Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Dec 31.
Published in final edited form as: Biol Blood Marrow Transplant. 2009 Oct 2;16(2):10.1016/j.bbmt.2009.09.023. doi: 10.1016/j.bbmt.2009.09.023

T helper17 Cells Are Sufficient But Not Necessary to Induce Acute Graft-Versus-Host Disease

Cristina Iclozan 1, Yu Yu 1, Chen Liu 2, Yaming Liang 1, Tangsheng Yi 3, Claudio Anasetti 1,4, Xue-Zhong Yu 1,4
PMCID: PMC3876952  NIHMSID: NIHMS455965  PMID: 19804837

Abstract

T helper (Th)1 cells were considered responsible for the induction of graft-versus-host disease (GVHD), but recently the concept has been challenged. Th17 cells play a critical role in mediating autoimmune diseases, but their role in the pathogenesis of GVHD remains unclear. Herein we compare the ability of in vitro generated Th1 and Th17 cells from C57BL/6 mice to induce GVHD in lethally irradiated BALB/c recipients. Allogeneic Th17 cells had superior expansion and infiltration capabilities in GVHD target organs, which correlated with their increased pathogenicity when compared with naïve or Th1 controls. Th17 cells caused no pathology in the syngeneic recipients, indicating that antigen-activation was required for their pathogenicity. Polarized Th17 cells could not maintain their phenotype in vivo as they produced a significant amount of interferon (IFN)-γ after being transplanted into allogeneic recipients; however, IFN-γ was not required for Th17 cell-induced GVHD. Further, we evaluated the pathogenesis of Th17 cells in GVHD by using polyclonal nonprimed CD4 T cells in a clinically relevant allogeneic bone marrow transplantation (BMT) setting. We found that disruption of Th17-differentiation alone by targeting RORγt (Th17-specific transcription factor) had no significant effect on GVHD development. We conclude that Th17 cells are sufficient but not necessary to induce GVHD.

Keywords: Th1, Th17, BMT, GVHD

INTRODUCTION

The therapeutic potential of allogeneic bone marrow transplantation (BMT) or hematopoietic cell transplantation (HCT) offers great promise for the treatment of various hematologic diseases. However, graft-versus-host disease (GVHD) remains the major complication, leading to high morbidity and mortality of the patient [1]. Mature donor T cells recognize disparate histocompatibility antigens of the recipient and cause injuries in skin, liver, gastrointestinal epithelium, lung, and elsewhere, initiating GVHD [2].

Th17 cells are a newly identified T cell lineage that develops via cytokine signals antagonized by products of the Th1 (interferon [IFN]-γ) and Th2 (interleukin [IL]-4) lineages [3,4]. IL-6 and tumor growth factor (TGF)-β induce Th17 differentiation [5–7], suggesting a potential kinship between these inflammatory mediators and regulatory T cells (Treg), which have a distinct anti-inflammatory role. TGF-β is known to suppress Th1 and Th2 differentiation while inducing generation of Tregs. IL-6, an inflammatory cytokine like tumor necrosis factor (TNF)-α and IL-1, acts as a switch upstream of TGF-β directing Th17 cell development by its presence or permitting Treg to develop in its absence. Blocking IL-6 signaling was recently found to shift the balance from proinflammatory T cells to Tregs [8]. At the transcription level, Th17 lineage is uniquely regulated by RORγt and Stat3 [9–11].

How various subsets of cytokine-producing T cells contribute to disease development is an active area in GVHD research [12,13]. Th1 cells are considered as the primary Th subset to induce GVHD, but Th2 cells are also involved [14,15]. Recently, Carlson et al. [16] reported that in vitro generated Th17 cells are able to induce acute GVHD(aGVHD). However, CD4 naïve T cells deficient for IL-17 caused ameliorated GVHD [17], but total T cells deficient for IL-17 have caused accelerated GVHD [18]. In the current study, we found that Th17 cells were highly pathogenic to induce GVHD, more so than naïve or Th1 controls. The high potency of Th17 cells in the induction of GVHD was associated with their superior ability to expand, survive, and infiltrate in vivo. However, we provide evidence that the Th17 subset alone is not necessary to cause GVHD.

MATERIALS AND METHODS

Mice

C57BL/6 (B6, H-2b), and BALB/c (H-2d) mice were purchased from the National Cancer Institute (Bethesda, MD). B6 congenic Ly5.1 (H-2b), DO11.10 TCR transgenic (Tg), IFN-γ KO, and RORγt KO mice were from the Jackson Laboratory. Luciferase-transgenic (Luc-Tg) strain on B6 background were kindly provided by Dr. R. Negrin (Stanford University, CA) [19]. All the mice used in this study were housed in a pathogen-free facility at H. Lee Moffitt Cancer Center & Research Institute (Tampa, FL). Experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee.

Generation of Th1 and Th17 Cells

Spleen and lymph node cells from B6 mice were depleted of CD8+ and CD25+ cells with magnetic beads. Residual CD4+CD25− cells (20%–25%) were stimulated with 2 μg/mL anti-CD3 monoclonal antibody (mAb) at 4 × 106/mL. Anti-IL-4 (10 μg/mL), IL-12 (10 ng/mL), and IFN-γ (1000 U/mL) were added to generate Th1 cells. Anti-IL-4, anti-IFN-γ mAb, IL-6 (10 ng/mL), and TGF-β (2–5 ng/mL) were added to generate Th17 cells. Cell culture was split 1:2 in media with mIL-2 (50 U/mL) on day 3. The cell phenotype was confirmed in day 4 after restimulation with Phorbol 12-myristate 13-acetate + ionomycin for 4 to 5 hours, adding GolgiStop for the last 2 hours. The cells were then stained for surface expression of CD4 and intracellular expression of IFN-γ and IL-17. In vitro generated Th cells were used 5 to 7 days after activation, unless specified.

Isolation of T Cells and Bone Marrow (BM) Cells from Donor Mice

CD4+ T cells were purified from pooled spleen and lymph node cells by negative selection to remove CD8+, CD25+ T cells and other non-T cells using MACS (Miltenyi Biotec, Auburn, CA). BM was harvested from tibia and femurs, and T cells were depleted through complement lysis of Thy1.2+ cells.

BMT and Bioluminescent Imaging (BLI)

In myeloablative (MA) BMT models, BALB/c mice at 8 to 10 weeks old were exposed to 800 to 900 cGy of total body irradiation (TBI). Antibiotics were added to the drinking water for the following 4 weeks. T cell-depleted BM (TCD-BM) cells alone or in combination with purified T cells from indicated donors were injected via the tail vein into recipients within 24 hours after irradiation. BM cells were typically isolated from B6 Ly5.1 congenic mice, whereas peripheral T cells were from normal B6 (Ly5.2) donors, so that BM-derived T cells and peripheral T cells can be distinguished in the recipient. Recipient mice were monitored every other day for clinical signs of GVHD, such as ruffled fur, hunched back, inactivity, or diarrhea, and mortality. Animals judged to be moribund were sacrificed and counted as GVHD lethality. In vivo BLI of BALB/c recipients transplanted with T cells from Luc-Tg B6 donors and BM from non-Tg B6 donors was performed as described previously [20], using an IVIS200 charge-coupled device imaging system (Xenogen, Alameda, CA).

Flow Cytometry Analysis

Multicolor flow cytometry was performed to measure the expression of surface molecules or intracellular cytokines according to standard techniques. All the fluorescence conjugated-mAbs were purchased from BD-Pharmingen or eBiosciences (San Diego, CA). Cell acquisition and analysis were performed by using a FACScan, FACSCalibur or LSR II, and CellQuest (BD Biosciences) or FlowJo software (Tree Star, Ashland, OR).

Histopathologic Analysis

Histopathology on small and large bowel, liver, and lung was assessed by an expert pathologist (C.L.) in a blinded fashion. Formalin-fixed tissues were embedded in paraffin, sectioned, and stained with H&E. The tissue sections were examined under a microscope, and the severity of the tissue damage was graded with a semiquantitative scoring system. Intestinal and liver tissues were scored for 19 to 22 different parameters and lung tissues were scored for 6 different parameters associated with GVHD as previously described [20].

Statistics

The log-rank test was used to detect statistical differences in recipient survival in GVHD experiments. Student’s t-test was used to compare percentages or numbers of donor T cells and pathologic scores.

RESULTS

In Vitro Polarized Th17 Cells Induce Severe aGVHD in Allogeneic Recipients

To test the pathogenicity of Th1 and Th17 cells in the induction of GVHD, we adapted previously established protocols for in vitro generating Th1 and Th17 cells from naïve T cells [5–7]. Under polarizing culture conditions for 5 days, we obtained CD4 T cells that produced IFN-γ and no IL-17 or IL-4 as Th1 cells, or produced IL-17, but no IFN-γ or IL-4 as Th17 cells (Figure 1A). The percentage of IFN-γ+ and IL-17+ cells typically ranged from 60% to 80% among Th1 and Th17 cells, respectively. IL-4 expression was minimal (<1%) for both cell types (data not shown).

Figure 1.

Figure 1

Open in a new tab

Potency and pathologic injures of Th1 and Th17 cells in GVHD induction. (A) Th1 and Th17 cells were generated from B6 naïve CD4+ T cells in vitro as described in Materials and Methods. Intracellular expression of IFN-γ and IL-17 on live CD4+ cells is shown on Th1 and Th17 cells in day 4 after in vitro polarization. Intracellular expressions of IL-4 on both cell populations were<1% (data not shown). Lethally irradiated BALB/c (B-D) or B6 (E) recipients were transplanted with B6 TCD-BM alone, or plus B6 CD4+ naïve (B), Th1 (C), or Th17 (D,E) cells as indicated. Data in graph B and E were from 1 experiment with 5 to 6 mice per group, and data in graph C and D were pooled from 3 to 4 replicate experiments with 10 to 18 mice per group.

To compare the ability of differentiated Th cells in the induction of GVHD, Th1 or Th17 cells were transplanted with TCD-BM into lethally irradiated BALB/c recipients. Recipients of TCD-BM alone were used as negative controls without GVHD, and recipients of TCD-BM plus naïve CD4 T cells were used as positive controls. Potency of Th17 cells in the induction of GVHD was assessed by titrating the numbers of naïve, Th1, and Th17 cells in allogeneic BMT. We found that Th17 cells were at least as pathogenic as naïve CD4 T cells, but more potent than Th1 cells in the induction of GVHD (Figure 1B–D). In syngeneic recipients Th17 cells failed to induce any sign of GVHD (Figure 1E), demonstrating that their pathogenic activity is antigen-specific.

We next studied the pathogenesis of Th17 cells in the development of GVHD by comparing them with Th1 cells. To evaluate the pathologic injuries in GVHD target organs caused by Th17 or Th1 cells in allogeneic recipients, we harvested liver, intestine, colon, and lung tissues 2 to 3 weeks after BMT and did the standard pathologic analysis (H&E staining). Recipients of BM alone had quite normal histology without evidence of inflammatory cell infiltration or cellular damage in the liver, intestine, and lung. In the recipients of BM+Th1 cells, there was minimal portal infiltration and scattered apoptosis in the liver, mixed inflammatory cell infiltration, apoptosis of epithelial cells and crypt dropout in the intestine, and mild perivascular infiltration in the lung. In contrast, in the recipients of BM+Th17 cells, there was severe inflammation, endotheliitis, and apoptosis in the liver, overt inflammation, apoptosis and crypt dropout in the intestine, and perivascular and peribronchial inflammatory infiltration in the lung (Figure 2A). Using more quantitative analysis, we found Th17 cells generated significantly more damage in recipient lung (P=.02) than Th1 cells (Figure 2B).

Figure 2.

Figure 2

Open in a new tab

Pathologic injuries caused by Th1 and Th17 cells. Lethally irradiated BALB/c mice were transplanted with B6 TCD-BM alone or together with Th1 or Th17 cells (1 × 106 each) generated from B6 CD4 T cells. Recipient mice were euthanized and organs were harvested 2–3 weeks after BMT. Formalin-fixed tissues were embedded in paraffin, sectioned, and stained with H&E. (A) Representative micrographs are from liver (upper), intestine (middle), and lung (lower) of BM alone, Th1, or Th17 groups (original magnification ×200). (B) Histopathology was assessed by an expert pathologist in a blind fashion. The data are pooled from 2 replicate experiments with 5 to 7 recipients per group.

Th17 Cells Have Superior Ability to Expand In Vivo

To further understand the pathogenicity of Th17 versus Th1 cells in GVHD, we generated them using naïve CD4 T cells from Luc-Tg mice on a B6 background [21]. TCD-BM from B6 donors were transplanted alone or together with luc+ Th1 or Th17 cells at 1 × 106 each into lethally irradiated BALB/c recipients. Because 1 × 106 Th17 cells will induce severe GVHD (Figure 1D), an additional group with 0.25 × 106 Th17 cells was included for comparison. The end point of this experiment was to follow donor T cell expansion and infiltration in vivo using the BLI technology. Based on the density and distribution of the BLI signals, Th17 cells expanded substantially more than Th1 cells (Figure 3). Both Th1 and Th17 cells migrated in multiple target organs such as the mouth, thymus, liver, lung, and intestines, but the overall signal intensity of Th17 cells was significantly higher than that of 1 × 106 Th1 cells (Figure 3A) (P < .01, at days 7 and 11). Furthermore, even 0.25 × 106 Th17 cells generated higher signal intensity than 1 × 106 Th1 cells at later time points (Figure 3B) (P=.005, at day 15). These data were consistent with the potency of Th1 and Th17 cells in the induction of GVHD (Figure 1), suggesting that the superior ability of Th17 cells in expansion and infiltration contributes to severe GVHD.

Figure 3.

Figure 3

Open in a new tab

Expansion and accumulation of Th1 and Th17 cells in allogeneic recipients. Lethally irradiated BALB/c mice were transplanted with B6 TCD-BM alone (5 × 106) or together with Th1 or Th17 cells in vitro generated from naïve CD4 T cells of Luc-Tg mice on B6 background. One million Th1, Th17, or 0.25 × 106 Th17 cells per recipient were transplanted. On day 4, 7, 11, and 15 after BMT recipient mice were injected i.p. with luciferin substrate. (A) Animals were imaged from the ventral position for quantification of donor T cells, and 2 representative mice per group are shown. (B) The average of relative signal intensity of 4 to 5 mice per group, and the data represent 1 of 3 replicate experiments.

GVHD Induced by Th17 Cells Is Refractory to a Treatment That Depletes Alloreactive T Cells

Our recent studies found that Th17 cells are resistant to activation-induced cell death (AICD) [22]. We further reasoned that such a resistance might not only render Th17 cells highly pathogenic, but also result in Th17-mediated GVHD refractory to certain treatments. To test this hypothesis, we selected a treatment with anti-CD3ε F(ab′)2, which was proven to prevent GVHD by selectively depleting alloreactive T cells through induction of apoptosis [23]. We found that anti-CD3-treatment effectively prevented GVHD induced by Th1, but not by Th17 cells (Figure 4). Given that we recently reported that Th17 cells were undergoing significantly less apoptosis than Th1 cells after TCR-ligation in vivo [22], these data suggest that the resistance of Th17 cells to AICD may contribute to high levels of expansion and subsequently high pathogenicity in GVHD development.

Figure 4.

Figure 4

Open in a new tab

Anti-CD3 treatment ameliorates GVHD induced by Th1 cells, but not by Th17 cells. Lethally irradiated BALB/c mice were transplanted with TCD-BM alone or plus polarized Th1 (4 × 106/mouse) or Th17 cells (2 × 106/mouse) from B6 donors. Half of the recipient mice received anti-CD3 treatment (20 μg/recipient i.p. every other day starting on day 0 for 10 doses). Percentages of survival (A) and body weight change (B) are shown, and 4 to 5 recipients were used in each group.

IFN-γ Is Not Required for GVHD Induced by Th17 Cells

One important question we asked was whether polarized Th17 cells could maintain their phenotype over time. We generated antigen-specific Th1 and Th17 cells in vitro under polarizing culture condition, and then measured their cytokine profiles upon restimulation without polarizing cytokines. Under this condition, previously polarized Th17 cells produced high levels of IL-17, but also significant amounts of IFN-γ. Conversely, previously polarized Th1 cells produced high levels of IFN-γ, but little or no IL-17 (Figure 5). These results suggest that phenotypes of Th17 cells were less stable than that of Th1 cells. To further address whether established Th17 cells could maintain their phenotype in vivo, we polarized naïve CD4 T cells toward the Th17 phenotype for 2 cycles in vitro and obtained a highly enriched IL-17+ population (>90%) with minimal contamination of IFN-γ+ cells. Those polarized Th17 cells could secrete considerable amounts of IFN-γ and TNF-α, in addition to IL-17 after in vivo transfer (Figure 6A, and data not shown). To test whether IFN-γ was also required for the effector function of Th17 cells to induce GVHD in vivo, we compared the ability of Th17 cells generated from WT or IFN-γ KO CD4 T cells in the induction of GVHD. As expected, most of the mice receiving WT Th17 cells died within 60 days after BMT, whereas 40% of the recipients of IFN-γ KO Th17 cells still survived (Figure 6B). Within the experimental constraints, weight loss (Figure 6C) and survival curves were similar after BMT with WT or KO Th17 cells (P > .05, Figure 6B and C). These data indicate that Th17 cells can produce Th1 cytokines in addition to IL-17 (IL-17+ IFN-γ+ TNF-α+) or redifferentiate into Th1 cells without IL-17 production (IL17− IFN-γ+ TNF-α+), but IFN-γ is dispensable for the development of GVHD caused by Th17 cells.

Figure 5.

Figure 5

Open in a new tab

Stability of Th1 and Th17 phenotype after restimulation in vitro. Naïve T cells from DO11.10 TCR Tg mice were activated with Ova323-339 peptide under polarizing conditions toward Th1 and Th17 for 4 days as described in Figure 1A. After polarization, phenotypes of DO11.10 Th1 and Th17 cells were confirmed as those in Figure 1A (data not shown). Two weeks after primary stimulation, rested Th1 (empty bars) and Th17 (filled bars) cells at 1 × 106/mL were restimulated with A20, A20 pulsed with OVA peptide, or OVA-transduced (low level OVA expression) A20 B cell lymphoma at 0.5 × 106/mL. Culture supernatants were harvested 24 hours later and measured for levels of IFN-γ (A) and IL-17 (B). The data represent 1 of 2 replicate experiments.

Figure 6.

Figure 6

Open in a new tab

Role of IFN-γ in Th17 cells induced GVHD. Lethally irradiated BALB/c mice were transplanted with TCD-BM alone, or plus 2 × 106/mouse Th17 cells generated from WT mice (A), and 0.5 × 106/mouse Th17 cells generated from WT or IFN-γ−/− B6 mice, respectively (B,C). (A) After 22 days intracellular expression of IL-17, IFN-γ, and TNF-α were measured. % mean ± 1 SD of intracellular expression of IFN-γ (upper panel) and TNF-α (lower panel) are shown together with IL-17 on donor CD4+H2b+ cells from recipient spleen and liver. GVHD severity assessed by changes over time in recipient survival (B) and body weight (C) is shown. Data are pooled from 2 experiments with 10 to 12 recipients per group.

Th17 Subset Alone Is Not Necessary for the Development of GVHD

The results presented so far indicate that polarized Th17 cells were highly pathogenic and sufficient to induce GVHD by themselves. These studies, however, did not reveal whether Th17-differentiation was necessary for GVHD development under more pathophysiologic situations. Therefore, we next asked whether Th17 subset is required for the development of GVHD. To address this question, we chose to examine whether T cells deficient for RORγt were able to induce GVHD, because RORγt is a necessary transcription factor for Th17-differentiation [9,11]. However, we found that RORγt−/− T cells had a comparable ability to induce GVHD (Figure 7), suggesting that Th17-differentiation was not required for GVHD development.

Figure 7.

Figure 7

Open in a new tab

RORγt−/− and WTCD4 T cells have comparable ability to induce GVHD. Lethally irradiated BALB/c mice were transplanted with TCD-BM alone or plus purified CD4+ T cells (1–2 × 106/mouse) isolated from WT or RORγt−/− mice on B6 background. Percentages of survival (A) and body weight change (B) are shown. Data cumulate 2 independent experiments with 8 to 9 mice per group.

DISCUSSION

For a long period of time, Th1 cells have been considered the most important CD4 T cell subset to induce GVHD through inflammatory cytokines including IFN-γ and TNF-α. However, the concept was seriously challenged by recent findings indicating that T cells deficient for Stat4 or IFN-γ caused severe or even exacerbated GVHD [24–26]. These data were interpreted as IFN-γ promotes apoptosis of antigen activated T cells and plays an essential role in T cell tolerance in transplantation. However, these data also demonstrate that Th1 subset is not necessary and other Th subsets are sufficient for the GVHD induction. Our current study unequivocally demonstrates that Th17 subset was sufficient to induce GVHD, and furthermore, Th17 cells were significantly more potent than Th1 cells (Figures 1–2) and Th0 cells (not shown). Preactivated T cells are known to have much reduced ability to cause GVHD. Previous studies showed that a large number of these T cells (ie, 10–20 × 106) were required to induce lethal GVHD and our data also indicated that >2 × 106 Th1 cells were needed to cause lethality (Figure 1C). The evidence that 0.25 × 106 activated and polarized Th17 cells were able to cause GVHD lethality on majority of allogeneic recipients demonstrates that Th17 subset is extremely pathogenic (Figure 1D).

We consider that a few factors make Th17 cells highly pathogenic. The level of Th17 cell expansion was markedly higher than Th1 cells, supported by stronger BLI signals in GVHD target organs (Figure 3). The high levels of expansion for Th17 cells could be because of faster rates of cell division and/or less cell death. We recently reported that Th17 cells are significantly less sensitive to AICD than Th1 cells in allogeneic recipients because of high levels of c-FLIP, which prevent Fas-mediated apoptosis [22]. Furthermore, Th17, but not Th1 cell-mediated GVHD did not respond to anti-CD3-treatment (Figure 4), which depletes alloreactive T cells primarily through induction of AICD [23]. Therefore, the Th17 cells resistance to AICD at least partially contributes to their high level of expansion and subsequently high pathogenicity in GVHD.

Th17 cells caused damages in overall target tissues as severe as Th1 cells, and more severe damage in lungs than Th1 cells (Figure 2A and B). This finding correlates with other studies showing that IFN-γ-deficient donor T cells caused augmented idiopathic pneumonia syndrome [27,28]. These results are also supported by severe lung inflammation in the transgenic mice that overexpress IL-17A in lung epithelial cells [4], likely by recruiting granulocytes and macrophages. In addition to pulmonary lesions, a recent study by Carlson et al. [16] also showed severe cutaneous pathology induced by Th17 cells, which was not observed in our study (not shown). We speculate that a different GVHD model might account for the discrepancy on cutaneous pathology.

Although in vitro generated Th17 cells essentially did not produce IFN-γ after polarization (Figure 1A), the same cells could produce IFN-γ upon restimulation without polarizing cytokines in vitro (Figure 5) and in vivo (Figure 6). These data suggest that unpolarized cells in the Th17 population could become IFN-γ-producing Th1 cells and/or polarized Th17 cells could start to produce IFN-γ together with IL-17 upon restimulation under no polarizing conditions. Highly enriched Th17 cells after adoptive transfer secreted considerable amounts of inflammatory cytokines IFN-γ and TNF-α besides IL-17 in the recipient (Figure 6A). These data are consistent to recent studies by others showing that in vitro polarized Th17 cells have a plastic or unstable cytokine expression profile [29,30]. Nonetheless, nonpolarized Th0 cells (data not shown) and polarized Th1 cells were less pathogenic than Th17 cells (Figure 1). Furthermore, Th17 cells deficient for IFN-γ were still able to induce GVHD (Figure 6B and C), demonstrating that IFN-γ is not required for, although it contributes to, pathogenicity of Th17 cells. The effector molecules responsible for Th17-mediated GVHD are not entirely clear. Most likely, IL-17 serves as an important effector cytokine in the development of GVHD, because IL-17 has been shown to cause direct tissue damages [31], and neutralizing IL-17 markedly decreased skin and lung GVHD [16,28,32]. Polarized Th17 cells produce significant levels of TNF-α (although lower than Th1 cells) after restimulation in vitro [33,34] as well as in vivo in allogeneic recipients (Figure 6A). Moreover, the contribution of TNF-α in Th17-mediated GVHD is further supported by the observations that administration of TNF-α antagonists significantly reduced GVHD induced by Th17 cells [16,28]. Finally, other cytokines produced by Th17 cells (ie, IL-21 and IL-22) may also contribute to Th17-mediated GVHD, but direct evidence remains to be found.

By using in vitro polarized Th17 cells, this study and the one by Carlson et al. [16] indicate Th17 cells are sufficient to induce GVHD, but does not exclude that Th1 cells or their cytokines can be important effectors. Because Th17 cells produce multiple distinct cytokines including IL-17A, IL-17F, IL-21, and IL-22, these data do not imply that IL-17A is sufficient or even necessary for the induction of GVHD. Moreover, these studies did not reveal whether Th17-differentiation was necessary for GVHD development under more pathophysiologic situations, because in vitro polarized Th17 cells may not truly represent the subset (ie, IL-17-producing T cells) of differentiated from naïve CD4 T cells in vivo after BMT. In further study, we have shown that RORγt−/− CD4 T cells were equally able to induce GVHD as WT counterparts (Figure 7). These results indicate that GVHD can occur in the absence of Th17-differentiation, which is consistent with the findings by others that Th1- or even Th2-subset alone is adequate to induce GVHD [25,35]. However, Th17 cells do differentiate from naïve T cells under allogeneic BMT, and Th17 cells are highly pathogenic. Thus, high levels of IL-17 or unbalanced T cell differentiation toward Th17-linage may correlate with high grades of GVHD after allogeneic HCT. Furthermore, because Th17 cells are resistant to apoptosis [22] and Th17-induced GVHD is refractory to treatment that induces T cell depletion (Figure 4), the current study also provides additional information to guide for the selection of proper treatment for Th17-mediated GVHD, for example, blocking Th17-effector function rather than inducing apoptosis of Th17 effectors.

Acknowledgments

The authors thank Drs. Amer Beg, Esteban Celis, and Lia Perez for their critical discussion on this project. They are grateful for the technical assistance provided by Ms. Francisca Beato, the Flow Cytometry and Mouse Core Facility at the Moffitt Cancer Center. This work was supported by the NIH Grants AI 63553 and CA 118116 (to X.-Z.Y.). X.-Z. Yu was a recipient of New Investigator Award supported by ASBMT.

Footnotes

Financial disclosure: The authors have nothing to disclose.

References

  • 1.Appelbaum FR. Haematopoietic cell transplantation as immunotherapy. Nature. 2001;411:385–389. doi: 10.1038/35077251. [DOI] [PubMed] [Google Scholar]
  • 2.Blazar BR, Murphy WJ. Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD) Philos Trans R Soc Lond B Biol Sci. 2005;360:1747–1767. doi: 10.1098/rstb.2005.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol. 2005;6:1123–1132. doi: 10.1038/ni1254. [DOI] [PubMed] [Google Scholar]
  • 4.Park H, Li Z, Yang XO, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol. 2005;6:1133–1141. doi: 10.1038/ni1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238. doi: 10.1038/nature04753. [DOI] [PubMed] [Google Scholar]
  • 6.Mangan PR, Harrington LE, O’Quinn DB, et al. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature. 2006;441:231–234. doi: 10.1038/nature04754. [DOI] [PubMed] [Google Scholar]
  • 7.Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 2006;24:179–189. doi: 10.1016/j.immuni.2006.01.001. [DOI] [PubMed] [Google Scholar]
  • 8.Chen X, Das R, Komorowski R, et al. Blockade of interleukin-6 signaling augments regulatory T cell reconstitution and attenuates the severity of graft versus host disease. Blood. 2009;114:891–900. doi: 10.1182/blood-2009-01-197178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Ivanov II, McKenzie BS, Zhou L, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17 + T helper cells. Cell. 2006;126:1121–1133. doi: 10.1016/j.cell.2006.07.035. [DOI] [PubMed] [Google Scholar]
  • 10.Yang XO, Panopoulos AD, Nurieva R, et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J Biol Chem. 2007;282:9358–9363. doi: 10.1074/jbc.C600321200. [DOI] [PubMed] [Google Scholar]
  • 11.Yang XO, Pappu BP, Nurieva R, et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity. 2008;28:29–39. doi: 10.1016/j.immuni.2007.11.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ferrara JL. Cytokine dysregulation as a mechanism of graft versus host disease. Curr Opin Immunol. 1993;5:794–799. doi: 10.1016/0952-7915(93)90139-j. [DOI] [PubMed] [Google Scholar]
  • 13.Krenger W, Hill GR, Ferrara JL. Cytokine cascades in acute graft-versus-host disease. Transplantation. 1997;64:553–558. doi: 10.1097/00007890-199708270-00001. [DOI] [PubMed] [Google Scholar]
  • 14.Via CS, Rus V, Gately MK, Finkelman FD. IL-12 stimulates the development of acute graft-versus-host disease in mice that normally would develop chronic, autoimmune graft-versus-host disease. J Immunol. 1994;153:4040–4047. [PubMed] [Google Scholar]
  • 15.Williamson E, Garside P, Bradley JA, Mowat AM. IL-12 is a central mediator of acute graft-versus-host disease in mice. J Immunol. 1996;157:689–699. [PubMed] [Google Scholar]
  • 16.Carlson MJ, West ML, Coghill JM, Panoskaltsis-Mortari A, Blazar BR, Serody JS. In vitro-differentiated TH17 cells mediate lethal acute graft-versus-host disease with severe cutaneous and pulmonary pathologic manifestations. Blood. 2009;113:1365–1374. doi: 10.1182/blood-2008-06-162420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kappel LW, Goldberg GL, King CG, et al. IL-17 contributes to CD4-mediated graft-versus-host disease. Blood. 2009;113:945–952. doi: 10.1182/blood-2008-08-172155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Yi T, Zhao D, Lin CL, et al. Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graft-versus-host disease. Blood. 2008;112:2101–2110. doi: 10.1182/blood-2007-12-126987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Edinger M, Hoffmann P, Ermann J, et al. CD4 + CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med. 2003;9:1144–1150. doi: 10.1038/nm915. [DOI] [PubMed] [Google Scholar]
  • 20.Liang Y, Liu C, Djeu JY, et al. Beta2 integrins separate graft-versus- host disease and graft-versus-leukemia effects. Blood. 2008;111:954–962. doi: 10.1182/blood-2007-05-089573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Negrin RS, Contag CH. In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease. Nat Rev. 2006;6:484–490. doi: 10.1038/nri1879. [DOI] [PubMed] [Google Scholar]
  • 22.Yu Y, Iclozan C, Yamazaki T, et al. Abundant c-FLIP expression determines resistance of Th17 cells to activation-induced cell death. Blood. 2009;114:1026–1028. doi: 10.1182/blood-2009-03-210153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Yu XZ, Bidwell SJ, Martin PJ, Anasetti C. Anti-CD3 epsilon F(ab′)2 prevents graft-versus-host disease by selectively depleting donor T cells activated by recipient alloantigens. J Immunol. 2001;166:5835–5839. doi: 10.4049/jimmunol.166.9.5835. [DOI] [PubMed] [Google Scholar]
  • 24.Murphy WJ, Welniak LA, Taub DD, et al. Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice. J Clin Invest. 1998;102:1742–1748. doi: 10.1172/JCI3906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Nikolic B, Lee S, Bronson RT, Grusby MJ, Sykes M. Th1 and Th2 mediate acute graft-versus-host disease, each with distinct end-organ targets. J Clin Invest. 2000;105:1289–1298. doi: 10.1172/JCI7894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Yang YG, Dey BR, Sergio JJ, Pearson DA, Sykes M. Donor-derived interferon gamma is required for inhibition of acute graft-versus-host disease by interleukin 12. J Clin Invest. 1998;102:2126–2135. doi: 10.1172/JCI4992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Burman AC, Banovic T, Kuns RD, et al. IFNgamma differentially controls the development of idiopathic pneumonia syndrome and GVHD of the gastrointestinal tract. Blood. 2007;110:1064–1072. doi: 10.1182/blood-2006-12-063982. [DOI] [PubMed] [Google Scholar]
  • 28.Mauermann N, Burian J, von Garnier C, et al. Interferon-gamma regulates idiopathic pneumonia syndrome, a Th17 + CD4 + T-cell-mediated graft-versus-host disease. Am J Respir Crit Care Med. 2008;178:379–388. doi: 10.1164/rccm.200711-1648OC. [DOI] [PubMed] [Google Scholar]
  • 29.Nurieva R, Yang XO, Chung Y, Dong C. Cutting edge: in vitro generated Th17 cells maintain their cytokine expression program in normal but not lymphopenic hosts. J Immunol. 2009;182:2565–2568. doi: 10.4049/jimmunol.0803931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Yang XO, Nurieva R, Martinez GJ, et al. Molecular antagonism and plasticity of regulatory and inflammatory T cell programs. Immunity. 2008;29:44–56. doi: 10.1016/j.immuni.2008.05.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Qian Y, Liu C, Hartupee J, et al. The adaptor Act1 is required for interleukin 17-dependent signaling associated with autoimmune and inflammatory disease. Nat Immunol. 2007;8:247–256. doi: 10.1038/ni1439. [DOI] [PubMed] [Google Scholar]
  • 32.Lohr J, Knoechel B, Wang JJ, Villarino AV, Abbas AK. Role of IL-17 and regulatory T lymphocytes in a systemic autoimmune disease. J Exp Med. 2006;203:2785–2791. doi: 10.1084/jem.20061341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Muranski P, Boni A, Antony PA, et al. Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood. 2008;112:362–373. doi: 10.1182/blood-2007-11-120998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Nakae S, Iwakura Y, Suto H, Galli SJ. Phenotypic differences between Th1 and Th17 cells and negative regulation of Th1 cell differentiation by IL-17. J Leukoc Biol. 2007;81:1258–1268. doi: 10.1189/jlb.1006610. [DOI] [PubMed] [Google Scholar]
  • 35.Yi T, Chen Y, Wang L, et al. Reciprocal differentiation and tissue-specific pathogenesis of Th1, Th2, and Th17 cells in graft versus host disease. Blood. 2009;114:3101–3112. doi: 10.1182/blood-2009-05-219402. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES