Abstract
TNF-TNFR2 interactions promote MHC class II-stimulated alloresponses while TNF-TNFR1 interactions promote MHC class I-stimulated alloresponses. The present studies were designed to evaluate whether TNF-TNFR2 interactions were involved in the in vivo generation of CD4+ T cell-mediated intestinal graft-versus-host disease (GVHD) in the (C57BL/6J (hereafter called B6)→B6 × B6.C-H-2bm12 (bm12))F1 GVHD model. Briefly, 5 × 106 splenic CD4+ T lymphocytes from B6.TNFR2−/− or control B6 mice were transferred with 1–2 × 106 T cell-depleted B6 bone marrow cells (BMC) to irradiated MHC class II-disparate (bm12 × B6)F1 mice. Weight loss, intestinal inflammation, and the surface expression of CD45RB (memory marker) on intestinal and splenic lymphocytes were assessed. IL-2 and IFN-α mRNA levels in intestinal lymphocytes were assessed by nuclease protection assays. A significant reduction in weight loss and intestinal inflammation was observed in recipients of the TNFR2−/−CD4+ SpC. Similarly, a significant decrease was noted in T cell numbers and in CD45RBlow (activated/memory) expression on intestinal but not CD4+ T cells in recipients of TNFR2−/−CD4+ spleen cells. IL-2 and IFN-α mRNA levels were reduced in the intestine in the recipients of TNFR2−/− splenic CD4+ T cells. These results indicate that TNF-TNFR2 interactions are important for the development of intestinal inflammation and activation/differentiation of Th1 cytokine responses by intestinal lymphocytes in MHC class II-disparate GVHD while playing an insignificant role in donor T cell activation in the spleen.
Allogeneic bone marrow transplantation (BMT) is now accepted as the treatment of choice for adults with chronic myeloid leukemia and in adults and children with acute myeloid leukemia and acute lymphoid leukemia. A major complication of BMT is graft-vs-host disease (GVHD). Gastrointestinal involvement is a major cause of morbidity and mortality in human GVHD (1). Animal models of GVHD have been used in determining the pathogenesis of intestinal GVHD (2, 3). In many murine models of intestinal GHVD, including the (DBA/2J (hereafter called B6D2F)1→B6D2)F1 GVHD model, CD4+ T cells play a prominent role in the pathogenesis (2, 3).
TNF-TNFR interactions appear to be important in the development of GVHD (4, 5, 6, 7). In previous studies, TNF blockade has been noted to ameliorate the development of intestinal GVHD in the DBA→B6D2F1 MHC class I- and II-disparate model (6) in which TNF and its family members appear to influence intestinal inflammation induced by GVHD by promoting a Th1 cytokine profile (IL-2, IFN-γ) (8). Additional studies have suggested that TNF signaling through the 55-kDa TNFR1 expressed on both donor and host cells is important in the initiation of GVHD (4, 5). For example, B6D2F1 recipients of TNFR1-deficient B6 spleen cells (SpC) have significantly reduced morbidity and mortality compared with recipients of control B6 or TNFR2-deficient (B6.TNFR2−/−) donor SpC in the B6→B6D2F1 MHC class I- plus II-disparate murine GVHD model (4). Moreover, in early acute GVHD induced by transfer of donor SpC and bone marrow cells (BMC) into fully MHC-disparate recipients deficient in the TNF-p55-kDa (TNFR1) receptor, there was an attenuation of lethal GVHD when compared with normal recipients, but no effect on development of intestinal GVHD (5). In contrast to studies implicating TNFR-TNFR1 interactions in selected aspects of GVHD pathogenesis in MHC class I- and II-disparate GVHD models, the 75-kDa TNFR (TNFR2) has not been implicated in the evolution of GVHD. However, in vitro, TNF-TNFR2 interactions have been found to promote MHC class II-stimulated T cell alloresponses while TNF-TNFR1 interactions promoted MHC class I-stimulated T cell alloresponses (9).
The purpose of these studies were to determine whether the expression of the 75-kDa TNFR2 located on donor CD4+ T cells is important for the initiation and progression of MHC class II-disparate intestinal GVHD. In brief, the results demonstrate that TNF-TNFR2 interactions on donor CD4+ T cells are important in the pathogenesis of the wasting associated with acute GVHD and in the generation of histopathological lesions of intestinal GVHD. Furthermore, reduced severity of GVHD generated by transfer of TNFR2-deficient donor CD4+ T cells is associated with diminished intestinal T cell activation and reduced intestinal Th1 cytokine responses.
Materials and Methods
Mice
C57BL/6J and B6.C-H-2bm12 (bm12) and C57BL/6-Tnfr2tm1Mwm (B6.TNFR2−/−) were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free environment. B6 males and bm12 females were bred to produce an F1 strain (B6 × bm12F1).
Transplantation
On the day of transplantation, the age- and sex-matched B6 × bm12F1 (H-2b/bm12) recipients were irradiated (900 cGy) and 2 h later were injected via the lateral tail vein with donor cells. Briefly, 4–5 × 106 CD4+ T cell-enriched SpC and 2–3 × 106 BMC from age- and sex-matched control B6 mice or B6.TNFR2−/− mice were injected into the lateral tail vein of these lethally irradiated allogeneic B6 × bm12F1 mice as previously described (1).This transplantation regimen has been documented previously to result in complete chimerism in this strain combination (10). In other experiments, 4 × 106 splenic CD4+ T lymphocytes from B6.TNFR2−/− and control B6 mice were transferred with 1–2 × 106 T cell-depleted B6 BMC to irradiated MHC class II-disparate B6 × bm12F1 mice. In other experiments, 4–5 × 106 splenic CD4 T lymphocytes from control B6 mice were transferred with 1–2 × 106 T cell-depleted B6 or B6.TNFR2−/− BMC to irradiated MHC class II-disparate B6 × bm12F1 mice. Recipients were maintained on acidified (pH 2) antibiotic (neomycin, 100 mg/L, and polymyxin B, 10 mg/L) H2O for 7 days after transplantation. Splenic B6 or B6.TNFR2−/−CD4+ T cells were enriched as previously described (1). Briefly, SpC were incubated with anti-CD8 Abs, anti-RBC, and B cell Abs (J11D.2) for 30 min at 4°C, then incubated with an adsorbed rabbit complement at 37°C for 30 min and then passed over a nylon wool column (11).
Histological scoring of intestinal GVHD
Individual recipients of either the control B6 or the experimental B6.TNFR2−/− SpC were evaluated according to the scoring system used by Snover (12) and Bottomly et al. (13). The large intestine was isolated by removing all of the intestine distal to the appendix and 2-cm sections were cut, fixed in Formalin, and then stained with H&E. Peyer’s patches were removed from the small intestine and then 2-cm sections from the entire small intestine were cut from the duodenum to the ileum, fixed in Formalin, and stained with H&E. Specifically, intestinal GVHD was reported as grade I with evidence of increased apoptosis, grade II with evidence of cystically dilated crypts containing necrotic debris with individual crypt loss, grade III with loss of multiple crypts with preservation of surface epithelium, and grade IV with complete loss of epithelium (data not observed in this study). The pathologist reviewing the slides was not aware of the types of SpC that the mouse had received.
Preparation of lamina propria lymphocytes
Mucosal lymphocytes were isolated and prepared according to a modification of a previously published method (8, 14, 15). Large intestines and small intestines were removed and placed in 15-ml tubes containing HBSS without Ca2+ and Mg2+ (Life Technologies, Gaithersburg, MD). Each intestine was carefully cleaned and flushed of its fecal content. Peyer’s patches were removed from the small intestines. The small and the large intestines were opened longitudinally and then cut into six 0.5-cm pieces. To isolate the lamina propria lymphocytes (LPL), the intestinal tissue was vigorously pipetted, transferred to 17 × 100-mm polypropylene tubes, and shaken for 20 min at 37°C in complete RPMI 1640 supplemented with 1% FCS containing dispase at 1.5 mg/ml (Sigma-Aldrich, St. Louis, MO). The tissue suspensions were collected and passed through nylon mesh, and the cells were pelleted by centrifugation at 600 × g. The cells were layered over Ficoll-Hypaque and the lymphocytes were removed from the interface, resuspended in staining buffer (0.5 L HBSS, 0.5 L PBS, 2% BSA, and 0.02% sodium azide), and counted using a Coulter counter (8).
Isolation of SpC
SpC were harvested by surgically removing the spleen and then mincing and disrupting the spleen over a funnel covered with nylon mesh and washing repeatedly with HBSS into a 50-ml conical tube (11). After the suspension was centrifuged at 600 × g for 10 min, the cell pellet was resuspended in staining buffer (0.5 L HBSS, 0.5 L PBS, 2% BSA, and 0.02% sodium azide) and cells were counted using a Coulter counter.
Flow cytometric analysis of splenic and intestinal lymphocytes
LPL were obtained from individual mice and in some experiments LPL from two to three mice from the same experimental group were pooled before flow cytometric analysis. Since we had six to nine mice in each experimental group, we were able to prepare two to three pools from each experimental group for analysis. These pooled lymphocytes were suspended in staining buffer (0.5 L HBSS, 0.5 L PBS, 2% BSA, and 0.02% sodium azide). mAb used to characterize LPL populations included fluorescein-conjugated anti-CD3ε clone 145-2C11, biotinylated anti-CD4 clone RM4-4, FITC anti-CD8α clone 53-6.7, FITC anti-CD45RB clone 16A, and PE anti-CD4 Ab (all from BD PharMingen, San Diego, CA). mAb were added to cell suspensions at 4°C and incubated for 20 min. After staining with the primary Ab, samples were washed in PBS at 4°C. Avidin fluorescein was added as a secondary staining reagent for the biotinylated mAb. In some experiments, the cells were doubled stained with PE anti-CD4 Ab and FITC anti-CD45 Ab. After mAb staining and washing, all samples were fixed in PBS containing 1% paraformaldehyde and stored at 4°C until flow cytometric analysis as previously described (11). Flow cytometric analysis was performed on a FACscan flow cytometer (BD Biosciences, Mountain View, CA).
Multiprobe RNase protection assay
A multiprobe RNase protection assay system (Riboquant; BD PharMingen) that generated a series of a cytokine templates (IL-4, IL-10, Il-13, IL-15, IL-9, IL-2, IFN-γ, and GAPDH standard), each of distinct length and each representing a sequence in a distinct cytokine mRNA species, was used in analyzing small and large intestine LPL RNA from B6 × bm12F1 mice that had received the control B6 CD4+ T lymphocytes and from mice that had received the B6.TNFR2−/−CD4+ T lymphocytes. The mouse cytokine templates utilized a T7 polymerase-directed synthesis of a high specific activity 32P-labeled antisense RNA probe set. The probe set was hybridized in excess to target small and large intestinal LPL RNA in solution, after which free probe and other ssRNA were digested with RNases (8). The remaining RNase-protected probes were purified, resolved on polyacrylamide gels, and quantified by phosphor imaging.
Statistical analysis
The results were analyzed by the Mann Whitney U rank sum nonparametric test.
Results
Absence of TNFR2 interactions on donor CD4+ T cells ameliorates MHC class II-disparate intestinal GVHD induced by the transfer of B6 CD4+ SpC into irradiated B6 × bm12F1 recipients
Previous publications have reported that intestinal GVHD is present after transfer of B6 SpC into lethally irradiated MHC class II-disparate B6 × bm12F1 recipients (10) and that TNF-TNFR2 interactions promote MHC class II-stimulated T cell alloresponses (9). To assess whether the wasting disease induced by B6→B6 × bm12F1 GVHD is dependent upon TNF-TNFR2 interactions on donor T cells, weights were assessed 11–14 days after transplantation. In the initial experiments, B6 CD4+ T cells and BMC and B6.TNFR2−/−CD4+ T cells and BMC were transferred to B6 × bm12F1 mice. A significant weight difference was noted 14 days after transplantation in recipients of B6.TNFR2−/− donor cells (15.3 +/− 1.22 g; n = 3) vs recipients of control B6 donor cells (11.95 +/− 1.5 g; n = 3). The initial weights of the animals were not different.
To assess whether the weight difference was associated with TNFR2 deficiency on the donor T cells or donor BMC, B6 CD4+ T cells or B6.TNFR2−/−CD4+ T cells were transferred with B6 T cell-depleted BMC. A significant weight difference was noted between the recipients of the B6.TNFR2−/−CD4+ SpC (20.33 ± 2.67 g (n = 6)) in which no significant decline from pretransplant weight was observed vs recipients of B6 CD4+ T cells (mean weight, 14.64 ± 3.53 g (n = 7)) in which an average of 10.82% weight loss was observed. (Fig. 1⇓).
Day 14 weights of the B6 × bm12F1 recipients of B6 CD4+ donor T cells were significantly lower than weights of recipients of B6.TNFR2−/−CD4+ donor T cells. Briefly, 4 × 106 splenic CD4+ T lymphocytes from B6.TNFR2−/− (n = 7) or control B6 mice (n = 7) were transferred with 2–3 × 106 B6 BMC to irradiated MHC class II-disparate B6 × bm12F1 mice. Weights were assessed at the indicated time point.
Furthermore, there were histological differences in the intestines of recipients of the B6.TNFR2−/− donor CD4+ T cells vs recipients of the B6 donor CD4+ T cells. The intestinal GVHD in B6 × bm12F1 recipients of control B6 donor CD4+ T cells was manifested by evidence of apoptosis, cystically dilated crypts containing necrotic debris with individual crypt loss, loss of multiple crypts with preservation of surface epithelium, and complete loss of epithelium in the large intestine (Fig. 2⇓). More of the large intestines isolated from the B6 × bm12F1 recipients of the B6 CD4+ T cells had evidence of grade II or higher GVHD (7 of 10) than recipients of the B6.TNFR2−/−CD4+ T cells (2 of 9; Fig. 3⇓). Histological changes in the B6→B6 × bm12F1 mice were clearly related to allogeneic responses as the large intestines of the syngeneic controls had no GVHD changes. These results indicate that TNF-TNFR2 interactions on alloreactive donor CD4+ T cells are important for the development of both intestinal GVHD and the wasting disease associated with GVHD in B6 →B6 × bm12F1 mice.
Histological appearance of colons from B6 × bm12F1 mice that had received either the control B6 CD4+ T cells or B6.TNFR2−/− CD4+ T cells. Histological features of grade I GVHD, apoptosis of crypt cells (filled arrow), were observed in the colonic tissue (A) obtained 9–15 days after transplantation in recipients of both the control B6 and the TNFR2−/−CD4+ T cells. Colonic tissue obtained 10–15 days after transplant of the control B6 CD4+ T cells displayed (B) cystically dilated crypt containing necrotic debris (filled arrow), partially destroyed crypt (arrowhead), and individual crypt loss (open arrow; grade 2 GVHD) as well as areas of (C) multiple crypts loss (filled arrow) with preservation of surface epithelium (grade 3 GVHD). Note normal crypt for comparison (open arrow).
Semiquantitative scoring of intestinal GVHD. Colonic tissue was harvested 9–15 days after transplantation from 10 recipients of control B6 CD4+ splenic T cells, 9 recipients of B6.TNFR2−/−CD4+ splenic T cells, and 3 recipients of syngeneic donor cells (two B6→B6, one B6 × bm12F1). The colonic tissue was fixed, sectioned, stained with H&E, and scored as detailed in Materials and Methods.
Absence of TNFR2 interactions on T cell-depleted BMC does not ameliorate MHC class II-disparate intestinal GVHD induced by the transfer of B6+CD4+ SpC and BMC into irradiated bm12 × B6F1 recipients
To determine whether lack of TNFR2-TNF interactions on non-T cell-derived donor BMC were important for the development of GVHD, 4–5 × 106 splenic CD4 T lymphocytes from control B6 mice were transferred with 1–2 × 106 T cell-depleted B6 or TNFR2−/− BMC to irradiated MHC class II-disparate B6 × bm12F1 mice. Twelve days after transplant, weights of recipients of B6 CD4+ T cells and either TNFR2−/− T cell-depleted BMC or B6 T cell-depleted BMC were assessed. The weights were similar for the TNFR2−/− BMC recipients (mean, 18.3 g (n = 3) (15.9% decrease) vs control B6 recipients (21.65 g (n = 2) (7.25% decrease). Intestinal histopathology also was similar in both groups (data not shown).
Fewer T cells were isolated from the small intestine in recipients of the B6.TNFR2−/−CD4+ T cells than from the recipients of the B6 CD4+ T cells
In addition to the analysis of colonic GVHD detailed in Fig. 3⇑, there was evidence of loss of villous architecture and gland drop-out in the small intestines of B6 →B6 × bm12F1 GVHD mice (Fig. 4⇓). To assess whether recipients of the B6.TNFR2−/−CD4+ T cells had lesser degrees of T cell infiltrates in the intestine than in the recipients of the control B6 CD4+ T cells, intestinal lymphocytes were removed from the small intestines of B6 × bm12F1 recipients. Of interest, recipients of the B6 CD4+ T cells had a large variance in intestinal lymphocyte counts (3.3 ± 3.7 × 106) while the recipients of the TNFR2−/−CD4+ T cells were consistently low (0.67 ± 0.59 × 106). The recipients of the syngeneic donor cells had counts similar to those of the recipients of TNFR2−/− donor cells (0.6 ± 0.57). To determine the absolute number of CD3 and CD4+ T cells, these counts were multiplied by the percentage of cells that stained positive with anti-CD3 and anti-CD4 Abs. As demonstrated by the data displayed in Table I⇓, significantly fewer CD3+ and CD4+ T cells were obtained from the B6 × bm12F1 recipients of the B6.TNFR2−/−CD4+ T cells than from recipients of the B6 CD4+ T cells ,whereas recipients of syngeneic donor cells had even fewer CD3 and CD4+ T cells isolated. These data indicate that TNF-TNFR2 interactions are critical for the infiltration and expansion of the CD4+ T cells in the small intestine.
Photomicrograph illustrating the severity of the small intestinal disease in the B6 × bm12F1 recipients of the control B6 CD4+ T cells. The small intestine was harvested from mice that received control B6 CD4+ T cells and B6 BMC. This is a representative photomicrograph of a section displaying loss of villous architecture and gland drop-out (A). In contrast, recipients of TNFR2−/−CD4+ T cells demonstrated normal villous architecture and no gland drop-out (B).
Total cell count and absolute CD3 and CD4 T cell count from small intestine of B6 × bm12F1 recipients
Fewer CD45RBlow CD3+ T cells are observed in the small intestines of the recipients of B6.TNFR2−/−CD4+ T cells
The level of expression of the CD45RB surface marker on T cells has been reported to be associated with prior T cell activation (memory T cells) (14, 15, 16, 17). Specifically, high levels of the CD45RB surface marker has been observed on T cells that have no prior exposure to Ags (naive), whereas those T cells with low levels of this marker on their surface have prior exposure to Ags (activated or memory cells). Intestinal LPL were isolated as described in Materials and Methods. FACS analysis revealed that >75% of these intestinal LPL were CD4+ T cells. The percentage of CD45RBlowCD4+ T cells in B6 × bm12F1 recipients of the B6 CD4+ T cells and of the B6.TNFR2−/−CD4+ T cells was examined to determine whether the lack of B6.TNFR2−/− on donor CD4+ T cells affected the percentage of memory T cells in the lamina propria of GVHD small intestines. A lower percentage and absolute number of CD4+CD45RBlow memory T cells was present in the recipients of the B6.TNFR2−/−CD4+ T cells (Fig. 5⇓, A and B), suggesting that TNFR2-TNF interactions on donor CD4+ T cells may be necessary for alloantigen-induced activation and/or memory T cell development in this model of intestinal GVHD.
A decreased percentage of CD45RBlow T cells was observed in small intestinal lymphocytes from B6 × bm12F1 recipients of the TNFR2−/− donor CD4+ splenic T cells. Small intestinal lymphocytes were harvested between day 8 and 14 after transplantation from B6 × bm12F1 recipients of the TNFR2−/− donor CD4+ T cells (n = 4) or B6 donor CD4+ T cells (n = 4). The histogram on the left of A displays CD45RB staining of LPL from a representative mouse that had received the B6 CD4+ donor T cells, and the histogram on the right displays CD45RB staining of LPL from a representative mouse that had received the TNFR2−/−CD4+ donor T cells. In B are displayed the percentage and the absolute number of intestinal lymphocytes (mean ± SEM) expressing low levels of CD45RB (memory).
There were no differences in splenic CD3+ T cell number or CD45RB expression in recipients of the B6.TNFR2−/−CD4+ donor T cells vs control B6 CD4+ donor T cells
To determine whether TNF-TNFR2 interactions were deterring the expansion or differentiation of donor T cells at extraintestinal sites, such as the spleen, total SpC counts were measured from mice that had received the B6.TNFR2−/−CD4+ T cells and mice that had received the control B6 SpC. Eleven to 14 days after transplant, similar number of cells (5.5 × 106 (n = 12 vs (6.47 × 106 (n = 12; Fig. 6⇓) and of CD3+ T lymphocytes (Table II⇓) were obtained from the spleens isolated from the recipients of the control B6 or B6. TNFR2−/−CD4+ donor T cells (p = 0.56). These results suggest that TNF-TNFR2 interactions on donor CD4+ T cells are not affecting the migration and expansion of donor T cell in the spleens of lethally irradiated B6 × bm12F1 mice.
Similar number of T cells are observed in the spleens isolated from the B6 × bm12F1 recipients of B6 or B6.TNFR2−/− donor CD4+ T cell. Briefly, 4 × 106 splenic CD4+ T cells from TNFR2−/− (n = 11) or control B6 mice (n = 11) were transferred with 2–3 × 106 control B6 BMC to irradiated MHC class II-disparate B6 × bm12F1 mice. Cells from recipient spleens were obtained on days 11–14 after BMT and counted by Coulter counter.
Absolute CD3 and CD4 T cell count from spleen of B6 × bm12F1 recipients
CD45RB surface expression on splenic CD4+ T cells was also not affected by lack of TNFR2-TNF interactions on donor CD4+ T cells. The majority of the splenic CD4+ T cells exhibited the CD45RBlow memory phenotype. In the spleen, there were similar numbers of CD4+ memory CD45RBlow T cells in recipients of control or TNFR2-deficient donor CD4+ T cells (data not shown). These results suggest that TNF is critical for the development of memory T cells in the intestine during GVHD but not in the spleen.
A significant reduction in intestinal IL-2 and IFN-α mRNA expression was observed in recipients of the TNFR2-deficient CD4+ T cells
To assess the level of in vivo cytokine expression during intestinal GVHD, mRNA isolated from small intestinal sections extracted from recipients of the TNFR2−/−CD4+ T cells or from recipients of B6 CD4+ T cells was probed with a multiprobe RNase protection assay system. After hybridization of the radioactive-labeled probe set to small intestinal mRNA, the radioactive counts for each cytokine mRNA were quantitated by phosphor imaging and then standardized to the control L32 RNA counts. Significantly lower levels of IL-2 (p = 0.019) and IFN-γ mRNA (p = 0.007) were observed in small intestine mRNA isolated from recipients of the TNFR2−/−CD4+ T cells than from controls (Fig. 7⇓).
Quantification of intestinal cytokine mRNA in GVHD mice. Quantification of cytokine mRNA in intestines from allogeneic bone marrow transplant recipients that received either the TNFR2−/− (n = 6) or the control B6 CD4+ T cell (n = 8) was performed as detailed in Materials and Methods. Cytokine mRNA levels were normalized to L32 mRNA levels in each mouse.
Discussion
These results indicate that TNFR2 signaling in donor CD4+ T cells is important for development of intestinal GVHD. Moreover, the present findings, in conjunction with the results of prior in vitro studies (9), indicate that TNF-TNFR2 (and/or lymphotoxin α) interactions amplify allospecific T cell responses to MHC class II differences and play a critical role in the activation and differentiation of Th1 cytokine responses by intestinal lymphocytes during MHC class II-disparate GVHD.
TNF is a highly pleiotropic cytokine that plays a role in both innate and adaptive immune responses via signaling mediated by two distinct receptors (18). Intracellular signaling through TNFR may lead to either apoptosis (programmed cell death) or to cell activation and/or growth mediated through NF-κB and c-Jun kinase activation (18). The role of TNF in GVHD has been recognized in multiple experimental models (4, 6, 8, 17, 19). Initial assessments of the mechanism underlying these observations focused on the role of TNF-α as an effector of host cytotoxicity during acute GVHD (4, 17). As TNF cytotoxicity as well as many additional acute inflammatory effects of TNFR signaling are predominately linked to TNFR1 signaling (5, 18), initial studies of the role of TNF in GVHD assessed the course of acute GVHD in TNFR1-deficient recipients of MHC class I- plus II- disparate donor cells (5). In such studies, absence of TNFR1 on host cells was associated with a delay in mortality from acute GVHD but had no effect on histological patterns of GVHD in the intestinal tract or liver (5).
Other, more recent, studies have focused on the role of TNFR signaling in donor T cells in the B6→B6D2F1 and B6→B6C3F1 class I plus II MHC-disparate mouse GVHD models (4) where CTL effector mechanisms play a prominent role in determining GVHD mortality rates (4, 20, 21). In these GVHD models, TNFR1 deficiency, but not TNFR2 deficiency on donor T cells, was associated with decreased GVHD mortality and clinical severity that correlated with decreased T cell proliferative IL-2 and CTL responses. Effects on gut GVHD in these studies were not reported.
Despite the large amount of data arguing for a predominant role for TNFR1 signaling in mediation of many of the TNF effects during acute GVHD, other findings have indicated that mechanisms underlying intestinal GVHD may be different from those leading to lethal GVHD in class I MHC-disparate murine models. In previous studies from our and other laboratories, CD4+ T cell responses have been found to play a major role in generating both small (22, 23) and large (2, 3, 6, 8) intestinal GVHD lesions. In addition, TNF inhibition during GVHD has been associated with both diminished severity of colonic mucosal injury (6) and with decreased intestinal CD4+ T cell activation and Th1 cytokine responses (8). In contrast to the prominent role that TNFR1 signaling has been found to play in propagation of CD8+ T cell allogeneic responses (4, 9), TNFR2 signaling has been found to enhance in vitro class II MHC alloantigen-stimulated CD4+ alloproliferative and Th1 cytokine responses (9). The present in vivo studies have confirmed the prominent role that TNFR2 signaling plays in CD4+ T cell responses to MHC class II alloantigens.
Furthermore, this is an important study in determining the role of TNFR2 in TNF-associated pathology. TNFR2 has been implicated in systemic toxicity of TNF and in TNF-mediated skin necrosis (24). Recently, the interaction between membrane-bound TNF and TNFR2 has been shown to be crucial in the development of experimental cerebral malaria (25). Our studies are the first to implicate TNFR2 signaling in the development of intestinal GVHD.
Of interest, however, when T cell responses in the spleen were examined for differences in control vs TNFR2-deficient T cell expansion and activation during GVHD, a lesser role for TNFR2 signaling was noted. CD4+ T cell numbers and activation markers were unaffected by lack of TNFR2 signaling (Fig. 7⇑ and Table II⇑ in present studies), and when Th1 cytokine responses were assessed (data not shown) levels of IL-2 and IFN-γ mRNA were not found to be consistently diminished in the spleens of recipients of TNFR2-deficient donor T cells. These results indicate that, whereas TNFR2 signaling is critical for the development of intestinal GVHD, TNFR2 signaling likely plays a minor or insignificant role in GVHD responses at other sites. Of note, in phase I and II trials of a monoclonal anti-TNF Ab for therapy of acute GVHD in humans, TNF inhibition was associated with more profound effects on intestinal GVHD than on GVHD in other organs (26). In other animal studies using a LPS antagonist to reduce but not totally ablate TNF responses during acute GVHD, significant improvement in intestinal pathology was noted following inhibition of LPS-induced TNF responses without impairment of either donor T cell responses in the spleen or systemic graft vs leukemia responses (17). In light of these observations, it is reasonable to speculate that therapies directed at selective blocking of TNF-TNFR2 actions during GHVD may prove beneficial in ameliorating intestinal GHVD while having lesser effects on graft vs leukemia responses or other GVHD responses that evolve in extraintestinal sites.
In conclusion, TNF-TNFR2 interactions are important for the differentiation and activation of donor CD4+ T cells in the intestine during the development of MHC class II GVHD. Furthermore, the present findings demonstrate that TNF-TNFR2 interactions on donor CD4+ T cells are important in the pathogenesis of the histopathological lesions of intestinal GVHD and the wasting associated with acute intestinal GVHD. Of note, however, TNF-TNFR2 interactions appear not to play a significant role in T cell activation in the spleen and thus therapeutic strategies that selectively target various TNF signaling pathways may have different effects on graft-versus-host alloimmune responses that evolve in various organ sites.
Footnotes
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↵1 This work was supported by National Institutes of Health Grant R01 AI-24639 and a Miles and Shirley Fiterman American Digestive Health Foundation grant.
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↵2 Address correspondence and reprint requests to Dr. Geri R. Brown, Department of Internal Medicine, Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9151. E-mail address: gbrown{at}chop.swmed.edu
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3 Abbreviations used in this paper: BMT, bone marrow transplantation; GVHD, graft-vs-host disease; SpC, spleen cell; BMC, bone marrow cell; LPL, lamina propria lymphocyte.
- Received November 6, 2001.
- Accepted January 10, 2002.
- Copyright © 2002 by The American Association of Immunologists