consisted of a reverse transcription step at 48°C for 30 min, followed by a 95°C step for 10 min, then 40 cycles of denaturation (95°C, 30 s) and annealing/extension (60°C, 1 min).

Quantification of perforin RNA

To facilitate quantification of perforin RNA, a standard perforin RNA template was constructed by ligating the perforin 118-bp amplicon, generated as described above, to a T7 promoter (Lig’nScribe; Ambion). Following a second round of amplification, the PCR products containing the T7 promoter were in vitro transcribed using T7 polymerase (Megashort transcription system; Ambion) to produce an RNA standard. To quantify the amount of perforin RNA in patient samples, the RNA standard was serially diluted at concentrations ranging from 100 to 107 copies/ml. The real-time PCR assay was linear throughout this range. Standard dilutions were amplified in triplicate. The CT values were plotted as a function of input template copy number and a least-squares regression was performed with the Prism 7700 software. Patient RNA samples were amplified in duplicate. Copy numbers for patient samples were calculated by interpolation of the experimentally determined CT values onto the control standard regression curve. Results were then normalized to total cellular RNA content as determined by rRNA analysis, and expressed as perforin RNA copies per nanogram of rRNA. rRNA analysis was performed in a separate assay using commercial primers and probes specific for rRNA (rRNA control kit; Applied Biosystems). Ribosomal RNA content was also confirmed by comparing to total RNA content using the Pico RNA 6000 Chip on an Agilent Bioanalyzer (Quantum Analytics, Foster City, CA).

Statistical analysis

Flow cytometry results were analyzed by standard statistical methods with the aid of Sigma Plot and Sigma Stat software packages (SPSS, Chicago, IL). For comparisons between groups, Student’s t tests were performed whenever the data were normally distributed and the variances of the two populations were equal. When these criteria were not satisfied, Mann-Whitney rank sum tests were performed in lieu of t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HIV-1-specific CD8+ T cells are present in rectal tissue during chronic infection

To assess the presence of HIV-1-specific CD8+ T cells in rectal tissue, we tested peripheral blood and rectal biopsy samples using MHC class I tetramers specific for the HLA-A*0201-restricted HIV-1 Gag SL9 epitope. Twelve individuals with chronic HIV-1 infection, as well as 6 healthy controls, were studied (26). Of these, 10 HIV-1-positive individuals and 3 controls were positive for the HLA-A*0201 allele. PBMC and gut lymphocytes from 7 of 10 HLA-A*0201-positive, HIV-1-positive individuals contained CD8+ T cells specific for the Gag SL9 tetramer (Table I). Numerical results for these tetramer studies were published in a previous study (26). Briefly, the frequency of SL9-specific CD8+ cells ranged from 0.17 to 1.53% (mean 0.44%) in PBMC, and from 0.28 to 2.6% (mean 0.71%) in rectal mucosa. None of the samples from HIV-1-negative controls recognized the Gag SL9 epitope (data not shown) (26). These data demonstrate the presence of HIV-1-specific CD8+ T cells in rectal tissue of infected subjects.

Rare expression of perforin in rectal CD8+ T cells

The lytic effector molecules perforin and granzyme A are central to the cytotoxic function of Ag-specific CD8+ T cells. We used intracellular staining to assess expression of perforin and granzyme A by CD8+ T cells in blood and rectal tissue. Fig. 1A shows the results of intracellular staining for representative HIV-1-positive and negative patients, and results for all patients are summarized in Fig. 1B. As previously reported, blood CD8+ T cells showed differential expression of perforin and granzyme A (25, 35). In PBMC from controls, perforin was expressed by 17.1% of CD8+ T cells, while granzyme A was expressed by 46.8%. In PBMC from HIV-1-positive individuals, a similar pattern was observed: perforin was expressed by 28.1% of CD8+ T cells, while granzyme A was expressed by 44.9%. Blood CD8+ T cells from HIV-1-positive individuals showed a trend toward greater perforin expression relative to controls (p = 0.061).



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  		FIGURE 1. Rare expression of perforin in intestinal CD8+ T cells. A, Intracellular expression of perforin and granzyme A by CD8+ T cells from blood and rectal tissue of a representative HIV-1-positive subject. B, Summary of results for 10 HIV-1-positive and 5 HIV-1-negative subjects. Bar graphs indicate mean expression of perforin (top) and granzyme A (bottom) in CD8+ T cells from PBMC and rectal tissue. In both patient groups, significantly fewer CD8+ T cells expressed perforin in rectal tissue than in blood (*, p = 0.010 in HIV-1-negative individuals, Student’s t test; {dagger}, p < 0.001 in HIV-1-positive patients, Mann-Whitney rank sum test). For A, numbers indicate the percentage of CD8+ T cells in each quadrant. C, Effects of collagenase treatment on intracellular staining for perforin and granzyme A. Duplicate PBMC samples were stained without prior treatment (left) or after digesting with collagenase for 30 min at 37°C (right). Plots are gated on CD3+/CD8+ lymphocytes.

 
In striking contrast to peripheral blood, granzyme A was detected in greater than 50% of rectal CD8+ T cells in healthy subjects (mean 52.8%, n = 5); however, only 1.9% of rectal CD8+ T cells expressed perforin (Fig. 1B). A similar pattern was observed in rectal tissue from HIV-1-positive subjects: granzyme A was expressed by >50% of CD8+ T cells (mean 57.4%, n = 10), while perforin was expressed by fewer than 5% (mean 2.9%). Thus, although granzyme A was abundantly expressed in rectal CD8+ T cells from both groups, perforin expression in rectal tissue was minimal. Furthermore, expression of perforin was significantly lower in rectal CD8+ T cells relative to PBMC in both groups of subjects (p < 0.001 for HIV-1-positive subjects, Mann-Whitney U test; p = 0.010 for HIV-1-negative control subjects, Student’s t test) (Fig. 1B).

To verify that our intracellular staining procedure was not adversely affected by treatment of cells with collagenase, we performed two sets of control experiments. First, collagenase-treated PBMC from eight individuals were tested for perforin and granzyme A expression in parallel with untreated PBMC. Results of perforin and granzyme A staining were similar for collagenase-treated and untreated PBMC (Fig. 1C). Second, rectal samples from four individuals were divided such that at least five tissue fragments from each individual were subjected to mechanical disruption while the remaining fragments were treated with collagenase. Again, results were comparable for rectal tissue that had been mechanically disrupted or treated with collagenase (not shown). Similar experiments also demonstrated that perforin RNA quantification was not affected by collagenase treatment (not shown).

Granzyme A, but not perforin, is present in intracellular granules of rectal CD8+ T cells

It was important to assess perforin expression in situ, to exclude the possibility that the procedure for isolating lymphocytes from gut biopsy samples might have resulted in partial degranulation or degradation of perforin. Accordingly, we performed immunohistochemical staining of rectal tissue. As shown in Fig. 2, the frequency of CD8+ cells was greatly increased in the lamina propria of HIV-1-positive, as compared with healthy, subjects (Fig. 2, A and D, respectively). Furthermore, numerous cells in rectal tissue expressed granzyme A intracellularly (Fig. 2, B and E). However, very few cells expressed perforin (Fig. 2, C and F). Granzyme A was frequently present in cytoplasmic granules, as shown by confocal microscopy (shown in green, Fig. 2G). Furthermore, staining with mAbs to CD8 (shown in red) revealed that granzyme A was often colocalized with CD8 near the plasma membrane, suggesting active exocytosis of granzyme-positive granules. Colocalization of granzyme A and CD8 appeared as yellow staining at the cell periphery (Fig. 2H). In contrast, staining for perforin revealed only rare perforin-positive CD8+ cells, and the intracellular distribution of positively stained granules within these cells was sparse (Fig. 2I). Like granzyme A, perforin was detected either in cytoplasmic granules (green) or colocalized with CD8 at the membrane (yellow). These findings demonstrate that cytotoxic granules in intestinal CD8+ T cells express abundant granzyme A, but little perforin.



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  		FIGURE 2. Immunohistochemistry. A–C, Rectal tissue from an HIV-1-positive subject, stained brown for CD8 (A), granzyme A (B), or perforin (C). Nuclei were stained blue. D–F, Sequential sections showing staining for CD8 (D), granzyme A (E), and perforin (F) in an HIV-1-negative control. Magnification in images A–F is x190. Note the increased frequency of CD8+ cells in HIV-1-positive compared with HIV-1-negative individuals. G–I, Scanning laser confocal images of granzyme A (green; G and H), perforin (green; I), and CD8 (red; G–I) in rectal tissue of an HIV-1-positive subject. Magnification is x800 in H and I, and x3000 in G. Areas containing both granzyme and CD8 (G) or perforin and CD8 (H) appear yellow. Nucleic acid appears blue. G highlights the granular staining pattern of granzyme A.

 
HIV-1-specific CD8+ T cells in gut are perforin low

As demonstrated above, fewer than 5% of rectal CD8+ T cells expressed perforin (Fig. 1B). In a previous study of the same group of HIV-1-positive individuals, we demonstrated that as many as 1–1.5% of rectal CD8+ T cells were specific for the HIV-1 Gag SL9 epitope (26). Taken together, these observations raised the possibility that the rare perforin-expressing cells in intestinal mucosa might be HIV-1-specific. To address this issue, we assessed perforin expression of tetramer-binding, HIV-1-specific CD8+ T cells. Results for a representative HIV-1-positive subject are shown in Fig. 3. Surprisingly, the SL9-specific cells present in rectal mucosa were uniformly perforin low (mean 3.1% positive, n = 5), yet granzyme A-positive (mean 91.7% positive, n = 3). Thus, HIV-1 Gag-specific CD8+ T cells in rectal tissue contained granzyme A, but little or no perforin. In blood, as many as 23% of SL9-specific CD8+ T cells expressed perforin (mean 10.2%, n = 5) (21), and the majority expressed granzyme A (mean 75.3%, n = 3). Taken together, these results demonstrate that expression of perforin and granzyme A is not coordinately regulated in Ag-specific CD8+ T cells. Furthermore, perforin expression in CD8+ T cells of the same antigenic specificity differs between blood and mucosa.



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  		FIGURE 3. HIV-1-specific CD8+ T cells in rectal tissue express granzyme A, but little perforin. Graphs show intracellular perforin and granzyme A expression in HLA-A*0201 Gag tetramer-binding CD8+ cells from a representative HIV-1-positive patient. Samples were gated on CD8+ T cells. Numbers indicate the percentage of CD8+ T cells in each quadrant.

 
Expression of perforin RNA in intestinal CD8+ T cells is altered by HIV-1 infection

In blood, perforin expression is regulated at the level of transcription and may be induced by IL-2 and other cytokines (36, 37, 38, 39). To determine whether perforin mRNA was expressed in mucosal tissue, we performed real-time PCR analysis of perforin RNA in CD8+ T cells from rectal tissue and PBMC of two HIV-1-positive and two control subjects. Perforin RNA was detected in rectal T cells and PBMC from all four individuals (Fig. 4). When normalized to rRNA, perforin RNA in blood and rectal CD8+ T cells ranged from 2.5 to 66.2 copies/ng of rRNA (Fig. 4B). In HIV-1-negative subjects, perforin expression was particularly low in rectal tissue (<10 copies/ng RNA) as compared with blood CD8+ cells (>50 copies/ng RNA). Thus, the ratio of perforin mRNA copies (i.e., per nanogram of rRNA) in rectal T cells vs PBMC for HIV-1-negative subjects ranged from 1:6 to 1:26. In contrast, in HIV-1-positive individuals, perforin transcripts were detected at similar frequencies in blood and rectal CD8+ cells, ranging from 20 to 25 copies/ng in both compartments. Thus, the ratio of perforin mRNA copies (per nanogram of rRNA) in rectal CD8+ T cells vs PBMC for the two HIV-1-positive subjects was nearly 1:1. Strikingly, in HIV-1-positive patients, the steady state level of perforin RNA in rectal CD8+ cells was 3- to 10-fold higher than in healthy controls.



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  		FIGURE 4. Perforin RNA is transcribed in rectal CD8+ T cells. A, Gels show TaqMan PCR products amplified using primers specific for granzyme A (GyA) or perforin (Perf). Results shown are from a representative HIV-1-negative control (top) and an HIV-1-positive individual (bottom). Predicted amplicon sizes are 56 bp (perforin) and 65 bp (granzyme A). B, Perforin mRNA copies per nanogram of rRNA, as determined by quantitative RT-PCR using an in vitro transcribed perforin standard, described in the text.

 
These findings demonstrate that perforin RNA expression occurs at a low but measurable level in mucosal CD8+ T cells from healthy individuals, and at a higher level in mucosal CD8+ cells from HIV-1-infected individuals. Additional studies will be required to assess the importance of transcriptional and/or posttranscriptional regulation of perforin expression in mucosal tissues.

HIV-1-specific intestinal CD8+ T cells express IFN-{gamma} upon peptide stimulation

To determine the capacity of rectal CD8+ T cells to express IFN-{gamma}, we stimulated cells from HIV-1-positive individuals with Gag SL9 peptide (Fig. 5). Duplicate samples were stained with Gag SL9 tetramer. The fraction of tetramer-binding cells secreting IFN-{gamma} was determined by comparing results of the two assays. In the patient shown in Fig. 5A, 0.18% of blood CD8+ T cells recognized tetramer and 0.06% (corresponding to 33% of SL9-specific cells as measured by tetramer staining) produced IFN-{gamma}. In rectal tissue, 0.55% of CD8+ T cells bound tetramer and 0.26% produced IFN-{gamma}. Thus, although rectal CD8+ T cells rarely express perforin, they do secrete cytokines upon stimulation with cognate peptide. Similar results were obtained in four separate experiments with samples from three HIV-1-positive subjects. These findings demonstrate that HIV-1-specific CD8+ T cells from blood and rectal tissue are similar in their ability to produce IFN-{gamma} in response to peptide stimulation despite differences in perforin expression.



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  		FIGURE 5. Intestinal CD8+ T cells secrete IFN-{gamma} in response to peptide stimulation. A, IFN-{gamma} production by CD8+ T cells from blood and rectal tissue of a representative HIV-1-positive individual (G25) in response to stimulation with SL9 Gag peptide (top) or SEB (bottom). Numbers indicate the percent of CD8+ T cells expressing IFN-{gamma} after subtracting background. B, Relationship between Gag tetramer-binding CD8+ T cells and IFN-{gamma}-producing cells. Numbers on the y-axis indicate the percentage of CD8+ T cells that bind tetramer ({blacksquare}) or secrete IFN-{gamma} (). Percentages refer to the relationship of IFN-{gamma}-producing cells to tetramer-binding cells.

 
Maturation phenotype of intestinal CD8+ T cells

Several studies have elucidated subsets of naive, memory, and effector T cells based on expression of CD27, CD28, CD45RO/RA, CCR7, and CD62L (20, 40, 41). We tested CD8+ T cells from rectal tissue and blood of five HIV-1-positive individuals and three controls for expression of CD45RO and CCR7 (Fig. 6, A and B). In healthy subjects, the majority of rectal CD8+ T cells expressed CD45RO (mean 88.7%), but not CCR7 (mean 20.4%), consistent with a previously activated phenotype. In blood, few CD8+ T cells expressed CD45RO (mean 32.9%) but approximately half expressed CCR7 (mean 51.9%). In rectal tissue from HIV-1-positive individuals, we observed a slight reduction in CD8+ T cells expressing CD45RO (mean 72.9%), and a slight increase in cells expressing CCR7 (mean 31.0%) (Fig. 6B). We also observed an increase in naive CD8+ T cells (CD45RO–, CCR7+) (mean 14.2% vs 3.0% in controls). These observations support the notion that intestinal CD8+ T cells may experience impaired or incomplete maturation in HIV-1-positive individuals (42). Accordingly, it will be of interest to determine whether these trends are accentuated in patients with advanced disease.



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  		FIGURE 6. Rectal CD8+ T cells are primarily effector memory cells. A, Expression of CCR7 and CD45RO on CD8+ T cells from PBMC and rectal tissue of representative HIV-1-negative (top) and positive (bottom) patients. Note the distinct population of CD45RO–, CCR7+ cells in HIV-1-positive rectal tissue. B, Summary of results for five HIV-1-positive and three HIV-1-negative control individuals. Bar graphs indicate mean expression of CCR7 (top) and CD45RO (bottom) on CD8+ T cells from PBMC and rectal tissue. C, HIV-1-specific CD8+ T cells in both blood and rectal tissue are primarily CCR7–, CD45RO+. Graphs show the cell surface phenotype of HLA-A*0201 Gag tetramer-binding CD8+ cells from a representative HIV-1-positive patient. For A and C, numbers indicate the percentage of CD8+ T cells in each quadrant.

 
Assessment of the memory/effector phenotype of HIV-1-specific CD8+ T cells revealed that the majority of SL9-specific cells in both rectal tissue and blood share an effector memory phenotype, expressing CD45RO but not CCR7 (20, 41) (Fig. 6C). This finding contrasts with the earlier observation that >95% of SL9-specific cells in intestinal tissue are perforin low, while up to 23% of SL9-specific cells in blood are perforin-positive. Thus, although HIV-1-specific CD8+ T cells in these two compartments differ in expression of cytotoxic effector proteins, the two populations share a similar differentiation profile.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Perforin-dependent lysis is a major effector mechanism of CD8+ T cells (16, 43). In this study, we have demonstrated that intestinal CD8+ T cells from healthy controls and HIV-1-infected individuals, regardless of antigenic specificity, contain abundant granzyme A but little or no stored perforin. The low levels of intracellular perforin detected in mucosal CD8+ T cells by flow cytometry suggested two possible interpretations: first, intestinal CD8+ T cells might represent a highly activated population of cytotoxic T cells that have already degranulated; second, perforin gene expression might be tightly regulated at the transcriptional and/or translational level in intestinal mucosa. However, subsequent experiments, using immunohistochemical staining and confocal microscopy, revealed that intracellular granules within rectal CD8+ T cells contain abundant granzyme A, but little or no perforin. In many cases, granzyme A was detected at the cell membrane in association with CD8. This surprising observation suggested that active granule exocytosis occurs in intestinal mucosa despite a relative absence of perforin. Accordingly, regulation of perforin expression in intestinal tissue may be subject to tight transcriptional and/or posttranscriptional control.

Real-time PCR analysis of samples from healthy individuals demonstrated that rectal CD8+ cells contained 6- to 26-fold less perforin mRNA than blood CD8+ cells. However, in the two HIV-1-positive individuals studied, similar amounts of perforin mRNA were detected in CD8+ cells from rectal tissue and blood. These data, although preliminary, suggest that some perforin transcription occurs in rectal CD8+ T cells, and that the level of perforin transcription in rectal tissue is increased in response to HIV-1 infection.

Our findings suggest that expression of cytotoxic effector proteins is regulated in a tissue-specific manner, and that expression and accumulation of perforin may be subject to stringent regulatory control in mucosal tissues. Prior studies in mice have supported a role for tissue-specific regulation of perforin expression, including negative control mechanisms that may be released upon T cell activation (37, 44). The murine perforin promoter is known to contain two enhancers, one of which may respond to TCR signals (39, 45, 46). Signaling is mediated by STAT5 and can be induced by IL-2 (44, 46, 47). Members of the ETS family of transcription factors have also been implicated in regulation of perforin expression (48, 49). However, regulation of perforin and granzyme expression has not been extensively studied in human cells.

Intestinal lymphocytes are poised to interact with numerous Ags from food and bacteria, as well as autoantigens. Accordingly, constitutively low levels of perforin expression might decrease the likelihood of inappropriate immune responses. The presence of perforin-expressing CD8+ T cells in intestinal mucosa might be compared with a "loaded shotgun": an effective weapon, yet dangerous if misfired. Tight regulation of perforin expression might thus allow the establishment of a provisional "perforin-free zone", serving to protect mucosal integrity from inflammatory damage.

This model is supported by the observation that perforin expression is increased in intestinal T cells from patients with inflammatory bowel disease, and has been implicated in autoimmune tissue damage. Intraepithelial lymphocytes from patients with untreated Celiac disease express perforin and granzymes (50, 51). In active stages of ulcerative colitis and Crohn’s disease, perforin mRNA was detected in 10–60% of CD8+ lamina propria lymphocytes, and 10–40% of CD8+ intraepithelial lymphocytes, as compared with fewer than 10% of intestinal CD8+ T cells in controls (52). Previous studies suggested that expression of perforin is low in other lymphoid tissues (i.e., peripheral lymph nodes) of HIV-1-positive individuals (33). Thus, certain lymphoid tissues may maintain tight transcriptional and/or posttranscriptional control of perforin expression to protect tissue integrity and limit inappropriate cytotoxic responses.

Previous studies indicated that perforin and granzyme A are colocalized in typical secretory lysosomes of cytotoxic T cells isolated from peripheral blood (43, 53). In HIV-1 infection, granule exocytosis, rather than Fas-mediated killing, is believed to be the major mechanism used by cytotoxic CD8+ T cells to eliminate infected target cells (3, 9, 15). Several recent studies support a clinically relevant correlation between perforin expression, as measured by intracellular perforin in CD8+ T cells, and immune control of HIV-1 infection (54, 55).

The apparent absence of armed effector cells in rectal mucosa may have important implications for the local immune response to viral pathogens. The acute phase of infection with HIV-1 or SIV of macaques (SIVmac) is accompanied by rapid depletion of CD4+ T cells, and influx of CD8+ T cells in intestinal mucosa (8, 56). In rhesus macaques, significant SIV-specific CD8+ T cells are not detected in the intestine until 2–3 wk postinfection (57). Thus, as recently suggested by Pope and Haase (58), the in vivo E:T ratio may be <1 in mucosal tissues. In the absence of immune control mediated by CD8+ cytotoxic T cells, replicating virus can quickly disseminate to other tissues. If, in addition, the CD8+ T cells present in intestinal mucosa do not produce adequate levels of perforin to efficiently kill virally infected target cells, they may be further hampered in their attempt to combat early infection. Thus, mucosal CD8+ T cells may provide "too little" effector function, "too late" after infection (58).

In summary, we have demonstrated that the vast majority of CD8+ T cells in rectal tissue, including HIV-1-specific T cells, fail to express the cytolytic protein perforin. Nevertheless, these cells do express other proteins associated with granule-mediated cytotoxicity, including abundant granzyme A. Limited expression of perforin may serve as an important autoprotective mechanism to preserve the integrity of rectal mucosa under normal conditions. However, the relative paucity of armed effector cells in intestinal tissue might provide an early advantage to HIV-1 and other sexually transmitted pathogens by creating a low in vivo E:T ratio.


   Acknowledgments
 
We thank the Flow Cytometry Core Laboratory of the J. David Gladstone Institutes, directed by Marty Bigos, for assistance with flow cytometry experiments. We also thank the Virology Core Laboratory, directed by Drs. Robert Grant and Teri Liegler, for assistance with real-time PCR, and the Genomics Core Laboratory, directed by Dr. Chris Barker, for use of the Agilent Bioanalyzer. We thank Annette Hofmann for assistance with confocal microscopy. We acknowledge Dr. Peter A. Anton for insightful discussions and for sharing unpublished data. We thank Dr. Judy Lieberman for supplying mAbs to granzyme A. We are grateful to the dedicated study volunteers for their participation in this research, and to the staff of the General Clinical Research Center, San Francisco General Hospital for clinical and administrative support.


   Footnotes
 
1 This research was supported in part by the National Institutes of Health (NIH) through the UCSF-GIVI CFAR, (NIH Grant P30-AI27763), which sponsored the CFAR Clinical Core and CFAR Developmental Award 2483SC (to B.L.S.). Additional funding was provided by NIH Grants R21-AI054235 and R01-AI057020 (to B.L.S.), and by the J. David Gladstone Institutes (to D.F.N.). The General Clinical Research Center (GCRC), SFGH, was supported by NIH M01-RR00083-41. J.K.S., M.F.Q., and J.A. received support from the Swedish Foundation for Strategic Research and the Swedish Research Council. D.F.N. is an Elizabeth Glaser Scientist supported by the Elizabeth Glaser Pediatric AIDS Foundation. Back

2 Address correspondence and reprint requests to Dr. Barbara L. Shacklett at the current address: Department of Microbiology and Immunology, School of Medicine, University of California, 1 Shields Avenue, Davis, CA 95616. E-mail address: blshacklett{at}ucdavis.edu Back

3 Abbreviations used in this paper: SEB, staphylococcal enterotoxin B; MGB-NFQ, minor groove binding nonfluorescent quencher; CT, threshold cycle. Back

Received for publication November 17, 2003. Accepted for publication April 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J. Kan-Mitchell, M. Bajcz, K. L. Schaubert, D. A. Price, J. M. Brenchley, T. E. Asher, D. C. Douek, H. L. Ng, O. O. Yang, C. R. Rinaldo Jr., et al.
Degeneracy and Repertoire of the Human HIV-1 Gag p1777-85 CTL Response.
J. Immunol., June 1, 2006; 176(11): 6690 - 6701.
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M. F. Quigley, K. Abel, B. Zuber, C. J. Miller, J. K. Sandberg, and B. L. Shacklett
Perforin Expression in the Gastrointestinal Mucosa Is Limited to Acute Simian Immunodeficiency Virus Infection
J. Virol., March 15, 2006; 80(6): 3083 - 3087.
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G. Zelinskyy, S. J. Robertson, S. Schimmer, R. J. Messer, K. J. Hasenkrug, and U. Dittmer
CD8+ T-Cell Dysfunction due to Cytolytic Granule Deficiency in Persistent Friend Retrovirus Infection
J. Virol., August 15, 2005; 79(16): 10619 - 10626.
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V. Monceaux, L. Viollet, F. Petit, R. H. T. Fang, M.-C. Cumont, J. Zaunders, B. Hurtrel, and J. Estaquier
CD8+ T Cell Dynamics during Primary Simian Immunodeficiency Virus Infection in Macaques: Relationship of Effector Cell Differentiation with the Extent of Viral Replication
J. Immunol., June 1, 2005; 174(11): 6898 - 6908.
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