Granzyme A–producing T helper cells are critical for acute graft-versus-host disease

Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4+ T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a potentially novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA+ Th cells were distinct from other Th lineages and exhibited a noncytolytic phenotype. In vitro, GrA+ Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA+ Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA+ Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graft-versus-leukemia effect. Our data indicate that GrA+ Th cells represent a distinct Th subset and are critical mediators of aGVHD.


Introduction
Acute graft-versus-host disease (aGVHD) is a deleterious alloimmune-mediated response that occurs in patients undergoing hematopoietic cell transplant (HCT) for treatment of hematopoietic malignancies or inborn errors. CD4 + T helper (Th) cells within the donor graft that recognize allogeneic HLA molecules are major effectors in aGVHD. During aGVHD, Th cells traffic to multiple organs, including the intestines, which is associated with worsened clinical prognosis and decreased ability to treat disease with steroids (1,2).
In this study, we identified a potentially novel subset of granzyme A-producing (GrA-producing) CD4 + T cells that were enriched within the intestines of mice with aGVHD and exhibited a phenotype distinct from other Th cells and cytotoxic lymphocytes. Further, GrA + Th cells required STAT3 and STAT6 for their development and lineage identity in vitro and in vivo. We showed that Th-derived GrA contributed to intestinal damage and lethality in multiple murine models of aGVHD. Importantly, GrA + Th cells were dispensable for the beneficial graft-versus-leukemia effect, indicating that GrA + Th cells may represent a novel therapeutic target for treatment of aGVHD.
Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4 + T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a potentially novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA + Th cells were distinct from other Th lineages and exhibited a noncytolytic phenotype. In vitro, GrA + Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA + Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA + Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graftversus-leukemia effect. Our data indicate that GrA + Th cells represent a distinct Th subset and are critical mediators of aGVHD.
as compared with cells cultured with IL-4 or IL-4+IL-6/21 (1206 with IL-4, 1001 with IL-4+IL-6/21; Figure  4, A and B). IL-4 and IL-6 induced expression of distinct sets of genes and addition of IL-6/21 to IL-4-cultured cells suppressed a subset of IL-4-induced genes while other IL-4-induced genes were relatively unchanged ( Figure 4A, Supplemental Figure 2A, and Supplemental Table 4). Conversely, IL-4 blunted the expression of IL-6/21-induced genes ( Figure 4A, Supplemental Figure 2B), suggesting that these cytokine-signaling pathways  Table 4). These data indicate that IL-6/21 signaling redirects cells from an IL-4-driven Th2 phenotype into a GrA-producing lineage by repressing specific aspects of the Th2 lineage and inducing a novel pattern of gene expression. However, adoptive transfer of Th cells polarized with IL-4 alone or with IL-4 and IL-6/21 was poor at inducing aGVHD. Both sets of recipient mice exhibited similar low clinical scores, weight loss, colon lengths, and lack of mortality after HCT (P = 0.41, Supplemental Figure 3, A-C). The poor pathogenicity of these cells is likely attributed to a loss of GrA production and large gains in IL-4 production after HCT (Supplemental Figure 3D).
Because IL-4 and IL-6/21 respectively activate STAT6 and STAT3, we examined targeted mRNA levels in IL-4+IL-6/21-cultured cells isolated from WT, Stat6 -/-, and Stat3 -/-(Stat3 fl/fl CD4-Cre ) mice by realtime PCR. Elevated expression of Gzma, Maf, Ikzf3, and Il21 in IL-4+IL-6/21-cultured cells required both STAT6 and STAT3 as cells from deficient mice exhibited significantly reduced (P < 0.006) expression of all these genes ( Figure 4, E and F). These data are consistent with our RNA-Seq analysis and demonstrate a necessity of both STAT6 and STAT3 in activating Gzma expression and the associated gene expression profile. Together, these data indicate that IL-4/STAT6 and IL-6/IL-21/STAT3 signals synergistically drive the GrA + Th cell gene signature.
As we identified both STAT3 and STAT6 to be critical for GrA + Th cell differentiation in vitro, we asked whether these signals were also required for their differentiation during aGVHD. In these studies, lethally irradiated BALB/c mice received T cell-depleted bone marrow and CD8 + T cells from WT C57BL/6 mice and purified CD4 + T cells from WT, Stat3 -/-, or Stat6 -/mice. After HCT, mice that received WT and Stat6 -/-CD4 + T cells exhibited similar increased clinical scores, but these were significantly reduced in Stat3 -/-CD4 + T cell recipients (P < 0.0001) and accompanied by increased colon length (P = 0.004) as compared with WT controls ( Figure 5, A and B). Mice that received either Stat3-or Stat6-deficient CD4 + T cells had similar CD4 + T cell numbers in the LI, SI, and liver as compared with WT recipients, suggesting that neither factor was required for the survival or expansion of Th cells at this time point ( Figure 5C). While both Stat3-and Stat6-deficient CD4 + T cells exhibited decreased GrA expression in the LI and SI, this was much more dramatic in Stat3-deficient Th cells (P < 0.0001 vs. P = 0.004 or P = 0.003) and also resulted in reduced GrA expression in the liver, where Stat6-deficient cells had similar GrA expression to WT cells in this organ (P = 0.33, Figure 6, A and B). Interestingly, while approximately 50% of LI-derived Representative FACS plots depicting GrA expression by CD3-gated human CD4 + and CD4 -(i.e., CD8 + ) T cells and quantitation of GrA + CD4 + and CD8 + T cells in each organ. (B) Representative FACS plots depicting human GrA, GrB, and perforin staining of gated CD3 + CD4 + or CD3 + CD8 + T cells. Error bars represent standard deviation of the mean. Human PBMC NRG mouse experiments represent 9 NRG mice that received PBMCs from 2 distinct donors. Statistical significance was determined by 1-way ANOVA with a Tukey's posttest for multiple comparisons.
GrA + CD4 + T cells expressed IFN-γ in these experiments, this was significantly increased (~80%) in Stat3and Stat6-deficient CD4 + T cells ( Figure 6, C and D; P < 0.0001 and P = 0.0003, respectively), indicating that both STAT3 and STAT6 play critical roles in suppressing a Th1-like phenotype in these cells and aid in maintaining the GrA + Th cell lineage identity.
Th-derived GrA is required for aGVHD. Having identified a population of intestinal GrA + Th cells in mice with aGVHD, we next questioned whether Th-derived GrA was important for development of disease. In these experiments, we used the C57BL/6 into BALB/c model of aGVHD because pathology in this system is dependent on CD4 + T cells (19). Briefly, mice received syngeneic or allogeneic cells as above, and additional groups of mice received allogeneic bone marrow and combinations of CD4 + and CD8 + T cells from WT or Gzma -/animals. As expected, mice that received Gzma -/-CD4 + T cells had normal percentages of GrA + CD8 + T cells but lacked GrA + Th cells ( Figure 7A and data not shown). Further, Gzma -/-Th cell recipients had similar intestinal total cell numbers, inflammatory cell numbers, frequencies of donor-derived (i.e., H-2K b+ ) Th cells, and frequencies of IFN-γ + and IL-17A + Th cells as compared with controls (Supplemental Figure  4, A-C; and data not shown). These data indicate equivalent proliferation, trafficking, and activation of WT and GrA-deficient Th cells in the intestines. In other analyses, mice that received WT Th cells exhibited significantly increased clinical scores and mortality by day 10 post-HCT as compared with syngeneic controls (P < 0.05, Figure 7B). Mice that received Gzma -/-Th cells exhibited significantly reduced clinical scores (P = 0.02-0.05) and mortality (P < 0.0001, Figure 7B) as compared with mice that received WT Th cells. Surviving Gzma -/-Th recipient mice persisted until 50 days post-HCT (Supplemental Figure 5, A and B). Mice that received Gzma -/-CD8 + T cells, but WT CD4 + T cells, exhibited similar clinical scores and mortality to mice that received WT CD4 + and CD8 + T cells ( Figure 7B). These data indicate that CD4 + T cell-derived, but not CD8 + T cell-derived, GrA is critical for the progression of aGVHD in this CD4 + T cell-dependent model. As a whole, GrA + Th cells contribute to disease without altering the recruitment of inflammatory cells into the intestines or altering the ability of Th cells to produce IFN-γ, IL-4, or IL-17A. Because only a fraction of HCTs performed clinically are from MHC major mismatched donors, we aimed to confirm the role of GrA + Th cells in a minor histocompatibility (miHA) mismatch model of GVHD. To this end, we induced GVHD in lethally irradiated C3.SW-H2 b /SnJ mice by HCT with bone marrow and T cells from C57BL/6 mice as per above. Similar to the allogeneic model, GrA + CD4 + T cells were significantly increased in the SI (P < 0.0001) in comparison with the liver in this model ( Figure 7C). GrA + Th cells from the SI of these animals also displayed a similar cytokine profile as observed in major mismatch recipients (Supplemental Figure 4D). Importantly, Gzma -/-Th recipients exhibited reduced clinical scores (days 8-15, P < 0.05) and enhanced survival as compared with WT recipients (P = 0.02, Figure  7D). Additionally, lack of GrA production by CD4 + T cells in these models had no effect on total LI infiltrating cells, total number of CD4 + T cells in the LI, numbers of inflammatory cells, or Th lineage-associated cytokine expression (Supplemental Figure 4, E-G). Interestingly, the role of GrA + Th cells in promoting aGVHD in the miHA-driven model was more modest than that observed in the major mismatch model of aGVHD. This may be due to a greater dependency of CD8 + T cells in the miHA-induced model as compared with the more CD4 + T cell-dependent nature of the major mismatch model (19). Regardless, these data indicate that GrA + Th cells contribute to disease severity in both MHC major mismatch-and miHA-driven aGVHD without altering immune cell infiltration or effector function.
CD4 + T cell-derived GrA is dispensable for the beneficial graft-versus-leukemia response. HCT is an effective therapy for treatment of hematological malignancies where allo-or miHA-specific T cells from the donor aid in suppression of tumor cell growth. We demonstrated above that GrA + Th cells play a pathogenic role in driving aGVHD, but whether they are required for the beneficial graft-versus-leukemia (GVL) response is unknown. To address this, we performed mixed GVHD/GVL experiments where irradiated BALB/c mice received syngeneic or allogeneic HCT with T cells from WT, Gzma -/-, or Gzma -/-CD4 + T cells and WT CD8 + T cells along with GFP + MLL-AF9 leukemia cells. Given that MLL-AF9 cells express MHC-II after transfer and are recognized by both CD4 + and CD8 + T cells in vitro and in vivo (20,21), we used this model to assess the relative contributions of GrA produced by all T cells (i.e., CD4 + and CD8 + T cells) versus that produced only by CD4 + T cells in both the GVHD and GVL response. After syngeneic HCT, despite minimal development of clinical score, we observed rapid death of 100% of these mice due to high GFP + leukemia burden in the bone marrow ( Figure 8, A-D). In contrast, mice with WT allo-HCT rapidly developed clinical scores and had a similar mortality rate as that of the syngeneic controls; however, the majority of deaths occurred from GVHD (67%) rather than leukemia (33%) (i.e., high clinical scores, lower GFP + tumor burden; Figure 8, A-D). Allogeneic HCT with Gzma -/-T cells resulted in reduced GVHD-associated clinical scores and mortality as compared with WT controls (P < 0.0001 and P = 0.005, respectively). Additionally, Gzma -/-T cell recipients had similar frequencies of GFP + tumor cells in the bone marrow (P > 0.05) and only modestly increased mortality due to leukemia (42%, P > 0.05 as compared with WT allo-HCT recipients; Figure 8, A-D). Interestingly, mice receiving an allo-HCT with Gzma -/-CD4 + T cells and WT CD8 + T cells exhibited the lowest clinical scores (P < 0.001 vs. Gzma -/-T cells and P < 0.0001 vs. WT T cells) and mortality as compared with all other allo-HCT recipients (P < 0.0001 vs. Gzma -/-T cells and P < 0.0001 vs. WT T cells). Further, Gzma -/-CD4 + T cell recipients exhibited no signs of GFP + leukemia cells in the bone marrow upon euthanization, and the only cause of death was GVHD (17%, Figure 8, A-D). Intriguingly, GrA-deficient Treg cells have reduced capacity to regulate allo-specific T cell responses during GVHD (22), and this reduced Treg function may explain the enhanced tumor cell clearance we observed here. Together, these data suggest that CD4 + and CD8 + T cell-derived GrA have different effects on the outcomes of GVHD and GVL and that CD4 + T cell-derived GrA is dispensable for GVL.
GrA + Th cells are associated with intestinal crypt damage. CD4 + T cells have been previously reported to localize to intestinal crypts and induce aGVHD by destroying cells within this niche (23-25). Consistent with this finding, we observed decreased colon length and increased goblet cell and crypt loss in aGVHD mice as compared with syngeneic controls (Figure 9, A-D; P < 0.004). In contrast, Gzma -/-Th cell recipient mice exhibited significantly increased colon lengths (P = 0.013, Figure 9B) and reduced loss of goblet cells and intestinal crypts as compared with WT controls (Figure 9, A, C, and D; P = 0.01 and P = 0.02, respectively). This was accompanied by reduced cellular infiltration near crypt bases, as indicated by arrows in Figure 9A. Liver portal inflammation was at similar elevated levels in both WT and Gzma-deficient CD4 + T cell recipients (P = 0.54, Figure 9, A and E), suggesting that CD4 + T cell-derived GrA may have a specific role in promoting intestinal aGVHD via destruction of intestinal crypts. Reduced intestinal pathology in Gzma -/-Th cell recipient mice did not lead to differences in gut permeability, as measured by serum FITC-dextran levels after oral gavage ( Figure 9F) or in intestinal cytokine production ( Figure 9G). However, reduced intestinal pathology in Gzma -/recipients did correlate with significantly reduced serum cytokine levels of IL-2, IL-5, and monocyte chemoattractant protein-1 (MCP-1) (P = 0.03, P = 0.04, P = 0.01, respectively) and a decrease in all other serum cytokines that were elevated in WT recipients (P > 0.05, Figure 9H).

Discussion
In this study, we identified a potentially novel subset of GrA-producing CD4 + T cells that accumulate specifically within the intestines and were critical for lethal GVHD. From our CyTOF analysis, GrA-producing Th cells were distinct from other Th subsets and did not express high levels of FOXP3 or IL-17A. However, a smaller population of cells exhibited some coexpression of GrB and IFN-γ that segregated independently from TNF/IL-2-coproducing IFN-γ-positive cells. Loss of IFN-γ, TNF, and IL-2 is a hallmark of chronically stimulated (e.g., exhausted) CD4 + and CD8 + T cells (26,27). In chronic infections, exhausted Th1 cells take on characteristics of T follicular helper cells (i.e., CXCR5, BCL6, PD-1) and have elevated expression of Batf, Hif1a, Maf, and Eomes. It is therefore possible that GrA + Th cells in the intestines might represent a product of chronic Th1 cell activation that has led to reduced IFN-γ production and loss of IL-2 and TNF. In vitro-derived GrA + Th cells also exhibited similar elevated expression of exhausted Th cell genes, including Il21, Maf, Batf, Hif1a, and Eomes ( Figure 4, Supplemental Figure 2, and data not shown). Importantly, while IFN-γ may play a protective role in some aGVHD models, Th cell-derived GrA was clearly detrimental. Therefore, this clearly distinguishes GrA-producing Th cells from IFN-γ-producing Th1 cells in aGVHD.
The cytokines that guide the differentiation of GrA + Th cells in vitro are distinct from other Th lineages. Synergistic signaling between IL-4 and IL-6/21 promoted optimal Gzma expression in vitro via alteration of the IL-4-induced gene expression profile in a STAT3-and STAT6-dependent manner. A similar phenomenon was also observed in both mouse and human macrophages polarized with IL-4 only (i.e., M2 macrophages) or in the presence of IL-6 that alters M2-polarized macrophage phenotype and function (28)(29)(30). In vivo, both STAT3 and STAT6 were involved in the generation GrA + Th cells during GVHD, with STAT3 having a greater effect. Interestingly, STAT3 and STAT6 had similar roles in maintenance of GrA + Th cell lineage identity as loss of either STAT resulted in increased IFN-γ production. While STAT3 is typically associated with Th17 and T follicular helper cell differentiation (31)(32)(33)(34), this pathway is shared by GrA-producing cells. Additionally, loss of STAT3 and STAT6 have each been associated with increased Th1 differentiation and IFN-γ production both in vitro and in vivo (34,35), indicating that these signaling pathways inhibit Th1 development. Although GrA-producing cells do coexpress IFN-γ, the differential requirement of STAT3 in promoting GrA production and limiting IFN-γ indicates that these cells are divergent from traditional Th1 cells. Further, these data suggest that part of the success of IL-6 blockade therapy for GVHD in mice (36-38) and humans (39, 40) may result from decreasing GrA + Th cells in the intestine.  During aGVHD, the intestines are one of the major target organs of donor Th cells, and their presence in this niche is associated with increased inflammatory cytokine production and lethality (1,2). In our study, transfer of Gzma-deficient CD4 + T cells resulted in reduced lethality and reduced goblet cell and intestinal crypt loss as compared with controls. Interestingly, this did not result in changes in intestinal permeability as measured by serum FITC-dextran after oral gavage. These data may indicate that overall barrier permeability is acutely similar, which is mirrored by similar clinical scores at this early time point. At later time points (i.e., >day 15 post-HCT), however, barrier function may be restored more rapidly in the absence of GrA, which may lead to enhanced survival. How GrA + Th cells mediate intestinal damage remains unclear. While GrA was initially shown to cause apoptosis when delivered through perforin-formed membrane pores, GrA was more recently shown to have little cytotoxic potential (41). Additionally, intestinal GrA + Th cells in aGVHD mice did not express perforin (data not shown). GrA has other reported roles in promoting inflammatory cytokine and chemokine production (IL-1β, IL-6, TNF, MCP-1) via interaction with TLRs (42)(43)(44)(45). However, these cytokines were not differentially expressed within the intestines, and only MCP-1 was significantly reduced in the serum of Gzma -/recipient mice. Although MCP-1 has documented roles in cellular trafficking during idiopathic pulmonary syndrome (46), we did not observe decreased recruitment of inflammatory cells to the intestines in the absence of GrA. These data indicate that GrA-mediated intestinal damage is likely independent of its role in inflammatory cytokine production and cytolysis.
While Th cells are required in a number of murine models for the development of GVHD, the exact subtypes involved and the effector molecules that they produce to induce disease have been difficult to determine. Treg cells play an important role in limiting intestinal pathology during GVHD. Interestingly, GrA expression is decreased in Treg cells in patients with aGVHD and was required for Treg cell protective function in murine GVHD (22,47). We observed few FOXP3 + Treg cells (<4% of the total CD4s) in the intestines of mice with aGVHD in our studies; however, approximately 20% to 30% of the intestinal Treg cells present did express GrA. While our studies were not designed to study the role of Treg cells in disease, it is possible that GrA in Treg cells and conventional Th cells may have opposite roles in regulating intestinal pathology.
Pathogenic Th subsets have been more difficult to define during aGVHD. Th1-derived IFN-γ has controversial roles in pathogenesis and depends highly on the conditioning regimen (8)(9)(10). Likewise, transfer of Th17 cells is sufficient to induce GVHD but with striking specificity for driving pathology in the lung and skin (3). Ullrich et al. (48) described a GM-CSF-producing Th cell subset dependent on basic leucine zipper transcription factor ATF-like (BATF) that was involved in intestinal inflammation. Mechanistically, BATF-deficient cells failed to accumulate within the intestines and induced pathology that is consistent with defects in BATFdeficient cells in inducing colitis (48)(49)(50). Batf was also highly expressed by GrA + Th cells induced in vitro by IL-4 and IL-6/21, and it is intriguing to speculate that these may be similar cell types. IL-23R + CD11c + Th cells were also recently shown to promote intestinal damage during GVHD (51). Il23r-deficient donor cells induced reduced liver, lung, and colon pathology scores and dramatically blunted Th1 and Th17 responses, suggesting that IL-23R is required for systemic GVHD effects and may not be specific to intestinal inflammation. In contrast to this report, Buchele et al. suggest that IL-23R on donor T cells may not be required for intestinal GVHD (49). Because IL-23/IL-23R activates STAT3, which in turn can activate BATF in other pathogenic Th cell lineages, it is possible that both IL-23R and BATF are critical regulators of GrA + Th cell differentiation.
In human HCT patients, granzymes have been used as biomarkers of aGVHD and a predictor of disease severity. In an 86-patient cohort, Kircher et al. (52) demonstrated that serum GrA was detected at higher levels than GrB and was a better predictor of disease severity than HLA mismatch or donor/ recipient age. Earlier studies by this same group indicated that GrA, but not GrB, produced in mixed lymphocyte reactions between donor and host PBMCs significantly correlated with GVHD grade posttransplant. Further, enhanced GrA expression in these mixed lymphocyte reactions correlated with increased numbers of MHC-II mismatches and outgrowth of CD4 + T cells (53). While the cellular source of GrA in actual patients with GVHD remains unclear, these data strongly indicate that CD4 + T cells are a potential producer of this protease. Our experiments in a human PBMC-induced NRG mouse model of GVHD support this hypothesis. While GrA was produced by both human CD4 + and CD8 + T cells in the spleen and liver, human CD4 + T cells in the intestines were the dominant producers of GrA and exhibited a noncytolytic phenotype as compared with GrB-and perforin-expressing CD8 + T cells in the same tissue. These data combined with the clinical data mentioned above indicate that GrA-producing CD4 + T cells may also contribute to human aGVHD. Further, these data indicate that specific GrA inhibitors or broader serine protease inhibitors may represent a novel treatment strategy for patients with GVHD.

Methods
Mice and initiation of aGVHD (including clinical scoring). C57BL/6 and BALB/c mice were purchased from The Jackson Laboratory or Envigo and housed in the animal facility at the Indiana University School of Medicine or within the Purdue University animal facility. Gzma -/mice (54) were housed as above. Stat6 -/- (55) and C3.SW-H2 b/ SnJ (stock 00438) mice were purchased from The Jackson Laboratory, and Stat3 fl/fl mice were originally provided by David Levy (New York University, New York, New York, USA) (56) and crossed to CD4-Cre mice to delete STAT3 in T cells. Both male and female mice were used between the ages of 8 and 13 weeks.
Clinical aGVHD scores were assessed weekly using a previously described scoring system (57). In short, clinical score was measured using the following parameters: weight loss, posture, mobility, skin integrity, stool score, and fur texture. Each parameter was scored 0-2, with 0 being normal and 2 being severe, and added to get a total GVHD clinical score. Mice were euthanized when the clinical score reached 9.0.
Induction and assessment of the GVL effect. BALB/c mice were lethally irradiated with 900 cGy on day -1 and received HCT from syngeneic mice (5 × 10 6 TCD bone marrow cells and 8 × 10 5 purified T cells) or allogeneic C57BL/6 donors (all received 5 × 10 6 bone marrow cells and 8 × 10 5 purified WT T cells, purified T cells from Gzma -/mice, or 5 × 10 5 purified Gzma -/-CD4 + T cells and 3 × 10 5 WT CD8 + T cells) along with 4 × 10 5 GFP + MLL-AF9 cells that have been described elsewhere (58). After HCT and inoculation with leukemia cells, mice were monitored for GVHD-associated clinical score and mortality, and cause of death was determined by accumulative analysis of GVHD score and the frequency of GFP + MLL-AF9 cells in the bone marrow upon euthanization as previously described (58).
Human PBMC NRG mouse model of GVHD. To examine GrA production from human CD4 + T cells in the context of GVHD, NRG mice (originally from The Jackson Laboratory, purchased from the Purdue Biological Evaluation Core) were injected i.v. with 5 × 10 6 to 20 × 10 6 human PBMCs that had been recovered the previous day (37°C, 5% CO 2 , complete RPMI) from frozen stocks isolated from 2 distinct donors (from ZenBio). After injection, mice were monitored daily until they reached 20% body weight loss or developed a significant clinical score (>3). Upon euthanization, immune cells were isolated from the spleen, liver, SI, and LI; were stained for cell surface expression of human CD3, CD4, and CD8; and were fixed, permeabilized, and stained for intracellular human GrA, GrB, and perforin (see below). The frequency of GrA-, GrB-, and perforin-expressing T cells was subsequently determined by flow cytometry.
Isolation of T cells and dissociation of tissues for cytometry. Naive CD44 -CD4 + T cells were isolated from the spleen and mesenteric lymph nodes (MesLNs) of C57BL/6 mice via negative magnetic enrichment (Miltenyi Biotec). CD4 + and CD8 + T cells were isolated from spleens and MesLNs using anti-CD4 and anti-CD8 microbeads following the manufacturer's instructions (Miltenyi Biotec).
Mononuclear cells were harvested from spleens and MesLNs using sterile frosted glass slides to disrupt the spleen, followed by removal of debris; in some experiments red blood cells were removed with red blood cell lysis buffer (BioLegend). Liver mononuclear cells were isolated by pressing perfused liver tissue through a wire mesh sieve (Bellco Glass) to make a single-cell suspension that was resuspended in 40% Percoll and centrifuged for 10 minutes at 4°C at 872g to remove debris and fat/connective tissue. Red blood cell lysis buffer was used to remove residual red blood cells. To isolate mononuclear cells from intestines, small and large intestines were harvested, and Peyer's patches were removed from small intestinal tissue. Intestines were cut longitudinally and washed with vigorous shaking 3 times with 40 mL of PBS to remove mucus. Washed intestinal tissue was then finely diced with scissors and transferred into digestion medium containing DMEM (Gibco, Thermo Fisher Scientific) with 2% BSA, 10 μg/mL of DNaseI (Roche), and 100 μg/mL of Liberase TL (Roche) or 1.5 mg/mL of type III collagenase (Worthington). Cells were incubated at 37°C for 1.5 hours in a shaking incubator (250 rpm) before straining through a 100 μm nylon sieve and washing with PBS containing 2% fetal calf serum. The resulting cells were resuspended in 40% Percoll and centrifuged as with liver-derived samples per above. Viable cells isolated from each tissue were enumerated via trypan blue staining and counted on a hemocytometer.
For CyTOF experiments, cells were stimulated and fixed as above; however, antibody reagents were developed specifically for use with CyTOF and purchased from Fluidigm unless otherwise noted (Supplemental Table 2). After incubation, cells were washed with PBS (Rockland Immunochemicals Inc.), and their viability was assessed by cisplatin (Fluidigm Inc.). Cells were then washed with Maxpar cell staining buffer (Fluidigm Inc.). Cells were fixed in PBS with 1.35% formaldehyde (MilliporeSigma) for 10 minutes at room temperature. Samples to be barcoded by Pd isotope were resuspended in barcode perm buffer (Fluidigm Inc.). Appropriate barcodes in barcode perm buffer were transferred into each individual sample and incubated for 30 minutes at room temperature. After washing with Maxpar cell staining buffer, all barcoded samples were combined into 1 tube. To prevent high nonspecific background signal, Fc blocking (BioLegend) was performed, followed by surface antibody cocktail staining. After permeabilization using the Nuclear Antigen Staining Buffer Set (Fluidigm Inc.), cells were stained with intracellular and transcription factors antibody cocktail. Cells were then stained with 193 Ir DNA-intercalator in Maxpar fix and perm buffer (Fluidigm Inc.) to identify all nucleated cells. In the final step, cells were washed by type I, ultrapure water (18.2 MΩ) and filtered into cell strainer cap tubes (40 μm). Prior to the data acquisition, cell concentration was adjusted to 5 × 10 5 /mL to obtain approximately 500 events/s. To normalize data, cells were diluted in ultrapure water containing 0.1x EQ Four Element Calibration Beads (Fluidigm Inc.). Samples were then injected into cyTOF2 mass cytometer machine (Fluidigm Inc.). Data were acquired from barcode channels (channels 102, 104, 105, 106, 108, and 110), EQ beads channels (channels 140, 151, 153, 165, and 175), Cisplatin channel (channel 195), cell intercalator 193 Ir, and corresponding channels to the antibody panel (Supplemental Table 2).
In vitro culture of Th cells. Naive CD4 + T cells were cultured in tissue culture-treated multiwell plates that were coated overnight with 2 μg/mL of anti-CD3ε (Bio X Cell) at 10 6 cells/mL. Th1, Th2, and Th9 were cultured as previously described (59), and in some cases IL-6 and/or IL-21 was added to these cultures in varying concentrations as noted. In cytokine screening experiments, cells were cultured in flat-bottom, 96-well plates with plate-bound anti-CD3 and soluble anti-CD28 as per above. Cytokines were added at day 0 of culture (IL-4, 10 ng/mL; IL-6, 20 ng/mL; IL-12, 10 ng/mL; IL-21, 100 ng/mL; Pam3CSK4, 2 μg/mL; TGF-β, 2 ng/mL; TNF 100 ng/mL; see Supplemental Table 3 for other cytokines used), and cells were expanded 1:4 in fresh media with no additional cytokines day 3 of culture. All cytokines were purchased from PeproTech, except TGF-β, which was purchased from R&D Systems, Bio-Techne. Pam3CSK4 was purchased from InvivoGen.
Serum/intestinal cytokine analysis, permeability, and histology. Serum was collected by cardiac puncture immediately after CO 2 asphyxiation and allowed to clot for 6 hours at 4°C. After clotting, samples were centrifuged for 10 minutes at 2000g, and serum was aliquoted into fresh tubes. For intestinal cytokine analysis, 2 cm sections of midcolon were collected into 1 mL of sterile complete RPMI medium and incubated for 24 hours at 37°C and 5% CO 2 . Medium was collected and centrifuged for 10 minutes at 2000g to remove debris, and clarified medium was frozen at -20°C until cytokine measurements. Cytokine levels were assessed by mouse cytokine Bio-Plex Pro (23-plex, Bio-Rad) following the manufacturer's directions at the Multiplex Analysis Core within the Indiana University Melvin and Bren Simon Cancer Center.
Intestinal permeability was measured by FITC-dextran permeability assays. In these studies, BALB/c mice received bone marrow and T cells from BALB/c (syngeneic) or WT or Gzma -/mice (allogeneic) as above.