Immunology

IL-22 deficiency in donor T cells attenuates murine acute graft-versus-host disease mortality while sparing the graft-versus-leukemia effect

Abstract

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation (allo-HCT), limiting the success of this therapy. Many proinflammatory cytokines secreted following the conditioning regimen have been linked to aGVHD initiation. Interleukin-22 (IL-22) is a cytokine related to IL-10 for its structure and is secreted by T helper type 17 (TH17) cells and innate immune cells. Given the paradoxical role of IL-22 in inflammation with both protective or proinflammatory functions, we investigated whether IL-22 could have a role in aGVHD pathophysiology in a mouse allo-HCT model. In this study, we show that IL-22 deficiency in donor T cells can decrease the severity of aGVHD, while limiting systemic and local inflammation in aGVHD target organs. In addition, we found that Foxp3+ regulatory T cells (Treg cells) were increased in recipient mice that received IL-22-deficient T cells, suggesting that Treg were involved in the reduced severity of GVHD. Finally, we found that the graft-versus-leukemia (GVL) effect mediated by donor T cells was preserved in the absence of IL-22. Overall, these data suggest that targeting of IL-22 may represent a valid approach towards decreasing aGVHD severity after allo-HCT while preserving the GVL effect.

Introduction

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation (allo-HCT), limiting the success of this therapy.1, 2 GVHD is the result of alloreactive donor T cells attacking host tissues, including, but not limited to, the skin, liver and gut.3 Many proinflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-12 and interferon (IFN)-γ secreted following the conditioning regimen have been linked to aGVHD initiation and pathophysiology.4, 5, 6 More recently, we and others reported that T helper type 17 (TH17)-related cytokines (especially IL-17A and IL-21) can contribute to aGVHD pathophysiology.7, 8, 9, 10 IL-22 is a cytokine structurally related to the IL-10 family and is secreted by TH17 cells, γδ T cells, natural killer cells and innate lymphoid cells (ILC).11, 12, 13 The IL-22 receptor (IL-22R) is mainly expressed by non-hematopoietic cells, including epithelial cells of the lung and of the gastrointestinal tract as well as keratinocytes,14, 15 found in the sites of aGVHD. In vitro studies showed that IL-22 can activate Stat3 (signal transducer and activator of transcription factor 3) inducing the expression of antimicrobial molecules S100A7, S100A8, S100A9 and β-defensins in keratinocytes.14, 16, 17 IL-22 can have either a protective or a pathogenic role in chronic inflammatory diseases depending on the nature of the affected tissue and the local cytokine milieu. IL-22 has been shown to be protective in several colitis models.18, 19, 20 By contrast, it has been reported that IL-22 is associated with the pathogenesis of rheumatoid arthritis21 or in the development of psoriasis.14, 22 Given the broad activity of IL-22, we hypothesized that donor-derived IL-22 could contribute to aGVHD pathophysiology. Thus, we examined the contribution of donor-derived IL-22 to aGVHD in experimental mouse models of allo-HCT using IL-22-deficient mice. We found that donor-derived IL-22 has a key role in exacerbating the inflammation in the gastrointestinal tract and contributes to the severity of aGVHD but does not significantly interfere with the graft-versus-leukemia (GVL) effect.

Materials and methods

Mice

BALB/c (H-2d) and B6D2F1 (H-2b/d) mice were purchased from Janvier (Genest-St-Isle, France). C57BL/6 IL-22−/− (B6.IL-22−/−, H-2b), which were originally generated in the 129 background and were subsequently backcrossed with C57BL/6 for 13 generations, and C57BL/6 (B6.WT, H-2b) mice were provided by J.C. Renauld (Ludwig Institute for Cancer Research, Brussel, Belgium). We next maintained breeding colonies in our animal facility. All mice were used at 8–12 weeks of age. All protocols were performed according to the approval of the ‘Services Vétérinaires de la Santé et de la Protection Animale’ delivered by the Ministry of Agriculture (Paris, France).

Hematopoietic cell transplantation procedures

Recipient mice were conditioned with total body irradiation administered at a single lethal dose on day −1 at 1100 cGy (B6D2F1) or 750 cGy (BALB/c). To induce aGVHD in BALB/c recipients, mice were transplanted with 5 × 106 allogeneic T-cell-depleted bone marrow (TCD BM) from B6.WT and 1 × 106 allogeneic donor splenic T cells from B6.WT or B6.IL-22−/− mice. Syngeneic control mice were obtained by transferring 1 × 106 splenic T cells from BALB/C mice. T-cell depletion was performed using the CD3 MicroBead Kit (Miltenyi Biotec, Paris, France) and purified populations of donor T cells were obtained using the Pan T Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s instructions. Treg (regulatory T cells; CD25)–T-cell depletion was performed using CD25-PE and anti-PE microbeads and AutoMacsPro (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of TCD BM and T cells in all the experiments exceeded 95%. CD25-depleted T cells’ purity were, respectively, 98.2±1.6% for WT T cells and 98.2±2.1% for IL-22−/− T cells. To induce aGVHD in B6D2F1 recipients, mice were transplanted with allogeneic splenocytes (10 × 106 cells, B6.WT or B6.IL-22−/−) or syngeneic splenocytes (10 × 106 cells, B6D2F1) isolated from donor mice previously treated by subcutaneous injection of 10 μg/animal recombinant human granulocyte colony-forming factor (Amgen, Thousand Oaks, CA, USA) on day −5. Cells were injected in the lateral tail vein.

GVL effect analysis

To examine the GVL effect, 5 × 105 luciferase-expressing A20 leukemic cells (A20-luc, H-2d) were given along with TCD BM in BALB/c recipients. Mice received 750 cGy total body irradiation on day −1. Delayed allogeneic T-cell infusions (1.106 T B6.WT or B6.IL-22−/−) were given on day 7 after allo-HCT. For GVL effect assessment by bioluminescence imaging (BLI), mice were injected intravenously with 150 μg/g body weight E-luciferin (Promega, Lyon, France) diluted in phosphate-buffered saline and anesthetized using isofluorane (2.5% vaporized in O2). For analysis, imaging was done 5 min later using NightOWL I imaging system (Berthold Technologies GmbH and Co. KG, Bad Wildbad, Germany) and total photon flux (photons per second) was measured from a fixed region of interest over the entire abdomen and thorax using WinLight software (Berthold Technologies). BLI was performed 1 day before and 3 days after delayed donor T-cell infusions, then once every week until day 40 after allo-HCT.

Assessment of GVHD

The degree of systemic aGVHD was assessed by a scoring system that sums changes in five clinical parameters: weight loss, posture (hunching), activity, fur texture and skin integrity (maximum index=10), as previously described.23 Recipient mice were graded weekly from 0 to 2 for each criterion without knowledge of the treatment group. Animals with severe clinical aGVHD (scores>6) were killed according to ethical guidelines. For histological analyses, samples of the skin, small and large intestine were removed immediately after killing, and fixed in 4% formalin. Paraffin sections were stained with hematoxylin and eosin. The histological grade of skin aGVHD was assessed according to the grading system, used routinely to assess the human skin aGVHD:24 grade 1, focal or diffuse vacuolar alteration of the basal cell layer; grade 2, grade 1 change plus dyskeratotic squamous cell in the epidermis; grade 3, grade 2 plus subepidermal vesical formation; and grade 4, complete separation of the epidermis from the dermis. The histological grade of small and large intestine aGVHD was assessed according to the grading system previously described.25 Scores were determined in a blinded fashion by two pathologists.

Flow cytometry analysis

At designated time points, animals were killed and organs were collected. Single-cell suspensions of the spleen, peripheral (PLN) and mesenteric lymph nodes (MLN) were prepared passing through sterile mesh filters. For detection of cytokine production, cells were stimulated for 6 h with 25 ng/ml phorbol-12-myristate-13-acetate (Sigma-Aldrich, Lyon, France) and 1 μg/ml ionomycin (Sigma-Aldrich), with 1 μl/ml Golgi Plug (BD Bioscience, Le Pont de Claix, France) before staining for flow cytometric analysis. Cells were first stained with eFluor780 Fixable Viability Dye (eBioscience, Paris, France) according to the manufacturer’s instructions. Surface staining was performed using FITC (fluorescein isothiocyanate)-conjugated CD3 (from BD Biosciences), Pacific Blue-CD4 and PE-CD8 antibodies (from Biolegend, Ozyme, Saint-Quentin-en-Yvelines, France), cells were then fixed and permeabilized using CytoFix/Perm Buffer (BD Biosciences) before staining with PE/Cy7-IFN-γ, PercP/Cy5.5-TNF-α (Biolegend) and APC (allophycocyanin)-IL-17A- (eBioscience) specific antibodies or the corresponding isotype controls. For the detection of IL-22-secreting cells, cells were stained with Horizon V500-conjugated CD3 (from BD Biosciences), Pacific Blue-CD4 and PercP-CD8 antibodies (from Biolegend, Ozyme), cells were then fixed and permeabilized using CytoFix/Perm Buffer (BD Biosciences) before staining with PE/Cy7-IFN-γ (Biolegend), PE-IL-22 and APC-IL-17A- (eBioscience) specific antibodies or the corresponding isotype controls. Analysis was performed using FACSCanto II and FACSDiva software (BD Biosciences). Treg cell analysis was performed by cell surface staining with FITC-CD3, Pacific Blue-CD4 (from Biolegend) and APC-CD25 (from BD Biosciences), cells were then fixed and permeabilized with Foxp3 Staining Buffer set (eBioscience) and stained with PE-Foxp3 (clone FKJ16, eBioscience).

Real-time PCR analysis

Before RNA extraction, tissue samples were homogenized using FastPrep-24 system (MP Biomedicals Europe, Illkirch, France). RNA was extracted using RNeasy Mini kit (Qiagen, Cergy-Pontoise, France) with RLT buffer (Qiagen) supplemented with β-mercaptoethanol (Sigma-Aldrich). Total RNA was subjected to reverse transcription (High Capacity RNA-to-cDNA Master Mix, Applied Biosystems, Courtaboeuf, France) and quantified by real-time quantitative PCR using commercially available primer/probe sets (Assay On Demand, Applied Biosystems). Real-time PCR were performed on the iCycler CFX96 real-time PCR system (Life Science, Bio-Rad, Marnes-la-Coquette, France). Relative expression for the mRNA transcripts was calculated using the ΔΔCt method and G6PDH mRNA transcript as reference.

Cytokines measurements

Blood samples were obtained from recipient mice at day 6 after allo-HCT. Cytokines were quantified in plasma using BD cytometric bead array kit (BD Biosciences) according to the manufacturer’s instructions. TNF-α and IL-22 levels in cell culture supernatants were measured using commercially available enzyme-linked immunosorbent assay kits (Biolegend) according to the manufacturer’s instructions.

Statistical analysis

Group comparisons of cell populations, gene expression, cytokine levels and pathology scores data were performed using the Student’s t-test. Survival curves were compared using the log-rank test. A value of P<0.05 was considered statistically significant in all the experiments. Data were computed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).

Results

Absence of IL-22 in donor T cells decreases acute GVHD severity

To examine the contribution of IL-22 produced by donor T cells in regulating development of aGVHD, we induced aGVHD by transferring T cells from wild-type C57BL/6 (B6.WT) or IL-22-deficient C57BL/6 (B6.IL-22−/−) mice with TCD BM into lethally irradiated allogeneic BALB/c-recipient mice. The absence of IL-22 in donor T cells led to a reduction from aGVHD-induced mortality as compared with recipients receiving WT allogeneic T cells (median survival time 12 versus 34 days, P<0.0001; Figure 1a) and to a reduced morbidity as shown by a significant lower clinical score up to day 24 (clinical aGVHD score on day 12 following allo-HCT: WT T cells 4.17±0.33 versus IL-22−/−T cells 1.11±0.36, P<0.0001, Figure 1b). Furthermore, we observed that the severity of aGVHD assessed by histological grade was significantly reduced in the skin and small intestine and colon of mice that received IL-22-deficient donor T cells compared with mice that received WT donor T cells (Figures 1c–f). As expected, we observed a significant increase of pathological scores in tissues from mice receiving allogeneic T cells as compared with syngeneic T cells. By contrast, for small intestine and colon, no significant differences were detected between mice receiving syngeneic T cells and IL-22−/− T cells, reflecting the reduced severity of aGVHD induced by IL-22−/− T cells. Moreover, we also observed a reduction in aGVHD-induced mortality in another aGVHD model26 consisting of transplanting lethally irradiated B6D2F1-recipient mice with donor splenocytes from B6.WT or B6.IL-22−/− mice that have received subcutaneous injections of granulocyte colony-forming factor (median survival: 9 days versus a median survival not reached, log-rank test P<0.01; Figure 1g). In this model, in which lethality related to GVHD was reduced as compared with the B6→BALB/C model, we confirmed the decreased severity of aGVHD in the absence of IL-22 from donor cells. These data demonstrated that IL-22 deficiency in donor T cells reduced aGVHD mortality and decreased aGVHD severity after allo-HCT, suggesting a potential pathological role of IL-22 in this allo-HCT setting.

Figure 1
figure1

Absence of T-cell-derived IL-22 decreases acute GVHD severity. Lethally irradiated BALB/c recipients were transplanted with 5 × 106 B6.WT TCD BM only or with 1 × 106 B6.WT T cells or 1 × 106 B6.IL22−/− T cells (n=30 per group and five independent experiments). (a) Survival data are shown (log-rank test, P<0.0001 for B6 TCD BM+B6.WT T cells versus B6.IL22−/− T cells). (b) GVHD clinical scores were determined as described in Methods and were decreased in B6.TCD BM+B6.IL22−/− T-cell recipients, at days 12, 14 and 19 post allo-HCT, P<0.0001. (ce) Pathology scores in the skin, small intestine and colon in mice 7 and 14 days after allo-HCT. Data are presented as the mean±s.e.m. from cumulative results from three experiments for day 7 and two experiments for day 14. Scores are significantly different between the syngeneic groups and the TCD BM+WT T cells groups (P<0.05), P-values are indicated on the graphs for comparison between the groups of syngeneic and TCD BM+WT T cells or between TCD BM+WT T cells and TCD BM+IL-22−/− T cells. (f) A representative example of the section of the small intestine of BALB/C mice transplanted with TCD BM and B6.WT T cells or B6.IL22−/− T cells at day 7 ( × 20 magnification). Intestinal mucosa of TCD BM+B6.WT T-cell recipient mice was characterized by an increased number of mononuclear inflammatory cells with extensive crypt loss and an increased number of apoptotic epithelial cells in resting crypts. Non-inflammatory intestinal mucosa was characterized by a normal architecture without crypt loss. Only some apoptotic epithelial cells were individualized in the bottom of some crypt. (g) Splenocytes from granulocyte colony-forming factor mobilized B6.WT or B6.IL-22−/− donor mice or syngeneic B6.D2F1 as control were transplanted into lethally irradiated B6.D2F1-recipient mice (n=12 per group). Survival data are shown (log-rank test, P<0.01 for B6.WT versus B6.IL-22−/− splenocytes).

Absence of T-cell-derived IL-22 leads to a reduction of inflammatory CD8 T cells in lymphoid organs

To investigate the contribution of T effector cell subsets in the development of aGVHD, we examined the proportion of CD4+ and CD8+ T cells in the spleen, PLN and MLN of recipient mice at day 6 after allo-HCT. As shown in Figure 2, absolute splenocytes and CD3+ T-cell numbers were decreased in recipient mice receiving B6.IL-22−/− T cells as compared with mice receiving B6.WT T cells (Figures 2a and b). Also, the proportion of CD8+ T cells was significantly decreased in the spleen, but not in MLN and PLN, of recipient mice receiving B6.IL-22−/−T cells as compared with mice receiving B6.WT T cells (Figures 2c–e), leading to an increased CD4/CD8 T-cell ratio (Figure 2f, P<0.001 in spleen). These differences observed in vivo were not related to an attenuated proliferative capacity of IL-22−/− T cells, as demonstrated by similar in vivo proliferation assessed by CFSE (carboxyfluorescein succinimidyl ester) staining (Supplementary Figure S1), as well as in vitro proliferation in response to the stimulation by anti-CD3 and anti-CD28 antibodies (Supplementary Figure S2A and B). We also observed a trend towards an increased CD4/CD8 ratio in the PLN and MLN of B6.IL-22−/− T cell allo-HCT-recipient mice (Figure 2f).

Figure 2
figure2

Absence of T-cell-derived IL-22 leads to decreased splenic CD8+ T cells. Lethally irradiated BALB/c recipients were transplanted with B6 TCD BM only or with B6.WT T cells or B6.IL22−/− T cells. Syngeneic controls were transplanted with BALB/c TCD BM and BALB/c T cells. Splenocytes, MLN and PLN from recipient mice were harvested at day 6 post allo-HCT. Absolute numbers of (a) total splenocytes and (b) CD3+ T cells are shown. The proportion of CD4+ and CD8+ T cells in the (c) spleen, (d) MLN and (e) PLN were analyzed by flow cytometry. Mean±s.e.m. is shown (n=12) and data are combined from at least three independent experiments. (f) CD4+/CD8+ ratio is indicated. ND, not determined.

We next investigated the profile of cytokines secreted by alloreactive T cells post allo-HCT. We observed that IL-22 secretion was induced in splenocytes as well as in cells isolated from MLN (Figure 3a) from B6.WT T-cell recipients, but not from B6.IL-22−/− T-cell recipients as expected, further suggesting that IL-22 contributes to aGVHD pathophysiology. In addition, we also observed that intracellular IL-22 staining was revealed in CD4+ T cells in the spleen and MLN in the B6.WT T-cell recipients, but not in syngeneic T-cell recipients, suggesting that IL-22 production resulted, at least in the spleen, from the alloreactive response of T cells (Figures 3b and c). Furthermore, IL-22 secretion was detected only after ex vivo restimulation of T cells (data not shown). We excluded that IL-22 was secreted by γ/δ T cells, because they were not detectable in the spleen and MLN after allo-HCT (data not shown). We next addressed whether the absence of IL-22 in donor T cells affected the generation of TH1 and TH17 cells in vivo 1 week after allo-HCT. We found no significant difference in IL-17A+ T cells among CD3+CD4+ splenocytes (Figure 3d), whereas a strong TH1 response was observed (Figures 3e and f). We could observe a trend for a decrease in the number of IFN-γ secreting CD8+ T cells in the spleen from B6.IL-22−/− allo-HCT recipients as compared with B6.WT recipients (Figures 3e and f), suggesting that IL-22 indirectly could be involved in the modulation of the TH1 response. Nevertheless, we did not observe differences in the capacity of T cells isolated from the spleen of B6.WT or B6.IL-22−/− mice and activated in vitro by anti-CD3/CD28 antibodies to secrete IFN-γ (Supplementary Figure S2C–E). The great majority of IL-22+ CD4+ T cells were also IFN-γ+ but not IL-17A+ (Supplementary Figure S3). We also analyzed the secretion of TNF-α by splenocytes isolated from allo-HCT recipients 1 week after allo-HCT. A significantly reduced number of TNF-α+ CD4+ and CD8+ T cells were detected in B6.IL-22−/− recipients (Figures 3g and h, P<0.01). Moreover, the level of TNF-α measured by enzyme-linked immunosorbent assay in the supernatant of splenocytes activated in vitro for 24 h by phorbol-12-myristate-13-acetate and ionomycin was significantly reduced in B6.IL-22−/−recipients (Figure 3i, P<0.05). Thus, the decreased TNF-α secretion could contribute to the diminished incidence of aGVHD in IL-22−/− T-cell recipients.

Figure 3
figure3

Absence of T-cell-derived IL-22 reduces secretion of inflammatory cytokines. Lethally irradiated BALB/c recipients were transplanted with BALB/c TCD BM with BALB/c T cells (syngeneic) or with B6 TCD BM only or with either B6.WT T cells or B6.IL-22−/− T cells. Splenocytes and MLN cells were harvested at day 6 post allo-HCT. (a) IL-22 level was measured by enzyme-linked immunosorbent assay (ELISA) in the supernatant of splenocytes and MLN cells activated for 48 h with phorbol-12-myristate-13-acetate (PMA) and ionomycin. Mean±s.e.m. are represented. (b, c) Splenocytes and MLN cells were activated for 6 h with PMA, ionomycin and Brefeldin A. Expression of IL-22 on gated CD3+CD4+ T cells is shown for a representative MLN sample (b) and mean±s.e.m. of the percentages of IL-22+ CD4+ T cells in the spleen and MLN is represented (c). (d) Expression of IL-17A on gated CD3+CD4+ T cells in the spleen is represented, mean±s.e.m. from three independent experiments. (ei) Intracellular IFN-γ and TNF-α levels of splenic CD4+ and CD8+ T cells were analyzed. Mean±s.e.m. of the percentage (e) and number (f) of T cells CD4+ and CD8+ secreting IFN-γ in the spleen is shown. Mean±s.e.m. of the percentage (g) and number (h) of T cells CD4+ and CD8+ secreting TNF-α in the spleen is shown. Data are combined from three independent experiments (n=12). (i) TNF-α level was measured by ELISA in the supernatant of splenocytes and MLN cells activated for 24 h with PMA and ionomycin. Mean±s.e.m. is represented from three independent experiments.

Absence of T-cell-derived IL-22 leads to an expansion of Treg cells in lymphoid organs

As IL-22 deficiency in donor T cells resulted in reduced aGVHD, we analyzed whether inhibition of aGVHD involved a Treg-dependent mechanism. On day 6 after allo-HCT, we found a significant increase in the proportion of CD25+Foxp3+ Treg among CD4+ T cells from the spleen (Figures 4a and b, P<0.005) as well as an increase of the numbers of Treg in the spleen of B6.IL-22−/− T-cell recipients as compared with B6.WT T-cell recipients (Figure 4c, P<0.005), suggesting that the increase in Treg could contribute to the regulation of the alloreactive response. This difference was not due to a difference in the graft content because the numbers of Treg CD4+CD25+Foxp3+ were similar in T cells from the spleen of B6.WT or B6.IL-22−/− mice (11.9%±0.7 for WT Treg and 9.8%±1.1 for IL-22−/− Treg, P=0.21). Moreover, we did not detect any differences in their suppressive capacity by evaluating in vitro using CD4+CD25− T cells stained with CFSE cultured in the presence of CD4+CD25+ Treg isolated from B6.WT or B6.IL-22−/− spleen cells (Supplementary Figure S4). To evaluate the contribution of Treg for the IL-22-mediated effects on aGVHD, we performed BMT experiments using CD25-depleted T cells from WT and IL-22−/− donor mice. On day 6 after allo-HCT, the numbers of CD25+Foxp3+ Treg among CD4+ cells were severely decreased in the groups of mice receiving CD25-depleted T cells (Figure 4c), suggesting that the majority of Treg observed at day 6 post allo-HCT resulted from the proliferation of natural Treg present in the graft. However, we could still observe an increase in the numbers of Treg in the spleen of B6.IL-22−/− CD25-depleted T-cell recipients, as compared with B6.WT CD25-depleted T-cell recipients (Figure 4c, P<0.005), suggesting that in the absence of IL-22, both expansion of natural Treg and conversion of naive CD4+CD25− T cells into induced Treg was more effective. We also observed that Treg cells were mainly of donor origin in the spleens of mice receiving B6.WT T cells. By contrast, the proportion of residual recipient Treg cells was significantly increased in mice receiving B6.IL-22−/− T cells, suggesting that they were spared from the alloreactive response (Figure 4d). Finally, when we compared the survival of recipient mice infused with B6.WT CD25-depleted T cells versus B6.IL-22−/− CD25-depleted T cells, we did not observe the beneficial effect of the absence of IL-22, suggesting that Treg cells were involved in the protection conferred by the absence of IL-22 in donor T cells (Figure 4e).

Figure 4
figure4

Absence of T-cell-derived IL-22 is associated with an increase of T regulatory cells. Lethally irradiated BALB/c-recipient mice were transplanted with B6 TCD BM alone or with unmanipulated or CD25-depleted B6.WT or IL22−/− T cells. Syngeneic controls were transplanted with BALB/c T cells. Splenocytes were harvested 1 week after allo-HCT (n=18) and were analyzed for the expression of CD4, CD25 and Foxp3. (a) Expression of CD25 and Foxp3 on gated CD4+ T cells is shown for a representative sample. (b) Mean±s.e.m. of the percentage and (c) absolute number of CD25+Foxp3+ in the spleen of at least three independent experiments with three mice/group are represented. Data comparing the three groups were significantly different using analysis of variance test, P<0.0001. (c) Origin of Treg cells was determined by the expression of H-2d and H2-b after gating on CD4+CD25+foxp3+ T cells. Mean±s.e.m. of the percentage of H-2b donor Treg and H-2d-recipient cells are represented. (e) Survival data after depletion of CD25+ T cells from the graft are shown (log-rank test, P<0.0001 for WT T cells versus IL22−/− T cells, P=0.109 for WT T cells versus IL-22−/− T cells–Treg, P=0.82 for WT T cells versus WT–Treg T cells, P=0.004 for IL-22−/− T cells versus IL-22−/− T cells–Treg, P=0.087 for WT T cells versus IL-22−/− T cells–Treg).

Absence of T-cell-derived IL-22 leads to a reduction of inflammatory mediators both systemically and in aGVHD target organs

To further investigate the underlying mechanism of reduced aGVHD severity in recipients of IL-22−/− T cells, we studied the role of IL-22 in controlling the systemic and local inflammatory response. We evaluated inflammatory cytokine levels in the plasma of recipient mice during aGVHD. A significant decrease of TNF-α, IFN-γ and MCP-1 in the plasma was detected in the first week following allo-HCT with B6. IL-22−/− T cells as compared with B6.WT T cells, whereas IL-6 level was unchanged (Figure 5). We detected neither IL-10 nor IL-12 in the plasma of treated mice (data not shown), suggesting that circulating IL-10, a negative regulator of inflammation, was not involved in the IL-22-deficiency-mediated decreased aGVHD severity.

Figure 5
figure5

Absence of T-cell-derived IL-22 decreases inflammatory cytokine levels in blood. Lethally irradiated BALB/c mice were transplanted with B6 TCD BM and either B6.WT or B6.IL-22−/− T cells. Plasma was collected at day 6 after allo-HCT and cytokines concentrations were measured using BD cytometric bead array set. Data from three independent experiments are shown. Mean is indicated by a bar.

We also examined the local inflammatory response induced in aGVHD target organs, the skin and the small intestine, given the fact that IL-22 has been associated with the pathogenesis of several inflammatory disorders in these organs. As shown in Figure 6, we observed a reduced expression of IFN-γ and the chemokines CXCL9 (C-X-C motif chemokine ligand 9), CXCL10, CXCL11 involved in the recruitment of TH1 lymphocytes, in the small intestine of allo-HCT recipients receiving B6.IL-22−/− T cells. The expression of β-defensin 3 and cathelicidin, both known to be induced by IL-22, were also decreased in the skin of B6.IL-22−/− T-cell allo-HCT recipients, thus likely contributing to a reduced recruitment of immune effector cells in GVHD target tissues. Expression of CXCL9 and IL-6 were also significantly reduced in the lung of B6.IL-22−/− T-cell allo-HCT recipients, indicating that IL-22 deficiency reduced proinflammatory mediators in multiple organs after allo-HCT.

Figure 6
figure6

Absence of T-cell-derived IL-22 decreases inflammatory gene expression in aGVHD target tissues. Lethally irradiated BALB/c mice were transplanted with B6 TCD BM and either B6.WT or B6.IL-22−/− T cells. Quantitative reverse transcriptase–PCR analysis for each indicated gene was performed using RNA isolated from the lung, skin and small intestine collected at day 6 after allo-HCT. Mean±s.e.m. of gene expression levels are represented. Data are combined from three independent experiments (n=12).

The GVL effect is preserved in donor IL-22-deficient T cells

Given the classical association between GVHD and GVL, we next sought to determine whether donor IL-22−/− T cells were able to retain their capacity to control tumor growth in relation to the GVL effect expected in the context of allo-HCT. To this end, we administered A20-luc leukemia cells to allo-HCT-recipient mice, which were subsequently monitored in vivo by BLI. As shown in Figure 7, a strong signal was observed at day 30 in recipient mice receiving TCD-BM and A20 cells, attesting for tumor growth (Figure 7a). By contrast, when recipient mice received both TCD-BM and A20 cells and then WT T cells, a low BLI signal was observed, indicating that tumor growth was controlled by WT T cells (Figures 7a–c). Ultimately, all recipient mice died from severe aGVHD, leading to an increased mortality (Figure 7d). A strong GVL effect was mediated by IL-22−/− T cells as shown by the absence of BLI signal in recipient mice (Figures 7a and b) and was comparable to that obtained with WT T cells (Figure 7c). Moreover, the majority of IL-22−/− T-cell recipients survived through the 40 days observation period without or with very little BLI signal (Figure 7d). Altogether, these data indicated that IL-22−/− T cells retained their capacity to mediate a GVL effect while preserving recipient mice from aGVHD mortality.

Figure 7
figure7

GVL activity is preserved in the absence of donor-T-cell-derived IL-22. Lethally irradiated BALB/c mice were administered with B6 TCD BM and A20-luc leukemic cells. On day 7 after allo-HCT, 1 × 106 B6.WT T cells or B6.IL-22−/− T cells were administered. Tumor growth in recipients was monitored by in vivo bioluminescent imaging (BLI). (a) One representative mouse from each group is shown at days 6 and 30 after administration of T cells. BLI signal intensity (photons/sec measured from the ventral view) is shown at (b) multiple time points or at (c) day 30 after administration of B6.WT or B6.IL-22−/− T cells. Data are obtained from two independent experiments with five recipient mice per group. (d) Recipient survival is shown for the three groups, median survival time 21 days versus undefined days, log-rank test P<0.01).

Discussion

Thus far, the role of IL-22, a TH17-related cytokine, has not been extensively addressed in the context of aGVHD pathophysiology. Our data show that donor-derived IL-22 contributes to the severity of aGVHD by participating into the systemic inflammation process and through amplification of local inflammation in aGVHD target tissues. This is in line with the critical role of IL-22, which can promote local or systemic inflammation.27, 28 Paradoxically, IL-22 has been found not only to promote pathological inflammation but also to prevent tissue destruction. Thus, IL-22 was tissue protective in mouse models of hepatitis,29 whereas IL-22 deficiency exacerbated tissue destruction in a dextran sodium sulfate-mediated colitis18 or a T-cell-transfer model of colitis.19, 20 Recently, Hanash et al.30 demonstrated that IL-22 produced by recipient ILC protected mice from aGvHD-induced tissue damage and mortality by protecting intestinal stem cells, which express IL-22R. Thus, these results highlight the fact that the same cytokine produced by donor versus recipient-derived sources may have opposing effects on clinical outcome of allo-HCT recipients. The inflammatory milieu resulting of the allo-HCT procedure and the induction of the so-called cytokine storm may favor the pathological function of IL-22 rather than its tissue-protective effect during the first week after allo-HCT. This is consistent with data showing that IL-22 was involved in immunopathology of the gut after Toxoplasma gondii infection and development of TH1 cytokine-mediated inflammation.31 The contribution of TH17 cells in aGVHD is somewhat controversial. In our study, we did not detect any differences in IL-17A production by T cells 1 week after allo-HCT. By contrast, we observed a significant increase of CD25+Foxp3+ Treg cells in recipients administered with B6.IL-22−/− T cells. The protective effect of Treg on aGVHD was already demonstrated in several models.32, 33 The mechanism through which IL-22 deficiency results in Treg expansion is not clear, but likely involves indirect factors as Treg cells do not express IL-22R.14, 15 Alternatively, Treg cell expansion could be the result of immune compensatory mechanisms in response to compromised containment of commensal bacteria in the absence of IL-22, leading to a change of the microbiota and the induction of Treg by bacteria.34 Of note, specific changes in the microbiota following allo-HCT have been recently reported and associated to aGVHD.35, 36 In our study, we did not completely abrogate aGVHD by using IL-22−/− T cells to induce GVHD. Although we did not observe a source of IL-22 other than T cells, we cannot exclude that radioresistant ILC from the recipient might persist and could contribute to the regulation of intestinal inflammation. However, Hanash et al30 showed that ILC were eliminated during aGVHD. The role of IL-22 in T-cell activation seems to be indirect given the restricted expression of IL-22R on non-hematopoietic cells.14, 15 However, we cannot exclude that antigen-presenting cells (APC) could modulate the alloreactive response in the absence of IL-22. Indeed, it has been reported that hematopoietic CD11b+ APCs expressed increased levels of IL-22R during induction of disease after immunization with uveitogenic antigen in an experimental model of autoimmune uveitis.37 Despite previous findings showing that MHC (major histocompatibility complex) class II-bearing host APCs are required to induce CD4+ T-cell dependent aGVHD,38 recent reports indicated that non-hematopoietic APCs are sufficient to induce lethal aGVHD.39, 40 In fact, MHC class II-expressing recipient non-hematopoietic cells of the gastrointestinal tract were shown to be able to induce aGVHD.39 By using well established BM chimeras, the group of Reddy and colleagues recently demonstrated that sex-mismatched H-Y antigens can induce GVHD even in the absence of radiosensitive host-derived APC.40 Thus, one may speculate that the modulation of IFN-γ in the gastrointestinal tract in the absence of IL-22 could impair the induction of class II expression on epithelial cells, leading to decreased incidence and severity of aGVHD. Interestingly, both TNF-α and IFN-γ have been shown to upregulate IL-22R expression in keratinocytes and fibroblasts, suggesting that these cytokines released after conditioning may amplify IL-22 activity on target tissues of aGVHD.14, 41 Notably, it has been recently reported that the C-type lectin regenerating-islet-3a (REG3α), which is produced by Paneth cells and induced by IL-22, is detected in the serum of aGVHD patients and validated as a biomarker of intestinal aGVHD.42 Therefore, REG3α expression likely increased upon inflammation and is then released into the systemic circulation as a consequence of breaches in the mucosal epithelial barrier caused by aGVHD. Interestingly, we also observed in our model an increase in antimicrobial peptide expression, such as β-defensin3 and cathelicidin, during aGVHD. As expected, in the absence of IL-22, the expression of these molecules was downregulated. Of note, another alarmin, namely Elafin, was also reported as a biomarker of human skin aGVHD.43 Finally, because of the critical role of IL-22 in keratinocyte differentiation and pathology of the skin,28, 44 we can also speculate that IL-22 could mediate a role in skin pathology during GVHD. Indeed, recent data demonstrated a crucial role of IL-22 in thymic regeneration after thymic injury. In the elegant study of Dudakov et al.,45 they observed that intrathymic IL-22 was produced by ILC after thymic insult and was essential to promote thymic recovery. Thus, this study emphasize the importance of IL-22 in allo-HCT by inducing a delicate balance between T-cell recovery and adverse effects on alloreactivity.

Overall, findings from this work suggest a potential important role of IL-22 in aGVHD pathophysiology. From the translational standpoint, the IL-22/IL-22R axis need to be further explored to delineate its inflammatory versus protective effects in GVHD. Data showing that blocking donor-derived IL-22 can attenuate aGVHD without impairing the GVL effect support the design of clinical approaches aiming to target IL-22 pathways in allo-HCT patients.

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Acknowledgements

We thank the technical support of D. Paris for animal care. We also thank the ‘Association pour la Recherche sur le Cancer (ARC; Grant No.3175 to MM and BG)’, the ‘Agence de Biomédecine’, the ‘Association Cent pour Sang la Vie’, the Conseil Régional de Franche-Comté (AutoMACS Pro), the Agence Nationale de la Recherche (Labex LipSTIC, ANR-11-LABX-0021) and the Etablissement Français du Sang (AO#2011-11) for their generous and continuous support for our research work.

Author contributions

All authors listed in the manuscript have contributed substantially to this work. BG designed experimental research, interpreted data and wrote the manuscript; MC and BL participated in experimental design, performed research, analyzed data and wrote the manuscript; JA, FB and SP participated in experimental work; JCR generated IL-22-deficient mice; FM and CB performed histological analyses; MM, PS and PT participated in experimental design, interpretation of data and helped in writing and revising the manuscript.

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Correspondence to B Gaugler.

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The authors declare no conflicts of interest.

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Couturier, M., Lamarthée, B., Arbez, J. et al. IL-22 deficiency in donor T cells attenuates murine acute graft-versus-host disease mortality while sparing the graft-versus-leukemia effect. Leukemia 27, 1527–1537 (2013) doi:10.1038/leu.2013.39

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Keywords

  • IL-22
  • GVHD
  • transplantation

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