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The Journal of Immunology, 2004, 173: 910-919.
Copyright © 2004 by The American Association of Immunologists

Differential Requirement for IFN-{gamma} in CTL Maturation in Acute Murine Graft-versus-Host Disease1

Roman Puliaev*, Phuong Nguyen*, Fred D. Finkelman2,{dagger} and Charles S. Via2,3,*

* Research Service, Baltimore Veterans Affairs Medical Center, and Division of Rheumatology and Clinical Immunology, University of Maryland School of Medicine, Baltimore, MD 21201; and {dagger} Immunology Division, Veterans Affairs Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH 45267


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although IFN-{gamma} is the archetypal Th1 cytokine, its role in CTL maturation is uncertain. We used an in vivo mouse model of CTL development, parent-into-F1 acute graft-vs-host disease (AGVHD), to evaluate this issue. In AGVHD, transfer of naive parental T cells into F1 hosts stimulates the development of allospecific CTL effectors that eliminate host lymphocytes, particularly B cells. Complete elimination of IFN-{gamma}, using IFN-{gamma}-deficient donors and administering anti-IFN-{gamma} mAb, suppressed B cell elimination, down-regulated TNF-{alpha} production, and enhanced Th2 cytokine production, but did not allow the B cell expansion characteristic of chronic GVHD (CGVHD). Because complete CTL inhibition results in full-blown CGVHD that is IFN-{gamma} independent, these observations indicate that IFN-{gamma} elimination only partially blocks CTL development. IFN-{gamma} elimination did not inhibit donor T cell engraftment or activation in the AGVHD model, but almost completely blocked Fas/Fas ligand (FasL) gene expression, protein up-regulation, and Fas/FasL-mediated CTL killing. In contrast, IFN-{gamma} elimination only partially inhibited perforin gene expression and perforin-mediated CTL activity. The contributions of IFN-{gamma} to CTL development were indirect, because IFN-{gamma} receptor-deficient donor cells differentiated normally into allospecific CTLs. Consistent with the view that the Fas/FasL and perforin pathways each mediate CTL killing in AGVHD, the absence of both perforin and IFN-{gamma} (perforin knockout donor cells and anti-IFN-{gamma} mAb) converted AGVHD to CGVHD. Thus, both IFN-{gamma}-dependent induction of Fas/FasL and IFN-{gamma}-independent induction of perforin contribute to CTL-mediated elimination of host B cells in AGVHD. Suppression of both pathways is required for typical CGVHD development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytotoxic T lymphocytes initiate target cell killing by two major pathways, a granule exocytosis pathway that requires perforin and an apoptotic, Fas-associated death domain-mediated pathway that requires target cell receptor binding. Fas is the most important surface receptor in the latter pathway, but TNF-{alpha} receptors also contribute (reviewed in Ref. 1). Naive CD8+ CTL precursors exhibit no cytotoxic activity and must undergo activation and maturation to become CTL effector cells. This period lasts several days and requires 1) an Ag-specific signal through the TCR, 2) costimulation provided by B7 ligation of CD28, and 3) binding of IL-2 to the T cell IL-2R (1). IFN-{gamma} and TNF-{alpha} are secreted by mature effector CTL (2, 3); however, recent evidence indicates that these cytokines also have a role in the inductive phase of CTL differentiation (4, 5).

The parent-into-F1 (P->F1)4 model of acute graft-vs-host disease (AGVHD) is useful for studying in vivo CTL development. In this model, naive parental strain T cells transferred into unirradiated F1 mice differentiate into donor anti-host CTL specific for host MHC class I that eliminate host lymphocytes through both the perforin and Fas-associated death domain pathways (6, 7, 8, 9). Host splenic B cells are particularly susceptible to elimination and by 2 wk after parental cell transfer are barely detectable by flow cytometry. Using this model, we have demonstrated that IL-2 and TNF-{alpha} are critical for anti-host CTL generation (4, 10). Agents that selectively block donor CD8+ T cell maturation into CTL, but do not block donor CD4+ T cell activation in this model (e.g., anti-IL-2 mAb and anti-TNF-{alpha} mAb), prevent host B cell elimination and result in significant B cell expansion at 2 wk of disease by converting AGVHD to chronic GVHD (CGVHD). Incomplete inhibition of donor CTL effector function by selective blockade of either perforin pathway killing or Fas pathway killing (e.g., transfer of either perforin-deficient or Fas ligand (FasL)-defective donor cells, respectively) results in an intermediate phenotype at 2 wk in which mice have B cell numbers that are neither as reduced as in wild-type (WT) AGVHD nor as increased as in WT CGVHD (9, 11). This is because the loss of a single effector CTL pathway impairs elimination of host B cells, but residual killing by the remaining pathway holds B cell expansion in check.

Our previous studies have demonstrated that P->F1 AGVHD is associated with massive increases in IFN-{gamma} serum levels and splenic IFN-{gamma} gene expression (4, 12) that are not seen in CGVHD, suggesting an important role for IFN-{gamma} in CTL maturation. However, the substitution of IFN-{gamma}-deficient for WT donor cells differs from treatment with anti-TNF-{alpha} or anti-IL-2 mAb in that it does not convert AGVHD into CGVHD; instead, it results in a long term disease phenotype that is neither distinctly AGVHD nor CGVHD, but, rather, has attenuated features of both (13). To directly determine the role of IFN-{gamma} in CTL maturation, we examined CTL in AGVHD mice that received both IFN-{gamma} knockout donor cells and a neutralizing anti-IFN-{gamma} mAb. Our results indicate that CTL develop to a limited extent in the absence of IFN-{gamma}, because the perforin pathway is only partially IFN-{gamma} dependent, whereas the Fas pathway is totally dependent on this cytokine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

All mice were purchased from The Jackson Laboratory (Bar Harbor, ME). WT C57BL/6J (B6 WT), IFN-{gamma} knockout B6.129S7-Ifngtm1Ts/J (IFN-{gamma}KO), IFN-{gamma} receptor knockout B6.129S7-IFN-{gamma}rtm1Agt (IFN-{gamma}RKO), and perforin knockout C57BL/6-Pfptm1Sdz (pfpKO) mice were used as donors, and B6D2F1 (BDF1) mice were used as recipients. All mice were males, aged 6–8 wk.

Induction of GVHD

Single-cell suspensions of splenocytes were prepared in RPMI 1640 medium from the spleens of donor strain mice. Cell suspensions were filtered through sterile nylon mesh, washed, and diluted to a concentration of 108 viable (trypan blue excluding) cells/ml. Unless otherwise noted, AGVHD was induced with 50 x 106 unfractionated splenocytes, and CGVHD was induced with 12 x 106 CD4+ (CD8+ T cell-depleted) splenocytes. After flow cytometric analysis, the number of donor CD4+ and CD8+ T cells was adjusted such that equal numbers of B6 WT (WT->F1), B6 IFN-{gamma}KO (KO->F1), and B6 IFN-{gamma}RKO (RKO->F1) were injected into recipient F1 mice. Depletion of donor CD8+ T lymphocytes was conducted using Dynabeads mouse CD8 (Lyt 2) from Dynal Biotech (Oslo, Norway). Flow cytometric analysis demonstrated <1% contaminating CD8+ T cells. Cell suspensions were injected i.v. into the tail vein of normal, unirradiated B6D2F1 recipients. Control mice consisted of uninjected age- and sex-matched F1 mice.

In vivo reagents

The effects of IFN-{gamma} were blocked in vivo by the administration of neutralizing doses of an anti-IFN-{gamma} mAb, XMG-6 (14), given at 1 mg/mouse i.v. on days 0 and 7 after parental cell transfer. A rat IgG1 control mAb, GL-113 (originally obtained from John Abrams, DNAX, Palo Alto, CA), was administered at the same dose and schedule. Additional controls consisted of untreated AGVHD mice and uninjected normal F1 mice.

Flow cytometric analysis

Spleen cells were incubated with anti-murine Fc{gamma}RII/III mAb, 2.4G2, for 20 min and stained with saturating concentrations of FITC-, biotin-, or PE-conjugated mAb against CD4, CD8, B220, H-2Kd, I-Ad, Pgp-1 (CD44), Fas (CD95), and FasL (CD178) purchased from BD Pharmingen (San Diego, CA). Three-color flow cytometric analyses were performed using a FACScan flow cytometer (BD Biosciences, San Jose, CA). Monocyte populations were excluded on the basis of forward and side scatter. Lymphocytes were gated by forward and side scatter, and fluorescence data were collected for 10,000 cells. Studies of donor T cells were performed on 5,000 gated cells that were CD4+ or CD8+ and did not stain positively for the MHC class I of the opposite parent.

In vivo cytokine capture assay (IVCCA)

The IVCCA (15, 16) was used to quantitate in vivo production of IFN-{gamma}, TNF-{alpha}, IL-10, and IL-4 in mice undergoing GVHD. The IVCCA increased the sensitivity of detection of each of the cytokines measured by a factor of ~100. Briefly, mice were injected i.v. with 10 µg of a biotin-labeled neutralizing mAb to IL-10, IL-4, TNF-{alpha}, or IFN-{gamma}, which bound some, but not all, of the respective cytokine shortly after it was secreted. The biotin-mAb-cytokine complexes formed had a much longer in vivo half-life than uncomplexed cytokines and accumulated in serum. Mice were bled 1 day after biotin-mAb injection, and concentrations of biotin-mAb-cytokine complexes were measured by ELISA using microtiter plate wells coated with an mAb to an epitope on the appropriate cytokine that was not blocked by the injected biotin-labeled mAb to the same cytokine. Biotin-labeled mAb-cytokine complexes in serum samples or standards (prepared by mixing recombinant cytokines (purchased from BD Pharmingen) with the appropriate biotin-anti-cytokine mAbs at a 1:100 weight ratio) were detected with streptavidin-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA), followed by a substrate solution (SuperSignal ELISA Femto Maximum Sensitivity Substrate; Pierce, Rockford, IL) that generated a luminescent compound when cleaved by HRP. Plates were read immediately after addition of substrate with a Fluoroskan Ascent FL luminometer (Labsystems, Helsinki, Finland). The IVCCA did not interfere with ongoing immune responses, because only a relatively small percentage of secreted cytokine was bound by the injected biotin-mAb and because biotin-mAb-cytokine complexes contain only one molecule of IgG mAb and thus do not fix, complement, or bind to Fc{gamma}R more avidly than endogenous serum IgG. The following pairs of anti-cytokine mAbs were used, all of which were obtained from Dr. D. Ernst (BD Pharmingen): for IL-4, inject biotin-BVD4-1D11 and coat wells with BVD6-24G2.3; for IFN-{gamma}, inject biotin-R46A2 and coat wells with AN-18; for TNF-{alpha}, inject biotin-TN3 and coat plates with G281-2626; and for IL-10, inject biotin-JES5-16E3 and coat wells with JES5-2A5.

Detection of anti-host CTL activity ex vivo

Effector CTL activity of freshly harvested splenocytes was tested in a 4-h 51Cr release assay without an in vitro sensitization period. Splenocytes from control and GVHD mice were tested for the ability to lyse Fas-dull P815 cells (H-2d, MHC class I positive, class II negative), Fas-positive L1210 cells (transfected to express surface Fas), Con A-stimulated (2.5 µg/ml, 48 h) DBA/2 blasts, and LPS-stimulated (10 µg/ml, 48 h) DBA/2 blasts. Using serial dilutions, effectors were tested in triplicate at four E:T cell ratios, beginning at 100:1 (2 x 106 effectors and 0.02 x 106 targets/well). The percentage of lysis was calculated according to the formula: (cpm sample – cpm spontaneous)/(cpm maximum – cpm spontaneous) x 100%. Results are shown as the mean percent lysis ± SEM at a given E:T cell ratio for each treatment group.

Cytokine expression by PCR

RNase-free plastic and water were used throughout the assay. Splenocytes (1 x 107) were homogenized in 1 ml of RNA-STAT-60 (Tel-Test, Friendswood, TX). RNA samples were reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA). Primers and probes were designed using the computer program Primer3 (17). Except for FasL, primers were always located in two different exons. RNA samples were treated with DNase I (Invitrogen) before reverse transcription to prevent contamination by chromosomal DNA. All cDNA sequences were obtained from the GenBank database. Sequences of PCR primers and specific probes (5'->3') are as follows: perforin: sense, GAT GTG AAC CCT AGG CCA GA; antisense, GGT TTT TGT ACC AGG CGA AA; probe, TCC AAG GTA GCC AAT TTT GC; IL-4: sense, GAA TGT ACC AGG AGC CAT ATC; antisense, CTC AGT ACT ACG AGT AAT CCA; probe, AGG GCT TCC AAG GTG CTT CGC A; IL-10: sense, CGG GAA GAC AAT AAC TG; antisense, CAT TTC CGA TAA GGC TTG G; probe, GAG CCA CAT GCT CCT AGA GC; and FasL: sense, TAG ACA GCA GTG CCA CTT CAT; antisense, AAC TCA CGG AGT TCT GCC AGT T; probe, CAT CAC AAC CAC TCC CAC TG.

For each gene product, the optimum number of cycles was determined experimentally. To verify that equal amounts of RNA were added in each RT-PCR within an experiment, primers for the housekeeping gene, 18S ribosomal RNA, were used in each experiment. Gene expression was quantitated by densitometry for individual mice and normalized to each individual rRNA value, and results for each cytokine were calculated as the fold increase over the respective cytokine expression in control F1 mice according to the ratio of normalized experimental group mean to normalized F1 mean.

Statistical analysis

Data was examined for normality and equal variance (Kolmogorov-Smirnov). If satisfactory, groups were compared by a two-tailed Student’s t test; if not, they were compared by the Mann-Whitney U rank-sum test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma} is produced by host cells in AGVHD, but not CGVHD

We have previously shown that, using a sensitive measure of serum cytokine production (IVCCA), striking elevations in serum IFN-{gamma} (≥103-fold over control) are observed in AGVHD mice as early as day 6 after parental cell transfer (4). In contrast, lesser elevations of serum IFN-{gamma} (≤10-fold) are observed in CGVHD mice. It has been previously reported that all detectable IFN-{gamma} produced in AGVHD mice in the P->F1 model is derived from donor cells (13). To determine whether host production of IFN-{gamma} contributes to serum levels in AGVHD and CGVHD mice, we induced GVHD using donor T cells unable to produce IFN-{gamma} (IFN-{gamma}KO). As shown in Fig. 1, circulating IFN-{gamma} is still detectable when IFN-{gamma}KO donor cells are used to induce AGVHD, although it is greatly reduced compared with WT->F1 AGVHD. In contrast, all the relatively small amount of IFN-{gamma} in CGVHD mice is produced by the donor cells, inasmuch as IFN-{gamma} levels are reduced to control levels (untreated F1 mice) when IFN-{gamma}KO mice are used as a source of donor cells.



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  		FIGURE 1. Both donor and host splenocytes contribute to elevated serum IFN-{gamma} levels in AGVHD, but not CGVHD. AGVHD and CGVHD were induced using either B6 WT or B6 IFN-{gamma}KO donor cells as described in Materials and Methods. Serum IFN-{gamma} was determined by IVCCA on day 6 after parental cell transfer, and values represent the group mean serum IFN-{gamma} concentration (±SEM; n = 5 mice/group). a, p < 0.001 compared with normal F1 or WT AGVHD; b, p < 0.05 compared with normal F1; c, p < 0.05 vs WT CGVHD; NS vs normal F1.

 
Neutralization of IFN-{gamma} impairs AGVHD development, but does not induce CGVHD

The above results indicate that it is necessary to neutralize both donor and host production of IFN-{gamma} to accurately determine the in vivo role of IFN-{gamma} in AGVHD. This was achieved by administering both neutralizing anti-IFN-{gamma} mAb and IFN-{gamma} KO donor cells to WT recipient F1 mice. Controls included F1 mice that received IFN-{gamma} KO donor cells and control mAb or WT donor cells with no mAb. As shown in Fig. 2, typical phenotypic parameters of AGVHD, i.e., engraftment of donor CD4+ and CD8+ T cells (Fig. 2A) and elimination of host B cells and splenocytes (Fig. 2B), were observed in WT->F1 mice 2 wk after donor cell transfer. In contrast, the transfer of IFN-{gamma} KO donor cells (with either control mAb or anti-IFN-{gamma} mAb) resulted in impaired elimination of host B cells and splenocytes, although the sparing of host B cells was significantly greater with anti-IFN-{gamma} mAb. It has been previously shown that elimination of host lymphocytes in this model is mediated by CD8+, donor anti-host CTL (7). Surprisingly, the impaired elimination of host lymphocytes seen when IFN-{gamma} was completely eliminated from the system (IFN-{gamma}KO and anti-IFN-{gamma} mAb) was not associated with impaired donor CD8 engraftment, but, rather, with normal donor T cell activation (CD44 up-regulation; Fig. 2C) and significantly increased engraftment of activated T cells (Fig. 2, A and D). These data indicate that 1) host production of IFN-{gamma} is functionally significant; 2) elimination of host B cells, a CTL-mediated activity, is a sensitive indicator of in vivo IFN-{gamma} activity; and 3) IFN-{gamma} appears to be critically important, not in donor T cell engraftment or expansion, but, rather, in maturation into CTL effector cells.



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  		FIGURE 2. Neutralization of IFN-{gamma} enhances donor CD4+ and CD8+ engraftment and activation, but prevents host B cell elimination in AGVHD mice. AGVHD was induced as described in Materials and Methods. IFN-{gamma}KO->F1 mice received either anti-IFN-{gamma} mAb (XMG-6) or control Ab (GL-113) i.v. on days 0 and 7 after parental cell transfer. Splenocytes were analyzed by flow cytometry on day 14 after parental cell transfer. A, Total number of engrafted donor CD4+ and CD8+ T cells; B, total splenocytes and host B cells; D, total number donor CD4+, CD44high, and CD8+, CD44high cells. Values are the group mean ± SEM x 10–6. C, The percentage of CD44high cells represents the mean group percentage of donor CD4+ and CD8+ cells staining positively with anti-CD44 fluorochrome-conjugated mAb (n = 5 mice/group for all groups). Donor cells were not detectable in uninjected F1 mice, and values represent the percentage of CD44high host T cells. a, p < 0.01 vs WT AGVHD; b, p < 0.05 vs WT AGVHD; c, p < 0.01 vs IFN-{gamma}KO->F1 and control Ab; d, p < 0.01 vs normal F1. Similar results were observed in two additional experiments.

 
Fas/FasL up-regulation is impaired in the absence of IFN-{gamma}

The foregoing data support the idea that donor anti-host CTL elimination of host lymphocytes is impaired when IFN-{gamma} is completely neutralized in vivo. Because anti-host CTL in this model eliminate host cells by both Fas and perforin pathways (9), further studies were performed to address the functional integrity of each of these pathways. We have previously demonstrated that AGVHD results in significant up-regulation of Fas and FasL on host and donor lymphocytes and that treatment with anti-IFN-{gamma} mAb significantly impairs this up-regulation (5). Similarly, as shown in Fig. 3, the use of IFN-{gamma}KO donor cells and either anti-IFN-{gamma} mAb or control mAb completely blocked both FasL up-regulation on donor CD4+ and CD8+ T cells and Fas up-regulation on host B cells. No significant differences in Fas or FasL expression were seen with the use of anti-IFN-{gamma} mAb compared with control mAb when combined with IFN-{gamma}KO donor cells. Moreover, the absolute number of FasL-positive CD4+ and CD8+ donor cells was profoundly reduced (p < 0.001) for IFN-{gamma}KO->F1 with or without anti-IFN-{gamma} mAb compared with WT->F1 mice (Fig. 3E). Importantly, this decrease was seen despite an increase in total number of donor cells engrafted in IFN-{gamma}KO->F1 and anti-IFN-{gamma} mAb (Fig. 2A), underscoring the sensitivity of FasL up-regulation to the presence of IFN-{gamma}.



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  		FIGURE 3. Neutralization of IFN-{gamma} blocks up-regulation of FasL on donor CD4+ and CD8+ cells and up-regulation of Fas on host B cells. AGVHD was induced and anti-IFN-{gamma} and control mAb were administered as described in Fig. 2. On day 10 after parental cell transfer, FasL expression was determined on gated donor CD4+ (A) and CD8+ (B) cells, and Fas expression was determined on gated host B cells (C). Group mean channel fluorescence values (mean ± SE) are shown for FasL (D) and Fas (F) expression (n = 5 mice/group). E, Total numbers of FasL-positive donor T cells. Normal, Uninjected B6 splenocytes for D and uninjected BDF1 splenocytes for F. a, p < 0.001 compared with WT AGVHD. Shaded curves represent naive uninjected B6 CD4+ T cells (A), B6 CD8+ T cells (B), and BDF1 B cells (C). Similar results were seen in an additional experiment on day 14.

 
The flow cytometric results correlate with FasL gene expression data (Fig. 4A). AGVHD is characterized by a significant increase in both FasL and perforin gene expression on day 10 (Fig. 4); however, IFN-{gamma}KO->F1 mice exhibit significant reductions in both FasL (~75% reduction) and perforin (~50% reduction) gene expression compared with WT->F1 mice (FasL, p < 0.001; perforin, p < 0.05). IFN-{gamma}KO->F1-treated groups did not differ significantly between themselves. These results have been confirmed in a second independent experiment. In neither instance did IFN-{gamma} neutralization result in complete inhibition of either FasL or perforin mRNA despite complete inhibition of FasL cell surface protein up-regulation. Additionally, perforin gene expression was always less impaired than FasL gene expression. Taken together, these data indicate that IFN-{gamma} is important for up-regulation of Fas/FasL and perforin in AGVHD and suggest that the Fas pathway is more severely affected than the perforin pathway.



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  		FIGURE 4. IFN-{gamma} is required for optimal perforin and FasL gene expression. Using the same experimental protocol as those described in Fig. 2, splenocytes were analyzed on day 10 after cell transfer, and cytokine gene expression was determined by PCR as described in Materials and Methods. Results are shown as the group mean ± SEM (n = 4–5 mice/group). a, p < 0.001 compared with WT AGVHD; b, p < 0.05 compared with WT AGVHD. Similar results were seen in an additional independent experiment.

 
IFN-{gamma} inhibition impairs anti-host CTL activity in AGVHD mice

To determine whether the significant reductions in Fas/FasL and perforin gene expression seen with IFN-{gamma} blockade are associated with functionally significant reductions in anti-host CTL activity, we used the same experimental protocol and assessed anti-host CTL activity against H-2d targets 10 days after parental cell transfer. As shown in Fig. 5, ex vivo anti-host CTL activity was readily detected using splenocytes from WT->F1 mice on all H-2d targets tested, including both Fas-negative targets (L1210– and P815 cells) and Fas-positive targets (DBA/2 Con A blasts, DBA/2 LPS blasts, and L1210+ cells). In contrast, detectable anti-host CTL activity in F1 mice receiving IFN-{gamma}KO donor cells (with or without anti-IFN-{gamma} mAb) was only observed on Fas-dull P815 targets (Fig. 5A). We have previously demonstrated that CTL killing of the Fas-dull target P815 is entirely perforin dependent and is, therefore, Fas independent (9). Thus, the residual 3-fold elevations of perforin mRNA over control in Fig. 4B correlate with the presence of residual, but significantly impaired, perforin-mediated CTL activity on P815 targets. Our inability to detect killing on other targets using IFN-{gamma} KO->F1 mice suggests that P815 cells are the most sensitive in vitro targets for detecting low level, perforin-dependent killing. Of note, in two independent experiments, we were unable to detect significant differences in P815 killing between IFN-{gamma}KO->F1 mice that received anti-IFN-{gamma} mAb vs mice that received control mAb, even though these mice exhibited significant differences in host B cell numbers (Fig. 2B). These results argue that host B cell number is a more sensitive indicator of in vivo anti-host CTL activity than are these ex vivo assays.



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  		FIGURE 5. IFN-{gamma} inhibition impairs anti-host CTL activity in AGVHD mice. Experimental groups are described in Fig. 2. Splenocytes were harvested on day 10 after parental cell transfer, and anti-host CTL were assessed directly ex vivo at the E:T cell ratio indicated. Mice were tested individually (n = 5/group), and results are shown as the group mean percent lysis of the following H-2d targets: P815 cells (A), Con A DBA/2 blasts (B), LPS DBA/2 blasts (C), L1210 Fas-positive cells (D), and L1210 Fas-negative cells (E). Similar results were seen in two additional experiments.

 
IFN-{gamma} does not act directly on T cells to promote CTL maturation

Our observation that IFN-{gamma} is required to optimally induce CTL activity in AGVHD was compatible with both the possibility that IFN-{gamma} acts directly on CD8+ T cells to promote their differentiation into CTL and the possibility that IFN-{gamma} acts on a different cell type, causing effects that indirectly induce CTL differentiation. To distinguish between these possibilities, we determined whether AGVHD would be induced when T cells from donor C57BL/6 IFN-{gamma}RKO, as opposed to WT or IFN-{gamma}-deficient mice, were transferred into WT B6D2F1 recipients. The IFN-{gamma}RKO donor cells would be able to produce IFN-{gamma}; however, IFN-{gamma} could not have any effect on the donor T cells. As shown in Fig. 6, the AGVHD phenotype in IFN-{gamma}RKO->F1 mice was remarkably similar to that in WT->F1 mice 2 wk after cell transfer. IFN-{gamma}RKO donor cells exhibited no defect in CD4+ and CD8+ expansion (Fig. 6A). Similarly, there was no defect in host B cell up-regulation of Fas or donor T cell up-regulation of FasL (Fig. 6B). Using elimination of host B cells as a surrogate marker for in vivo anti-host CTL activity, there was a minimal, but significant (p = 0.044), decrease in elimination of host B cells using IFN-{gamma}RKO donor cells compared with WT donor cells (Fig. 6C). Thus, the role played by IFN-{gamma} in the maturation of CTL killing pathways is predominantly indirect.



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  		FIGURE 6. IFN-{gamma} does not act directly on T cells to promote CTL maturation. AGVHD was induced using either WT donor cells or IFN-{gamma}RKO donor cells, and splenocytes were assessed 14 days after cell transfer for donor CD4+ and CD8+ T cell engraftment (A), FasL up-regulation on donor T cells and Fas up-regulation on host B cells (percent FasL or Fas bright cells, respectively; B), and total splenocytes and host B cells (C). Mice were tested individually, and results shown are the group mean ± SE (n = 5 mice/group). NS, not significant vs WT AGVHD; a, p < 0.01 vs normal F1; b, p < 0.05 vs WT AGVHD.

 
IFN-{gamma} blockade in AGVHD skews cytokine production to resemble that in CGVHD

To determine the effect of IFN-{gamma} neutralization on cytokine production in AGVHD mice, serum IL-4, IL-10, and TNF-{alpha} were determined by IVCCA using the experimental protocol outlined above. Compared with uninjected F1 mice, WT->F1 AGVHD mice exhibit a small increase in IL-4 mRNA, but no detectable increase in serum IL-4 (Fig. 7). Both serum and mRNA IL-4 levels were significantly boosted by neutralization of IFN-{gamma}. In contrast, striking elevations were seen for IL-10 (both serum and mRNA expression) in WT->F1 AGVHD mice compared with control mice. This increase in IL-10 was significantly reduced with complete IFN-{gamma} neutralization (both protein and gene expression), but was not completely blocked to control levels. Thus, complete IFN-{gamma} neutralization changes the cytokine profile of AGVHD to resemble that reported for CGVHD (e.g., elevated IL-4, low level elevations of IL-10, and minimal IFN-{gamma}) (12). However, neutralization of IFN-{gamma} mice in AGVHD mice did not result in the host B cell expansion characteristic of CGVHD (Fig. 2B), suggesting that the presence of residual anti-host CTL is sufficient to limit host B cell expansion unless IFN-{gamma} is required for B cell expansion.



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  		FIGURE 7. Suppression of IFN-{gamma} in AGVHD skews cytokine production to resemble that in CGVHD. The experimental protocol is outlined in Fig. 2. After parental cell transfer, mice were bled on day 6 for serum values of IL-4 (A) and IL-10 (C), and splenic mRNA was harvested on day 14 for IL-4 (B) and IL-10 (D). Mice were tested individually by IVCCA for serum cytokines and by PCR for cytokine gene expression as described in Materials and Methods. Results are shown as the group mean ± SE (n = 5 mice/group). a, p < 0.01 compared with WT AGVHD; b, p < 0.05 compared with WT AGVHD; NS vs IFN-{gamma} KO GVHD and control Ab.

 
IFN-{gamma} is not required for B cell expansion

To determine whether IFN-{gamma} is required for host B cell expansion, we induced CGVHD using CD8+-depleted WT or IFN-{gamma}KO donor T cells in conjunction with anti-IFN-{gamma} mAb or control mAb (Fig. 8). F1 mice receiving IFN-{gamma}KO donor CD4+ T cells with or without anti-IFN-{gamma} mAb exhibited increased host B cell numbers 2 wk after cell transfer that did not differ significantly from those observed in WT->F1 CGVHD. These results do not support a role for IFN-{gamma} in B cell expansion.



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  		FIGURE 8. IFN-{gamma} is not required for B cell expansion in CGVHD mice. CGVHD was induced as described in Materials and Methods, and experimental groups are described in Fig. 2, with the exception that CGVHD is substituted for AGVHD. Host B cells were determined by flow cytometry as described in Materials and Methods, and results are shown as the group mean ± SEM (n = 5 mice/group). NS, not significant vs WT CGVHD.

 
IFN-{gamma} is absolutely required for Fas/L mediated CTL activity in vivo

Our results demonstrate an important role for IFN-{gamma} in CTL maturation in vivo and indicate that the FasL pathway may be more IFN-{gamma} dependent than the perforin pathway. To test whether the FasL pathway of CTL effector function is entirely dependent on IFN-{gamma}, we tested the ability of pfpKO donor cells to eliminate F1 host B cells in the absence of IFN-{gamma}. We have previously demonstrated that 2 wk after the transfer of pfpKO donor cells into normal F1 mice (pfpKO-> F1), there was a strong IFN-{gamma} response and the development of donor anti-host CTL that act solely through the FasL pathway, resulting in incomplete elimination of host B cells compared with WT->F1 GVHD mice (9). As shown in Fig. 9A, F1 mice receiving pfpKO B6 strain parental cells with or without control mAb exhibited impaired elimination of host B cells at 2 wk, resulting in values that were mildly elevated compared with those in uninjected F1 mice (≤ 135% B cells and ≤156% total spleen cells). These results are consistent with the idea that residual donor anti-host killing by the FasL pathway prevents the expansion of B cells to levels characteristic of CGVHD. This idea is confirmed by the results seen for mice receiving pfpKO donor cells and neutralizing doses of anti-IFN-{gamma} mAb, in which significantly greater B cell expansion and total splenocyte numbers were observed compared with either uninjected F1 mice (~200 and ~300%, respectively) or pfp->F1 mice given control mAb (~150 and ~220%, respectively). This increase in B cell expansion and total splenocytes for pfp->F1 and anti-IFN-{gamma} GVHD mice compared with pfp->F1 mice with or without control mAb is seen despite engraftment of greater numbers of donor CD8+ T cells in pfp->F1 and anti-IFN-{gamma} GVHD mice (Fig. 9B). Thus, donor CD8+ T cells are engrafted in adequate numbers, but the loss of the perforin pathway by targeted gene disruption and the loss of the FasL pathway by IFN-{gamma} neutralization completely blocks the development of anti-host CTL effectors, resulting in host B cell expansion commensurate with that in CGVHD.



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  		FIGURE 9. IFN-{gamma} is absolutely required for Fas/FasL-mediated CTL activity in vivo. AGVHD was induced using either 50 x 106 WT or 75 x 106 pfpKO donor cells, and anti-IFN-{gamma} and control mAb were injected as described in Fig. 2. Splenocytes were assessed by flow cytometry on day 14 after parental cell transfer. Results are shown as the group mean ± SEM for donor CD8+ T cell engraftment (A) and total spleen cells and host B cells (B; n = 5 mice/group). a, p < 0.001 vs pfpKO GVHD and control mAb.

 
Complete blocking of IFN-{gamma} during AGVHD only partially inhibits TNF-{alpha} production

We have previously shown that TNF-{alpha} is critical for anti-host CTL development in the P->F1 model (4). To some extent, the TNF-{alpha} contribution to CTL development may result from TNF-{alpha} induction of IFN-{gamma} production, inasmuch as IFN-{gamma} is required for the FasL pathway of CTL killing, and TNF-{alpha} neutralization suppresses IFN-{gamma} production by >99% (4). These considerations suggest that TNF-{alpha} induces FasL-independent, perforin-dependent CTL killing through a mechanism that is at least partially IFN-{gamma} independent; however, TNF-{alpha} production can result in a positive feedback loop and promote Th1 cytokines, such as IFN-{gamma}, which, in turn, result in greater TNF-{alpha} production (18). Hence, it was important to determine the extent to which suppression of IFN-{gamma} might influence the production of TNF-{alpha} during GVHD. As shown in Fig. 10, neutralization of IFN-{gamma} in AGVHD mice significantly reduced TNF-{alpha} production, as measured by IVCCA; however, this reduction was limited to 50–60%, in contrast to the nearly complete reduction in IFN-{gamma} production when TNF-{alpha} was neutralized (4). Thus, it appears likely that sufficient TNF-{alpha} is produced, even in the absence of IFN-{gamma}, to elicit the perforin pathway of CTL killing and it is possible that the partial IFN-{gamma} dependence of this pathway is an indirect effect of IFN-{gamma} enhancement of TNF-{alpha} production.



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  		FIGURE 10. Neutralization of IFN-{gamma} in AGVHD mice does not result in complete abrogation of serum TNF-{alpha}. Experimental groups are described in Fig. 2. Mice were bled on day 6, and serum TNF-{alpha} was determined in individual mice by IVCCA. Results are shown as the group mean ± SE (n = 5 mice/group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IFN-{gamma} is a pleiotropic cytokine with a significant role in antiviral defense. Additionally, IFN-{gamma} is important in mediating acute GVHD (19) and graft-vs-tumor effects (20) in the bone marrow transplant setting. NK and T cells, particularly Th1 CD4+ and Tc1 CD8+ T cells, are major sources of IFN-{gamma} (21, 22). An inductive role for IFN-{gamma} in CD8+ T cell maturation into effector CTL in vivo has not been previously demonstrated. Using the P->F1 model of AGVHD as an in vivo model of CTL development in the absence of ionizing radiation, we demonstrate that IFN-{gamma} is absolutely required for maturation of FasL-dependent CTL effector function. Maturation of perforin-dependent CTL effector function is significantly, but not exclusively, dependent on IFN-{gamma}.

Previously we have demonstrated that both IL-2 and TNF-{alpha} are absolutely required for CTL maturation in the P->F1 model, and that AGVHD is converted to CGVHD in their absence (4, 10). The conversion of AGVHD to the CGVHD phenotype reflects a selective and complete block of anti-host CTL effector cell development that occurs in conjunction with no inhibition of the cytokines important in donor T cell-driven host B cell expansion. By contrast, GVHD developing in the absence of IFN-{gamma} has an intermediate phenotype. That is, neither complete host B cell elimination, characteristic of AGVHD, nor significant host B cell expansion, characteristic of CGVHD, is observed. Instead, host B cells remain at near normal levels. This result probably represents a balance between two competing forces; B cell stimulation characteristic of CGVHD drives B cell expansion, whereas residual, but impaired, anti-host CTL characteristic of AGVHD hold B cell expansion in check.

These results support the idea that host B cell numbers 2 wk after parental cell transfer are a sensitive correlate of in vivo anti-host CTL activity. The near complete elimination of host B cells in WT->F1 AGVHD mice was associated with readily detectable and robust anti-host CTL activity ex vivo on a variety of H-2d target cells. In contrast, the near normal numbers of B cells seen in IFN-{gamma}-KO->F1 AGVHD mice was accompanied by detectable ex vivo anti-host CTL activity against only one target, P815 cells, which are killed exclusively through a perforin-dependent mechanism (9). However, assays with this cell line were unable to detect the effects of small, but functionally significant, changes in circulating IFN-{gamma}. That is, neutralization of both donor and host sources of IFN-{gamma} resulted in additional sparing of host B cells compared with that seen when host-produced IFN-{gamma} was still present, yet this in vivo change was not accompanied by a detectable change in ex vivo CTL activity using P815 cells as targets.

The consequences of the complete loss of Fas/FasL pathway killing seen in the absence of IFN-{gamma} are 2-fold. Not only does IFN-{gamma} induce up-regulation of FasL on donor T cells, making them better killer cells, but IFN-{gamma} also promotes Fas up-regulation on host B cells, making them better targets. These results are consistent with previous reports that IFN-{gamma} promotes Fas/FasL expression in vitro and in vivo (23, 24) and with recent observations in murine irradiated recipient bone marrow transplant models that IFN-{gamma} contributes to optimal acute GVHD expression (25) and promotes donor CD4+ T cell-mediated pathology in the presence of sublethal irradiation (26). Moreover, IFN-{gamma}, Fas/FasL and perforin all contribute to CTL-mediated liver damage in a transgenic model of CD8+ T cell-mediated liver disease (27). Our results also demonstrate that IFN-{gamma} does not act directly on T cells to induce FasL up-regulation as IFN-{gamma}RKO donor T cells differentiated into potent anti-host CTL when transferred into WT recipient F1 mice, as measured by host B cell elimination and Fas/FasL up-regulation. More likely, IFN-{gamma} acts through one or more intermediates. Although the number of IFN-{gamma}-regulated genes is large, e.g., ≥200 (28), it is likely that one of the intermediates is TNF-{alpha}. IFN-{gamma} enhances both the production of TNF-{alpha} and the up-regulation of TNF-{alpha} receptors (29). Previously, using the same P->F1 model of AGVHD, we reported that the absence of TNF-{alpha} during the first few days of disease converts the disease phenotype to CGVHD by preventing anti-host CTL generation and by completely inhibiting IFN-{gamma} production, but not inhibiting B cell stimulatory cytokines such as IL-6, IL-4, and IL-10. In contrast, complete neutralization of IFN-{gamma} results in an ~50% reduction in TNF-{alpha} production and partial inhibition of the anti-host CTL response. These results are consistent with those reported by Brown et al. (30), demonstrating that TNF-{alpha} promotes CD4+ T cell-mediated GVHD and IFN-{gamma} production across murine MHC class II donor/recipient disparity. Taken together, our results indicate that TNF-{alpha} is absolutely required for IFN-{gamma} production in vivo, whereas IFN-{gamma} contributes to, but is not required for TNF-{alpha} production. We hypothesize that the residual perforin-dependent anti-host CTL activity seen with IFN-{gamma} blockade is mediated by the residual TNF-{alpha} and that IFN-{gamma} contributes indirectly to CTL induction through a pathway(s) additional to its effect on the TNF-{alpha} response.

In summary, our results indicate that IFN-{gamma} blockade converts AGVHD to an attenuated form of CGVHD through indirect, but complete, inhibition of the Fas/FasL pathway of CTL killing and by partial inhibition of the perforin pathway that may involve inhibition of TNF-{alpha} production. The attenuated nature of the CGVHD that develops in the absence of IFN-{gamma} results from incomplete inhibition of the perforin pathway, rather than from any requirement for IFN-{gamma} in the development of typical CGVHD.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI47466 and AI33882 and Department of Veterans Affairs Merit Review grants (to C.S.V. and F.D.F.). Back

2 F.D.F. and C.S.V. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Charles S. Via, Division of Rheumatology and Clinical Immunology, MSTF Room 8-34, 10 S. Pine Street, University of Maryland School of Medicine, Baltimore, MD 21201. E-mail address: cvia{at}umaryland.edu Back

4 Abbreviations used in this paper: P->F1, parent-into-F1; AGVHD, acute GVHD; CGVHD, chronic GVHD; FasL, Fas ligand; GVHD, graft-vs.-host disease; IVCCA, in vivo cytokine capture assay; KO, knockout; WT, wild type. Back

Received for publication February 20, 2004. Accepted for publication May 5, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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