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Nonlymphoid peritoneal cells suppress the T cell response to Mls
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Immunobiology 209 (2004) 575–584 www.elsevier.de/imbio
Nonlymphoid peritoneal cells suppress the T cell response to Mls Laura Rosini1, Robin Matlack, Justin Taylor2, Koko F. Howell, Kenneth Yeh, Anthony Pennello, James E. Riggs Department of Biology, Rider University, Lawrenceville, NJ 08648-3099, USA Received 4 May 2004; accepted 13 July 2004
Abstract Comparative analyses of the ability of lymphoid tissue to present the minor lymphocyte stimulatory (Mls) superantigen Mls-1a in vitro revealed that all tissues containing mature B cells, except peritoneal cavity (PerC) cells, induced Mls-1a-specific T cell activation. Irradiation and mitomycin C treatment, addition of IL-2 and IL-12, and neutralization of IL-10 and TGF-b did not restore Mls-1a antigen presentation by PerC cells. Co-culture studies revealed that PerC cells actively suppress the T cell response to Mls-1a. PerC cells from severe-combined immunedefective (SCID) mice also suppressed this response indicating that nonlymphoid cells mediate this effect. These results suggest that in addition to antigen processing and presentation, resident peritoneal cavity cells may temper lymphocyte activation. r 2004 Elsevier GmbH. All rights reserved. Keywords: Mls; Superantigens; Suppression
Introduction Superantigens (SAgs) induce CD4+ T lymphocyte activation and permit assessment of cellular cooperation in immunity. SAgs may be exogenous microbial proteins or gene products of retroviruses endogenous to the murine genome (e.g., the minor lymphocyte stimulatory (Mls) gene product [Mls-1a] in DBA/2J mice) (Choi et al., 1991). These molecules can activate a large portion Abbreviations: APC; Antigen presenting cell; BM; Bone marrow; LN; Lymph node; Mls; Minor lymphocyte stimulatory; PB; Peripheral blood; PerC peritoneal cavity; PP; Peyer’s patch; SAg; Superantigen; SCID; Severe-combined immune-defective; SP; Spleen; THY; Thymus. Corresponding author. E-mail address: [email protected] (J.E. Riggs). 1 Present address: Ortho-McNeil Pharmaceutical Research Institute, Skillman, NJ, USA. 2 Present address: Immunology Program, U. of Pennsylvania, Philadelphia, PA, USA. 0171-2985/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2004.07.002
of the CD4+ T cell pool by crosslinking the TCR Vb chain on Th cells with the Class II MHC molecule expressed by antigen presenting cells (APCs) (Luther and Acha-Orbea, 1997; MacDonald et al., 1988). Investigation of SAg activation of T cells has advanced understanding of T cell differentiation, tolerance, and deletion. Most recently, investigation of the role of APCs in promoting these functions in T cells has intensified (Steinman et al., 2003; Unanue, 2002). Diverse cell types present Mls. T and B lymphocytes, and several of their subsets, have been shown to present Mls, in some cases promoting T cell activation, in others anergy or deletion (Larsson-Sciard et al., 1990; Luther and Acha-Orbea, 1997; Molina et al., 1989; Riggs et al., 2004; Webb and Sprent, 1990; Webb et al., 1989). Macrophages and dendritic cells have been variously reported to present, or not present, Mls (Ardavin et al., 1996; Jarvis et al., 1994; Molina et al., 1989; Webb et al., 1989). The variability of these results is being clarified by
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a growing understanding of APC diversity (Steinman et al., 2003). Although mature APCs were thought to activate T cells and immature APCs to induce tolerance, a simple, age-dependent delineation of APC function remains dubious (Steinman et al., 2003). There is also growing comprehension of the role of APCs in the generation of regulatory lymphocytes (Fallarino et al., 2003; Groux et al., 2004). Investigation of APC heterogeneity, both among and within cell types, is essential for understanding the events that determine T cell function. In this report, experiments initially designed to determine why peritoneal cavity (PerC) cells failed to serve as Mls APCs revealed that these cells actually suppress T cell activation. These observations are discussed relative to the potential role of macrophages resident in body cavities as suppressors of lymphocyte activation.
Materials and Methods Mice Two to four-month old male and female C.B-17.scid (SCID), BALB.xid (XID), BALB/c, DBA/2J, and (BALB.xid DBA/2J [XD2J])F1 mice, bred and maintained at Rider University, were studied. All mice were handled in accord with NIH, Animal Welfare Act, and Rider University IACUC guidelines.
Preparation of cell suspensions and adoptive transfer Lymph node (LN), spleen (SP), thymus (THY), and Peyer’s patch (PP) cell suspensions were obtained by gentle disruption of the organ between the frosted ends of sterile glass slides. PerC cells were obtained by flushing the peritoneum with 10 ml warm (37 1C) HBSS supplemented with 3% FBS. Bone marrow (BM) cells were obtained by flushing femurs with sterile HBSS. Peripheral blood (PB) cells were collected by retro-orbital bleeding and washing in Alsever’s solution. RBCs were depleted by treatment by hypertonic lysis. NK cells were depleted by i.p. injections of rabbit anti-asialo GM1 antibody 24 h prior to PerC cell harvest as recommended by the manufacturer (Wako Chemical, Richmond, VA). Viable cell counts were determined by Trypan blue exclusion. Equal numbers (107) of cells were injected i.v. into the lateral tail vein of BALB/c recipients. One week after injection the recipients were sacrificed and their SP cells collected for flow cytometric analyses.
Mixed lymphocyte response (MLR) and cell culture Responder (4 106/ml) and various dilutions (0.5–4.0 106/ml) of stimulator cells, in RPMI 1640
culture media (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT), 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml gentamicin, 2 mM Lglutamine, antibiotics, 2 10 5 M 2-ME, and 10 mM HEPES, were incubated in a humidified atmosphere of 5% CO2 at 37 1C in 96 well microtiter plates (Costar, Cambridge, MA). For anti-CD3 stimulation, tissue culture plates were coated with 10 mg/ml hamster antimouse CD3e MAb (BD-Pharmingen, San Diego, CA) for 5 h and rinsed 5 times with sterile PBS prior to addition of cell preparations. Recombinant human IL-2 and murine IL-12 (PeproTech , Rocky Hill, NJ) were used as described by the supplier. In radiation experiments, SP and PerC cells, kept on ice, were subjected to 1000 rad (137Cs) in a GammaCell radiator generously shared by Bristol–Myers Squibb Pharmaceutical Research Institute, Lawrenceville, NJ as described (Kruisbeek and Shevach, 1991). Mitomycin C (Sigma Chemical, St Louis, MI) treatment was conducted as described (Kruisbeek and Shevach, 1991). The caspase inhibitors Z-DEVD-FMK and Z-VAD-FMK, (Calbiochem, San Diego, CA) were used as described by the supplier. Neutralizing MAbs to TGF-b, IL-10, IL-10R, and IL-12 (R&D Systems, Minneapolis, MN) were added at the initiation of culture. After 44 (anti-CD3) or 68 (Mls MLR) h, 1 mCi of [3H] thymidine (Amersham, Boston, MA) was added to each well. The wells were frozen 4 h after labeling, then thawed for analysis using a semi-automated cell harvester (Skatron Instruments, Richmond, VA). Radioactivity was measured by scintillation spectrometry. In each experiment, five microtiter wells were established for each test group.
Immunofluorescence staining and flow cytometric analyses Cell suspensions stained for B cell composition had titered amounts of FITC-labeled, affinity-purified goat anti-mouse IgM (Southern Biotechnology, Birmingham, AL) added or PE-labeled rat anti-mouse CD45R (B220) MAb (BD-Pharmingen, La Jolla, CA). SP cell suspensions stained for Mls-reactive T cells had titered amounts of PE-labeled mouse anti-mouse Vb6 TCR MAb (BD-Pharmingen) added concurrent with CYlabeled rat anti-mouse CD4 MAb (BD-Pharmingen). Isotype- and fluorochrome-matched, nonspecific MAb controls were employed to establish gates. The percentage of lymphocytes or myeloid cells co-expressing sets of these markers were determined via multiparameter flow cytometric analyses on a FACSCaliburTM flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) by FSC/SSC gating of the lymphoid or myeloid populations using CellQuest software.
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Statistical analyses Data sets were compared using the student’s t-test.
Results Except for PerC cells, B cell-rich lymphoid tissues present Mls in vitro B cells are the primary Mls APC in vitro (Webb et al., 1989). We recently showed that B lymphocyte subsets differ in their ability to present Mls, specifically that B-1 cells, enriched in the PerC, were less stimulatory than B-2 cells (Riggs et al., 2004). In addition, we found that PerC cells actively suppress the T cell response to Mls (Riggs et al., 2004). This observation led to a comprehensive survey of the ability of a variety of lymphoid tissues to serve as Mls APCs (Fig. 1). PP and SP cells presented Mls in a cell-dosedependent fashion; LN cells presented this SAg, but only at the highest cell concentration tested. Like PerC cells, THY, BM, and PB, were poor Mls APCs (Figs. 1A and B). However, unlike PerC cells, THY, LN, BM, and PB cells have low numbers of mature B cells (Fig. 1C). Intravenous injection of cells from these tissues led to the in vivo expansion of Vb6+ Mls–reactive CD4+ T cells in all recipients except those receiving THY cells (Fig. 2). These data show that PerC cells are unique in having significant B cell composition but poor in vitro presentation of Mls.
Irradiation, mitomycin C, and caspase inhibitor treatments fail to rescue the PerC cell Mls MLR In attempts to enhance their Mls APC function, PerC cells were irradiated or subjected to mitomycin C
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treatment. Both treatments reduced Mls presentation by SP cells and both failed to enhance presentation by PerC cells (Figs. 3A and B). To determine if apoptosis of responder T cells or stimulatory B cells was a factor in the lack of an Mls response to PerC cells, the caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK were included in the cultures. Neither of these treatments enhanced Mls APC function by PerC cells (Fig. 3C).
IL-2 and IL-12 addition fail to rescue the PerC cell Mls MLR Activation and proliferation of Th1 cells is a hallmark of the response to Mls (Gollob et al., 1993). As the generation and expansion of Th1 cells is dependent upon IL-2 and IL-12 production, these cytokines were added to PerC cell cultures to determine if the response to Mls could be recovered. The addition of IL-2 led to a modest (o3-fold) increase in the response (Fig. 4A). Addition of IL-12 reduced the T cell response to Mls as presented by PerC cells (Fig. 4B). In contrast, IL-12 enhanced the Mls response in SP cell cultures. These results illustrate that neither IL-2 or IL-12 enhance the T cell response to Mls when presented by PerC cells.
Role of suppressive cytokines in the failure of PerC cells to present Mls To determine if endogenous IL-12 production was functioning as a suppressive cytokine in PerC cell cultures, a neutralizing anti-IL-12 MAb was added. In separate cultures, antibodies that neutralize IL-10 and TGF-b, cytokines known to block Th1 activation (Gorham et al., 1998; Moore et al., 2001), were tested. Although anti-IL-10 had no effect, a modest response
Fig. 1. Mls presentation by lymphoid tissues and PerC cells in vitro. (A) Graded numbers of SP, PerC , LN, PB, PP, BM, and THY cells from (XD2J)F1 female mice were co-cultured with Mls-responsive BALB.xid LN cells as described in Methods. Each point represents the average of five wells +/ SEM. (B) Peak proliferative responses (2.0 105) of the tissues tested in (A) are listed in rank order. (C) Percent B cell composition (average [n=3] +/ SEM) of tissues listed in (A) as determined by FSC/SSC gating of the lymphoid population. Data presented are representative of three separate experiments.
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Fig. 2. Mls presentation by lymphoid tissues and PerC cells in vivo. Spleen cells from BALB/c mice reconstituted i.v. with 107 DBA/ 2J BM, SP, LN, PP, PerC, PB, or THY cells 1 week previously were subjected to FACS analyses. Data are presented as the average (n=3–5) percentage (upper histogram) or number (lower histogram) of splenic CD4+ Vb6+ T lymphocytes +/ SEM. Data presented is representative of two separate experiments.
Fig. 3. Irradiation, mitomycin C treatment, and inhibition of apoptosis fail to enhance Mls presentation by PerC cells in vitro. Each data point represents the average (n=5 wells) CPM +/ SEM. (A) Graded numbers of untreated or irradiated SP and PerC cells were co-cultured with Mls-responsive BALB.xid LN cells as described in Methods. (B) Same as (A) with mitomycin C treatment of SP and Per Cells. (C) Same as (A) with the apoptosis inhibitors Z-VAD-FMK or Z-DEVD-FMK included. Data presented are representative of three separate experiments.
(o2.5-fold), particularly at low PerC cell numbers, was recovered when an anti-IL-10R MAb was included in the culture (Fig. 5). Anti-TGF-b was modestly effective in a cell-dose dependent pattern, enhancing APC function (o1.5-fold) the best at the lowest cell dose. Anti-IL-12 was the most effective blocking antibody at the highest concentration of PerC cells (2.5-fold). For all antibodies tested, the enhancement of PerC APC function was always modest, particularly relative to responses obtained with SP cells serving as Mls APCs.
PerC cells inhibit the T cell response to Mls presented by SP cells We have recently shown that PerC cells actively suppress the SP cell-induced Mls MLR (Riggs et al., 2004). To determine if a similar, suppressive function is present in other lymphoid organs, BM, LN, THY, PP, and PB cells were tested for their ability to inhibit the SP cell-induced Mls MLR. Only PerC and BM cells could inhibit Mls-induced T cell proliferation in a cell-dosedependent fashion (Fig. 6).
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Fig. 4. IL-2 and IL-12 addition fail to recover Mls presentation by PerC cells in vitro. Each data point represents the average (n=5 wells) CPM +/ SEM. (A) Graded numbers of SP or PerC cells were cultured with Mls-responsive BALB.xid LN cells in the presence of 10, 100, or 1000 mg/ml of IL-2. (B) 1.0 105 SP or Per cells were cultured with BALB.xid LN cells in the presence of graded doses of IL-12. Data presented are representative of four separate experiments.
Nonlymphoid PerC cells from SCID mice inhibit Mls presentation by SP cells Flow cytometry was employed to provide an initial assessment of what properties BM and PerC cells might share that could explain their ability to suppress the T cell response to Mls. FSC/SSC analyses of all tissues tested for suppression revealed that BM and PerC cells had the lowest lymphoid composition (Fig. 7). To determine if mature, functional lymphocytes are necessary for suppression, PerC cells from severe-combined immune-defective (SCID) mice were tested for their ability to inhibit the SP cell-induced Mls response. The results show that SCID PerC cells, in a dose-dependent fashion, suppress the response to Mls (Fig. 8A). Evidence that NK cells are not responsible for suppression and that these observations are not solely restricted to the response to Mls is shown by PerC cell inhibition of T cells stimulated via anti-CD3 (Fig. 8B). These data illustrate that the PerC harbors a nonlymphoid cell with the capacity to suppress T cell activation.
Discussion Mls-1a is a well-characterized retroviral SAg expressed by APCs in the DBA/2J strain of mice (Choi
et al., 1991). Cells with the capacity to express or present this molecule include dendritic cells, B cells, macrophages, CD8+ T cells, and activated CD4+ T cells (Larsson-Sciard et al., 1990; Luther and Acha-Orbea, 1997; Molina et al., 1989; Riggs et al., 2004; Webb and Sprent, 1990; Webb et al., 1989). B cells are the primary Mls APC in vitro (Webb et al., 1985; 1989). In this study, comparative analyses of the ability of a variety of lymphoid tissues to present Mls-1a demonstrated that all tissues that contained significant numbers of mature B cells, except peritoneal cavity cells, induced Mls-specific T cell activation. A variety of treatments designed to facilitate APC function including irradiation, mitomycin C treatment, addition of IL-2 or IL-12, and neutralization of putative inhibitory cytokines did not enhance Mls presentation by PerC cells. Co-culture studies revealed that limiting numbers of PerC or BM cells could suppress the T cell response to Mls. That PerC cells from SCID mice depleted of NK cells were suppressive indicated that non-lymphoid cells mediate this effect. One question generated by these results is why PerC cells present Mls in vivo but not in vitro. We have previously shown that cell-sorter-purified PerC B cells present Mls in vitro and that these cells, based on antibody production and cellular proliferation, present this SAg in vivo (Riggs et al., 2004; Tocce et al., 2000). In toto these observations suggest that the cell density
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Fig. 5. Neutralization of suppressive cytokines fails to enhance Mls presentation by PerC cells. Each histogram represents the percent response of the MAb-treated PerC cell culture relative to the untreated control PerC cell response (=100%). Graded numbers of PerC cells were cultured (n=5 wells) with Mls-responsive BALB.xid LN cells in the absence (control) or presence of neutralizing MAbs to IL-10, IL-10R, IL-12, and TGF-b as described in Methods. The results presented are representative of three to six separate experiments conducted with each MAb.
created by in vitro culture could be a key factor in the suppression of T cell activation. This is evidenced by titration experiments showing the loss of suppression as the number of PerC cells is reduced (Figs. 6 and 8). The ‘‘crowding’’ of cells inherent to cell culture is unlikely to be generated after adoptive transfer. Distinct cell types will have different homing properties, particularly after the relocation of cells normally resident in a body cavity to the confines of the vascular system and its associated lymphoid tissue. This is not to infer, however, that this observation is unique to cell culture as recent work has shown that high APC density can temper immunity in vivo (Alaniz et al., 2004). What cell type is responsible for suppression? Historically, suppression has been associated with lymphocytes, most notably in the form of T suppressor (Ts) cells (Gershon and Kondo, 1970). Although the existence of Ts cells was rigorously refuted, there has been a resurgent interest in ‘‘natural suppressor’’ or
regulatory T (Tr) cells (Chatenoud et al., 2001). However, the ability of PerC cells from SCID mice to suppress T cell activation indicates that the effector cell is not a mature, functional lymphocyte. Based on anatomic location, FSC/SSC properties, CD11b and F4/80 expression, macrophages are likely the cell mediating suppression (not shown; Lagasse and Weissman, 1996). Bone marrow cells, also capable of suppression, contain a significant proportion of macrophages, particularly immature cells that exhibit antiinflammatory properties (Imhof and Aurrand-Lions, 2004; van Furth et al., 1970). Poor Mls presentation by macrophages has been postulated to be due to low or absent expression of a molecule notoriously difficult to detect (Ardavin et al., 1996; Jarvis et al., 1994; Molina et al., 1989). Such experiments could be revisited in light of macrophage suppression of the Mls response. Macrophage suppression of T cell activation has been reported in other systems (Ferrick and Herscowitz, 1981; Munn
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Fig. 6. PerC and BM cells inhibit the T cell response to Mls presented by SP cells. Graded numbers of PerC, LN, PB, PP, BM, and THY cells from DBA/2J mice were added to BALB.xid LN plus DBA/2J SP cells cultured as described in Methods. Each point represents the average of five wells +/ SEM. The data presented are representative of three separate experiments.
et al., 1999; Shimizu et al., 2004; Stevenson and Battisto, 1986). Since macrophages and dendritic cells have been shown to promote Tr cell function, the possibility that Tr cells might be generated in these cultures cannot be discounted (Groux et al., 2004; Lohr et al., 2003). Flow cytometric analyses of the myeloid cells in the PerC and of the T cells persisting in culture will allow definition of the cells mediating suppression. How do PerC cells suppress T cell activation? Potential mechanisms including the production of inhibitory cytokines or prostaglandins, by APC consumption of critical metabolites e.g., tryptophan or arginine, or by induction of apoptosis (Chace et al., 1995; Fuss et al., 2002; Munn et al., 1999; Rodriguez et al., 2003). Although IL-10 and TGF-b production by Tr cells have been shown to temper T cell activation, they do not appear to be involved with the results described here (Fig. 5). Indomethacin does not release suppression suggesting that prostaglandins are not responsible (not shown). Preliminary studies using 1-methyl tryptophan
to block indoleamine dioxygenase function suggest that tryptophan consumption and T cell apoptosis are factors in PerC cell-mediated suppression (not shown; Munn et al., 1999). The anti-CD3 stimulation system, which has shown that this effect is not restricted to Mlsspecific T cell activation, will facilitate defining how PerC cells regulate immunity. In summary, nonlymphoid cells resident in the PerC are capable of potent suppression of the in vitro T cell response. Regulation of T lymphocyte activation can be added to the list of housekeeping duties attributed to the phagocytes resident in the PerC. In addition to representing a first-line response to sporadic microbial transgressions, these cells appear to maintain a default anti-inflammatory mode, potentially coincident with their routine scavenging of apoptotic debris. Such a function is logical, particularly in sites proximal to a significant microbial burden where the potential for excessive lymphocyte activation exists (Savill et al., 2003).
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Fig. 7. Lymphocyte composition of various tissues. The average (n=5-9 tissues) percentage +/ SEM of lymphocytes, as defined by FSC/SSC gating, found in PerC, LN, PB, SP, PP, BM, and THY cell preparations was determined by flow cytometry.
Fig. 8. PerC cells from SCID mice suppress the T cell response to Mls and anti-CD3. (A) Graded numbers of DBA/2J or SCID PerC cells were added to BALB.xid LN plus DBA/2J SP cells as described in Methods. (B) Graded numbers of total SCID or NK cell-depleted SCID PerC cells were added to BALB.xid LN in wells coated with anti-CD3e as described in Methods. Each point represents the average of five wells +/ SEM. Data presented are representative of four separate experiments.
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Acknowledgements This work was supported by grants from the NIH AREA program (R15 CA77814-01, AG19631-01) and the Fannie E. Rippel Foundation. We are grateful to Faith Archer, Diana Gonzalez, and Theron Jenifer for excellent maintenance of our mouse colonies.
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Immunobiology 209 (2004) 575–584 www.elsevier.de/imbio
Nonlymphoid peritoneal cells suppress the T cell response to Mls Laura Rosini1, Robin Matlack, Justin Taylor2, Koko F. Howell, Kenneth Yeh, Anthony Pennello, James E. Riggs Department of Biology, Rider University, Lawrenceville, NJ 08648-3099, USA Received 4 May 2004; accepted 13 July 2004
Abstract Comparative analyses of the ability of lymphoid tissue to present the minor lymphocyte stimulatory (Mls) superantigen Mls-1a in vitro revealed that all tissues containing mature B cells, except peritoneal cavity (PerC) cells, induced Mls-1a-specific T cell activation. Irradiation and mitomycin C treatment, addition of IL-2 and IL-12, and neutralization of IL-10 and TGF-b did not restore Mls-1a antigen presentation by PerC cells. Co-culture studies revealed that PerC cells actively suppress the T cell response to Mls-1a. PerC cells from severe-combined immunedefective (SCID) mice also suppressed this response indicating that nonlymphoid cells mediate this effect. These results suggest that in addition to antigen processing and presentation, resident peritoneal cavity cells may temper lymphocyte activation. r 2004 Elsevier GmbH. All rights reserved. Keywords: Mls; Superantigens; Suppression
Introduction Superantigens (SAgs) induce CD4+ T lymphocyte activation and permit assessment of cellular cooperation in immunity. SAgs may be exogenous microbial proteins or gene products of retroviruses endogenous to the murine genome (e.g., the minor lymphocyte stimulatory (Mls) gene product [Mls-1a] in DBA/2J mice) (Choi et al., 1991). These molecules can activate a large portion Abbreviations: APC; Antigen presenting cell; BM; Bone marrow; LN; Lymph node; Mls; Minor lymphocyte stimulatory; PB; Peripheral blood; PerC peritoneal cavity; PP; Peyer’s patch; SAg; Superantigen; SCID; Severe-combined immune-defective; SP; Spleen; THY; Thymus. Corresponding author. E-mail address: [email protected] (J.E. Riggs). 1 Present address: Ortho-McNeil Pharmaceutical Research Institute, Skillman, NJ, USA. 2 Present address: Immunology Program, U. of Pennsylvania, Philadelphia, PA, USA. 0171-2985/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2004.07.002
of the CD4+ T cell pool by crosslinking the TCR Vb chain on Th cells with the Class II MHC molecule expressed by antigen presenting cells (APCs) (Luther and Acha-Orbea, 1997; MacDonald et al., 1988). Investigation of SAg activation of T cells has advanced understanding of T cell differentiation, tolerance, and deletion. Most recently, investigation of the role of APCs in promoting these functions in T cells has intensified (Steinman et al., 2003; Unanue, 2002). Diverse cell types present Mls. T and B lymphocytes, and several of their subsets, have been shown to present Mls, in some cases promoting T cell activation, in others anergy or deletion (Larsson-Sciard et al., 1990; Luther and Acha-Orbea, 1997; Molina et al., 1989; Riggs et al., 2004; Webb and Sprent, 1990; Webb et al., 1989). Macrophages and dendritic cells have been variously reported to present, or not present, Mls (Ardavin et al., 1996; Jarvis et al., 1994; Molina et al., 1989; Webb et al., 1989). The variability of these results is being clarified by
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a growing understanding of APC diversity (Steinman et al., 2003). Although mature APCs were thought to activate T cells and immature APCs to induce tolerance, a simple, age-dependent delineation of APC function remains dubious (Steinman et al., 2003). There is also growing comprehension of the role of APCs in the generation of regulatory lymphocytes (Fallarino et al., 2003; Groux et al., 2004). Investigation of APC heterogeneity, both among and within cell types, is essential for understanding the events that determine T cell function. In this report, experiments initially designed to determine why peritoneal cavity (PerC) cells failed to serve as Mls APCs revealed that these cells actually suppress T cell activation. These observations are discussed relative to the potential role of macrophages resident in body cavities as suppressors of lymphocyte activation.
Materials and Methods Mice Two to four-month old male and female C.B-17.scid (SCID), BALB.xid (XID), BALB/c, DBA/2J, and (BALB.xid DBA/2J [XD2J])F1 mice, bred and maintained at Rider University, were studied. All mice were handled in accord with NIH, Animal Welfare Act, and Rider University IACUC guidelines.
Preparation of cell suspensions and adoptive transfer Lymph node (LN), spleen (SP), thymus (THY), and Peyer’s patch (PP) cell suspensions were obtained by gentle disruption of the organ between the frosted ends of sterile glass slides. PerC cells were obtained by flushing the peritoneum with 10 ml warm (37 1C) HBSS supplemented with 3% FBS. Bone marrow (BM) cells were obtained by flushing femurs with sterile HBSS. Peripheral blood (PB) cells were collected by retro-orbital bleeding and washing in Alsever’s solution. RBCs were depleted by treatment by hypertonic lysis. NK cells were depleted by i.p. injections of rabbit anti-asialo GM1 antibody 24 h prior to PerC cell harvest as recommended by the manufacturer (Wako Chemical, Richmond, VA). Viable cell counts were determined by Trypan blue exclusion. Equal numbers (107) of cells were injected i.v. into the lateral tail vein of BALB/c recipients. One week after injection the recipients were sacrificed and their SP cells collected for flow cytometric analyses.
Mixed lymphocyte response (MLR) and cell culture Responder (4 106/ml) and various dilutions (0.5–4.0 106/ml) of stimulator cells, in RPMI 1640
culture media (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT), 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml gentamicin, 2 mM Lglutamine, antibiotics, 2 10 5 M 2-ME, and 10 mM HEPES, were incubated in a humidified atmosphere of 5% CO2 at 37 1C in 96 well microtiter plates (Costar, Cambridge, MA). For anti-CD3 stimulation, tissue culture plates were coated with 10 mg/ml hamster antimouse CD3e MAb (BD-Pharmingen, San Diego, CA) for 5 h and rinsed 5 times with sterile PBS prior to addition of cell preparations. Recombinant human IL-2 and murine IL-12 (PeproTech , Rocky Hill, NJ) were used as described by the supplier. In radiation experiments, SP and PerC cells, kept on ice, were subjected to 1000 rad (137Cs) in a GammaCell radiator generously shared by Bristol–Myers Squibb Pharmaceutical Research Institute, Lawrenceville, NJ as described (Kruisbeek and Shevach, 1991). Mitomycin C (Sigma Chemical, St Louis, MI) treatment was conducted as described (Kruisbeek and Shevach, 1991). The caspase inhibitors Z-DEVD-FMK and Z-VAD-FMK, (Calbiochem, San Diego, CA) were used as described by the supplier. Neutralizing MAbs to TGF-b, IL-10, IL-10R, and IL-12 (R&D Systems, Minneapolis, MN) were added at the initiation of culture. After 44 (anti-CD3) or 68 (Mls MLR) h, 1 mCi of [3H] thymidine (Amersham, Boston, MA) was added to each well. The wells were frozen 4 h after labeling, then thawed for analysis using a semi-automated cell harvester (Skatron Instruments, Richmond, VA). Radioactivity was measured by scintillation spectrometry. In each experiment, five microtiter wells were established for each test group.
Immunofluorescence staining and flow cytometric analyses Cell suspensions stained for B cell composition had titered amounts of FITC-labeled, affinity-purified goat anti-mouse IgM (Southern Biotechnology, Birmingham, AL) added or PE-labeled rat anti-mouse CD45R (B220) MAb (BD-Pharmingen, La Jolla, CA). SP cell suspensions stained for Mls-reactive T cells had titered amounts of PE-labeled mouse anti-mouse Vb6 TCR MAb (BD-Pharmingen) added concurrent with CYlabeled rat anti-mouse CD4 MAb (BD-Pharmingen). Isotype- and fluorochrome-matched, nonspecific MAb controls were employed to establish gates. The percentage of lymphocytes or myeloid cells co-expressing sets of these markers were determined via multiparameter flow cytometric analyses on a FACSCaliburTM flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) by FSC/SSC gating of the lymphoid or myeloid populations using CellQuest software.
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Statistical analyses Data sets were compared using the student’s t-test.
Results Except for PerC cells, B cell-rich lymphoid tissues present Mls in vitro B cells are the primary Mls APC in vitro (Webb et al., 1989). We recently showed that B lymphocyte subsets differ in their ability to present Mls, specifically that B-1 cells, enriched in the PerC, were less stimulatory than B-2 cells (Riggs et al., 2004). In addition, we found that PerC cells actively suppress the T cell response to Mls (Riggs et al., 2004). This observation led to a comprehensive survey of the ability of a variety of lymphoid tissues to serve as Mls APCs (Fig. 1). PP and SP cells presented Mls in a cell-dosedependent fashion; LN cells presented this SAg, but only at the highest cell concentration tested. Like PerC cells, THY, BM, and PB, were poor Mls APCs (Figs. 1A and B). However, unlike PerC cells, THY, LN, BM, and PB cells have low numbers of mature B cells (Fig. 1C). Intravenous injection of cells from these tissues led to the in vivo expansion of Vb6+ Mls–reactive CD4+ T cells in all recipients except those receiving THY cells (Fig. 2). These data show that PerC cells are unique in having significant B cell composition but poor in vitro presentation of Mls.
Irradiation, mitomycin C, and caspase inhibitor treatments fail to rescue the PerC cell Mls MLR In attempts to enhance their Mls APC function, PerC cells were irradiated or subjected to mitomycin C
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treatment. Both treatments reduced Mls presentation by SP cells and both failed to enhance presentation by PerC cells (Figs. 3A and B). To determine if apoptosis of responder T cells or stimulatory B cells was a factor in the lack of an Mls response to PerC cells, the caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK were included in the cultures. Neither of these treatments enhanced Mls APC function by PerC cells (Fig. 3C).
IL-2 and IL-12 addition fail to rescue the PerC cell Mls MLR Activation and proliferation of Th1 cells is a hallmark of the response to Mls (Gollob et al., 1993). As the generation and expansion of Th1 cells is dependent upon IL-2 and IL-12 production, these cytokines were added to PerC cell cultures to determine if the response to Mls could be recovered. The addition of IL-2 led to a modest (o3-fold) increase in the response (Fig. 4A). Addition of IL-12 reduced the T cell response to Mls as presented by PerC cells (Fig. 4B). In contrast, IL-12 enhanced the Mls response in SP cell cultures. These results illustrate that neither IL-2 or IL-12 enhance the T cell response to Mls when presented by PerC cells.
Role of suppressive cytokines in the failure of PerC cells to present Mls To determine if endogenous IL-12 production was functioning as a suppressive cytokine in PerC cell cultures, a neutralizing anti-IL-12 MAb was added. In separate cultures, antibodies that neutralize IL-10 and TGF-b, cytokines known to block Th1 activation (Gorham et al., 1998; Moore et al., 2001), were tested. Although anti-IL-10 had no effect, a modest response
Fig. 1. Mls presentation by lymphoid tissues and PerC cells in vitro. (A) Graded numbers of SP, PerC , LN, PB, PP, BM, and THY cells from (XD2J)F1 female mice were co-cultured with Mls-responsive BALB.xid LN cells as described in Methods. Each point represents the average of five wells +/ SEM. (B) Peak proliferative responses (2.0 105) of the tissues tested in (A) are listed in rank order. (C) Percent B cell composition (average [n=3] +/ SEM) of tissues listed in (A) as determined by FSC/SSC gating of the lymphoid population. Data presented are representative of three separate experiments.
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Fig. 2. Mls presentation by lymphoid tissues and PerC cells in vivo. Spleen cells from BALB/c mice reconstituted i.v. with 107 DBA/ 2J BM, SP, LN, PP, PerC, PB, or THY cells 1 week previously were subjected to FACS analyses. Data are presented as the average (n=3–5) percentage (upper histogram) or number (lower histogram) of splenic CD4+ Vb6+ T lymphocytes +/ SEM. Data presented is representative of two separate experiments.
Fig. 3. Irradiation, mitomycin C treatment, and inhibition of apoptosis fail to enhance Mls presentation by PerC cells in vitro. Each data point represents the average (n=5 wells) CPM +/ SEM. (A) Graded numbers of untreated or irradiated SP and PerC cells were co-cultured with Mls-responsive BALB.xid LN cells as described in Methods. (B) Same as (A) with mitomycin C treatment of SP and Per Cells. (C) Same as (A) with the apoptosis inhibitors Z-VAD-FMK or Z-DEVD-FMK included. Data presented are representative of three separate experiments.
(o2.5-fold), particularly at low PerC cell numbers, was recovered when an anti-IL-10R MAb was included in the culture (Fig. 5). Anti-TGF-b was modestly effective in a cell-dose dependent pattern, enhancing APC function (o1.5-fold) the best at the lowest cell dose. Anti-IL-12 was the most effective blocking antibody at the highest concentration of PerC cells (2.5-fold). For all antibodies tested, the enhancement of PerC APC function was always modest, particularly relative to responses obtained with SP cells serving as Mls APCs.
PerC cells inhibit the T cell response to Mls presented by SP cells We have recently shown that PerC cells actively suppress the SP cell-induced Mls MLR (Riggs et al., 2004). To determine if a similar, suppressive function is present in other lymphoid organs, BM, LN, THY, PP, and PB cells were tested for their ability to inhibit the SP cell-induced Mls MLR. Only PerC and BM cells could inhibit Mls-induced T cell proliferation in a cell-dosedependent fashion (Fig. 6).
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Fig. 4. IL-2 and IL-12 addition fail to recover Mls presentation by PerC cells in vitro. Each data point represents the average (n=5 wells) CPM +/ SEM. (A) Graded numbers of SP or PerC cells were cultured with Mls-responsive BALB.xid LN cells in the presence of 10, 100, or 1000 mg/ml of IL-2. (B) 1.0 105 SP or Per cells were cultured with BALB.xid LN cells in the presence of graded doses of IL-12. Data presented are representative of four separate experiments.
Nonlymphoid PerC cells from SCID mice inhibit Mls presentation by SP cells Flow cytometry was employed to provide an initial assessment of what properties BM and PerC cells might share that could explain their ability to suppress the T cell response to Mls. FSC/SSC analyses of all tissues tested for suppression revealed that BM and PerC cells had the lowest lymphoid composition (Fig. 7). To determine if mature, functional lymphocytes are necessary for suppression, PerC cells from severe-combined immune-defective (SCID) mice were tested for their ability to inhibit the SP cell-induced Mls response. The results show that SCID PerC cells, in a dose-dependent fashion, suppress the response to Mls (Fig. 8A). Evidence that NK cells are not responsible for suppression and that these observations are not solely restricted to the response to Mls is shown by PerC cell inhibition of T cells stimulated via anti-CD3 (Fig. 8B). These data illustrate that the PerC harbors a nonlymphoid cell with the capacity to suppress T cell activation.
Discussion Mls-1a is a well-characterized retroviral SAg expressed by APCs in the DBA/2J strain of mice (Choi
et al., 1991). Cells with the capacity to express or present this molecule include dendritic cells, B cells, macrophages, CD8+ T cells, and activated CD4+ T cells (Larsson-Sciard et al., 1990; Luther and Acha-Orbea, 1997; Molina et al., 1989; Riggs et al., 2004; Webb and Sprent, 1990; Webb et al., 1989). B cells are the primary Mls APC in vitro (Webb et al., 1985; 1989). In this study, comparative analyses of the ability of a variety of lymphoid tissues to present Mls-1a demonstrated that all tissues that contained significant numbers of mature B cells, except peritoneal cavity cells, induced Mls-specific T cell activation. A variety of treatments designed to facilitate APC function including irradiation, mitomycin C treatment, addition of IL-2 or IL-12, and neutralization of putative inhibitory cytokines did not enhance Mls presentation by PerC cells. Co-culture studies revealed that limiting numbers of PerC or BM cells could suppress the T cell response to Mls. That PerC cells from SCID mice depleted of NK cells were suppressive indicated that non-lymphoid cells mediate this effect. One question generated by these results is why PerC cells present Mls in vivo but not in vitro. We have previously shown that cell-sorter-purified PerC B cells present Mls in vitro and that these cells, based on antibody production and cellular proliferation, present this SAg in vivo (Riggs et al., 2004; Tocce et al., 2000). In toto these observations suggest that the cell density
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Fig. 5. Neutralization of suppressive cytokines fails to enhance Mls presentation by PerC cells. Each histogram represents the percent response of the MAb-treated PerC cell culture relative to the untreated control PerC cell response (=100%). Graded numbers of PerC cells were cultured (n=5 wells) with Mls-responsive BALB.xid LN cells in the absence (control) or presence of neutralizing MAbs to IL-10, IL-10R, IL-12, and TGF-b as described in Methods. The results presented are representative of three to six separate experiments conducted with each MAb.
created by in vitro culture could be a key factor in the suppression of T cell activation. This is evidenced by titration experiments showing the loss of suppression as the number of PerC cells is reduced (Figs. 6 and 8). The ‘‘crowding’’ of cells inherent to cell culture is unlikely to be generated after adoptive transfer. Distinct cell types will have different homing properties, particularly after the relocation of cells normally resident in a body cavity to the confines of the vascular system and its associated lymphoid tissue. This is not to infer, however, that this observation is unique to cell culture as recent work has shown that high APC density can temper immunity in vivo (Alaniz et al., 2004). What cell type is responsible for suppression? Historically, suppression has been associated with lymphocytes, most notably in the form of T suppressor (Ts) cells (Gershon and Kondo, 1970). Although the existence of Ts cells was rigorously refuted, there has been a resurgent interest in ‘‘natural suppressor’’ or
regulatory T (Tr) cells (Chatenoud et al., 2001). However, the ability of PerC cells from SCID mice to suppress T cell activation indicates that the effector cell is not a mature, functional lymphocyte. Based on anatomic location, FSC/SSC properties, CD11b and F4/80 expression, macrophages are likely the cell mediating suppression (not shown; Lagasse and Weissman, 1996). Bone marrow cells, also capable of suppression, contain a significant proportion of macrophages, particularly immature cells that exhibit antiinflammatory properties (Imhof and Aurrand-Lions, 2004; van Furth et al., 1970). Poor Mls presentation by macrophages has been postulated to be due to low or absent expression of a molecule notoriously difficult to detect (Ardavin et al., 1996; Jarvis et al., 1994; Molina et al., 1989). Such experiments could be revisited in light of macrophage suppression of the Mls response. Macrophage suppression of T cell activation has been reported in other systems (Ferrick and Herscowitz, 1981; Munn
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Fig. 6. PerC and BM cells inhibit the T cell response to Mls presented by SP cells. Graded numbers of PerC, LN, PB, PP, BM, and THY cells from DBA/2J mice were added to BALB.xid LN plus DBA/2J SP cells cultured as described in Methods. Each point represents the average of five wells +/ SEM. The data presented are representative of three separate experiments.
et al., 1999; Shimizu et al., 2004; Stevenson and Battisto, 1986). Since macrophages and dendritic cells have been shown to promote Tr cell function, the possibility that Tr cells might be generated in these cultures cannot be discounted (Groux et al., 2004; Lohr et al., 2003). Flow cytometric analyses of the myeloid cells in the PerC and of the T cells persisting in culture will allow definition of the cells mediating suppression. How do PerC cells suppress T cell activation? Potential mechanisms including the production of inhibitory cytokines or prostaglandins, by APC consumption of critical metabolites e.g., tryptophan or arginine, or by induction of apoptosis (Chace et al., 1995; Fuss et al., 2002; Munn et al., 1999; Rodriguez et al., 2003). Although IL-10 and TGF-b production by Tr cells have been shown to temper T cell activation, they do not appear to be involved with the results described here (Fig. 5). Indomethacin does not release suppression suggesting that prostaglandins are not responsible (not shown). Preliminary studies using 1-methyl tryptophan
to block indoleamine dioxygenase function suggest that tryptophan consumption and T cell apoptosis are factors in PerC cell-mediated suppression (not shown; Munn et al., 1999). The anti-CD3 stimulation system, which has shown that this effect is not restricted to Mlsspecific T cell activation, will facilitate defining how PerC cells regulate immunity. In summary, nonlymphoid cells resident in the PerC are capable of potent suppression of the in vitro T cell response. Regulation of T lymphocyte activation can be added to the list of housekeeping duties attributed to the phagocytes resident in the PerC. In addition to representing a first-line response to sporadic microbial transgressions, these cells appear to maintain a default anti-inflammatory mode, potentially coincident with their routine scavenging of apoptotic debris. Such a function is logical, particularly in sites proximal to a significant microbial burden where the potential for excessive lymphocyte activation exists (Savill et al., 2003).
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Fig. 7. Lymphocyte composition of various tissues. The average (n=5-9 tissues) percentage +/ SEM of lymphocytes, as defined by FSC/SSC gating, found in PerC, LN, PB, SP, PP, BM, and THY cell preparations was determined by flow cytometry.
Fig. 8. PerC cells from SCID mice suppress the T cell response to Mls and anti-CD3. (A) Graded numbers of DBA/2J or SCID PerC cells were added to BALB.xid LN plus DBA/2J SP cells as described in Methods. (B) Graded numbers of total SCID or NK cell-depleted SCID PerC cells were added to BALB.xid LN in wells coated with anti-CD3e as described in Methods. Each point represents the average of five wells +/ SEM. Data presented are representative of four separate experiments.
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Acknowledgements This work was supported by grants from the NIH AREA program (R15 CA77814-01, AG19631-01) and the Fannie E. Rippel Foundation. We are grateful to Faith Archer, Diana Gonzalez, and Theron Jenifer for excellent maintenance of our mouse colonies.
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