Abundant Expression of Granzyme A, but Not Perforin, in Granules of CD8  T Cells in GALT: Implications for Immune Control of HIV-1 Infection 1 Barbara L. Shacklett, 2 * Catherine A. Cox,* Ma ´ ire F. Quigley, § Christophe Kreis,* Neil H. Stollman, ‡ Mark A. Jacobson, †‡ Jan Andersson, § Johan K. Sandberg,* § and Douglas F. Nixon* ‡ 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- 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. The Journal of Immunology, 2004, 173: 641– 648. C ell-mediated immune responses to HIV-1 can be vigor- ous 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 orga- nized 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 ma- lignancies, 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 pro- pria (8). HIV-1 infection is characterized by significant intestinal inflammation, as evidenced by increased production of MIP-1, MIP-, 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 exocy- tosis (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 –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- (20 –23), MHC class I-restricted killing (20 –22), signal transduc- tion, 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 mole- cules. Our results suggest that rectal CD8 + T cells express gran- zyme A and secrete cytokines in response to antigenic stimulation, yet rarely express perforin. These findings demonstrate an impor- tant difference between CD8 + T cells in blood and mucosa, and *Gladstone Institute of Virology and Immunology (GIVI), † University of California San Francisco (UCSF)-GIVI and Center for AIDS Research (CFAR), San Francisco General Hospital (SFGH), and ‡ Department of Medicine, University of California, San Francisco, CA 94141; and § Center for Infectious Medicine, Department of Med- icine, Karolinska Institutet, Stockholm, Sweden Received for publication November 17, 2003. Accepted for publication April 23, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 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.). Addi- tional 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 Re- search and the Swedish Research Council. D.F.N. is an Elizabeth Glaser Scientist supported by the Elizabeth Glaser Pediatric AIDS Foundation. 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@ucdavis.edu The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00