Graft-versus-host disease (GVHD) induced by donor-derived T cells remains the major limitation of allogeneic bone marrow transplantation (allo-BMT). We previously reported that the pan-Notch inhibitor dominant-negative form of Mastermind-like 1 (DNMAML) markedly decreased the severity and mortality of acute GVHD mediated by CD4+ T cells in mice. To elucidate the mechanisms of Notch action in GVHD and its role in CD8+ T cells, we studied the effects of Notch inhibition in alloreactive CD4+ and CD8+ T cells using mouse models of allo-BMT. DNMAML blocked GVHD induced by either CD4+ or CD8+ T cells. Both CD4+ and CD8+ Notch-deprived T cells had preserved expansion in lymphoid organs of recipients, but profoundly decreased IFN-γ production despite normal T-bet and enhanced Eomesodermin expression. Alloreactive DNMAML T cells exhibited decreased Ras/MAPK and NF-κB activity upon ex vivo restimulation through the TCR. In addition, alloreactive T cells primed in the absence of Notch signaling had increased expression of several negative regulators of T cell activation, including Dgka, Cblb, and Pdcd1. DNMAML expression had modest effects on in vivo proliferation but preserved overall alloreactive T cell expansion while enhancing accumulation of pre-existing natural regulatory T cells. Overall, DNMAML T cells acquired a hyporesponsive phenotype that blocked cytokine production but maintained their expansion in irradiated allo-BMT recipients, as well as their in vivo and ex vivo cytotoxic potential. Our results reveal parallel roles for Notch signaling in alloreactive CD4+ and CD8+ T cells that differ from past reports of Notch action and highlight the therapeutic potential of Notch inhibition in GVHD.
Notch signaling is a highly conserved cell-to-cell communication pathway with multiple functions in health and disease (1). Notch receptors interact with Jagged or Delta-like ligands, leading to proteolytic release of intracellular Notch (ICN). ICN translocates into the nucleus to interact with CSL/RBP-Jk (encoded by Rbpj) and a Mastermind-like (MAML) transcriptional coactivator. ICN, CSL/RBP-Jk, and MAML assemble a large complex mediating gene activation. In hematopoiesis, Notch was first identified for its essential role during early T cell development (2). Other functions in B cell and myeloid lineages were subsequently reported (3–5). Moreover, emerging evidence indicates that Notch has important context-dependent effects on mature T cell differentiation and function (6).
Using genetic models of Notch inhibition, we discovered an essential role of Notch signaling in CD4+ T cells mediating graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT) (7). GVHD is the most significant complication limiting the success of allo-BMT (8). Current strategies to control GVHD rely on global immunosuppression. These strategies are incompletely effective and decrease graft-versus-tumor activity. To improve allo-BMT outcome, it is essential to identify new approaches that limit GVHD without eliminating potent anticancer effects. To inhibit Notch signaling downstream of all Notch receptors, we expressed a Cre-inducible dominant-negative form of Mastermind-like 1 (DNMAML) under control of the ROSA26 locus. Upon Cd4-Cre expression, pan-Notch inhibition was achieved in mature CD4+ and CD8+ T cells without interference with early stages of T cell development (7, 9, 10). DNMAML blocks the Notch transcriptional activation complex downstream of all Notch receptors, with similar effects to those observed in the absence of CSL/RBP-Jk (the DNA-binding transcription factor that mediates all the effects of canonical Notch signaling). In mouse allo-BMT models, pan-Notch blockade in donor CD4+ T cells led to markedly reduced GVHD severity and improved survival (7). Notch-deprived alloreactive CD4+ T cells had decreased production of inflammatory cytokines, including IFN-γ, TNF-α, IL-17A, IL-4, and IL-2. Concomitantly, Notch inhibition led to increased accumulation of regulatory T cells (Tregs). However, Notch-deprived CD4+ alloreactive T cells were capable of extensive proliferation, allowing for their enhanced accumulation in lymphoid tissues. Despite reduced cytokine production, Notch-deprived CD4+ T cells retained potent cytotoxic potential in vivo. Thus, Notch signaling in T cells is a new attractive therapeutic target to control GVHD after allo-BMT.
Both CD4+ and CD8+ T cells have pathogenic effects during GVHD. Although we reported an essential function of Notch in CD4+ alloreactive T cells, no information has been available about Notch in CD8+ T cell–driven GVHD. Furthermore, it is unclear if the profound effects of Notch signaling in acute GVHD can be explained by its previously reported effects in T cells or if new mechanisms are involved. Past work highlighted independent effects of Notch signaling in CD4+ and CD8+ T cells. Notch was shown to regulate Il4 and Gata3 expression during Th2 differentiation (9–12). In Th1 cells, pharmacological inhibitors and a Notch1 antisense strategy suggested that Notch controlled expression of Tbx21, encoding T-bet, a transcription factor regulating Ifng transcription (13). Notch signaling was also shown to influence Th17 and Treg differentiation, as well as CD4+ T cell longevity, at least in vitro (14–17). In CD8+ T cells, Notch was suggested to act directly at the Eomes and Gzmb loci, with an impact on differentiation and function (18–20). However, these findings originate from heterogeneous experimental systems, different immune contexts, and variable strategies to manipulate Notch signaling, including gain-of-function approaches and pharmacological inhibitors. These results can be confounded by off-target effects and may not reflect the physiological functions of Notch in T cells.
In this study, we investigated the cellular and molecular mechanisms underlying the effects of Notch signaling in alloreactive CD4+ and CD8+ T cells during GVHD. Our strategy relied on in vivo priming of donor T cells in the presence or absence of all canonical CSL/RBP-Jk and MAML-dependent Notch signals specifically in T cells, ensuring that T cells were exposed to relevant Notch ligands in the posttransplantation environment. Notch-deprived alloreactive CD4+ and CD8+ T cells shared a profound defect in IFN-γ production, suggesting parallel effects of Notch in both T cell subsets. Decreased IFN-γ was observed despite preserved or enhanced expression of the transcription factors T-bet and Eomesodermin, consistent with the absence of a classical Th1 or effector CD8+ T cell differentiation defect. Notch-deprived alloreactive CD4+ and CD8+ T cells acquired a hyporesponsive phenotype with decreased Ras/MAPK and NF-κB signaling. Notch inhibition led to increased expression of selected negative regulators of T cell activation. Some of these characteristics have been observed in anergic T cells, suggesting that Notch-inhibited CD4+ and CD8+ T cells acquire an anergy-like phenotype after allo-BMT, resulting in decreased production of inflammatory cytokines. Despite these changes, Notch inhibition preserved alloreactive T cell expansion in vivo and only had modest effects on their proliferative potential, while increasing expansion of pre-existing natural Tregs and preserving high cytotoxic potential. Altogether, our data demonstrate a novel, shared mechanism of Notch action in alloreactive CD4+ and CD8+ T cells during allo-BMT that differs from all previous reports of Notch activity in T cells. Understanding these effects is essential to harness the therapeutic benefits of Notch blockade to control GVHD after allo-BMT.
Materials and Methods
BALB/c (H-2d) and C57BL/6 (B6, H-2b, CD45.2+) mice were from Harlan (Indianapolis, IN); C57BL/6.Ptprca (B6-SJL, H-2b, CD45.1+) from the National Cancer Institute (Frederick, MD); and BALB/b (H-2b) and Foxp3–internal ribosome entry site–RFP (FIR) from The Jackson Laboratory (Bar Harbor, ME) (21). NF-κB reporter mice (NF-κB–GFP–Luciferase [NGL]) were described previously (22). B6.129S6-Tbx21tm1Glm/J mice were provided by Dr. B. Segal (University of Michigan, Ann Arbor, MI) (23); Eomesf/f × Cd4-Cre mice by Dr. S. Reiner (Columbia University, New York, NY) (24); and Rbpjf/f mice by Dr. T. Honjo (Kyoto University, Kyoto, Japan) (4). ROSA26DNMAMLf mice (DNMAML) contain a Cre-inducible cassette encoding the DNMAML-GFP pan-Notch inhibitor (9, 25). DNMAML and Rbpjf/f mice were crossed to Cd4-Cre mice. All mice were backcrossed to the B6 background (more than eight generations). The University of Michigan Committee on Use and Care of Animals approved all experiments.
Induction and assessment of GVHD
Mice underwent allo-BMT as described (7). T cell–depleted bone marrow (TCD BM) was prepared with anti-Thy1.2 Abs and complement (Cedarlane Laboratories, Burlington, NC; >95% depletion). BALB/c recipients were irradiated (850–900 rad, [137Cs]) 4 h before allo-BMT. We transplanted donor B6-SJL TCD BM (4 to 5 × 106) with/without B6 splenocytes (5–10 × 106). Where indicated, CD4+ and CD8+ T cells were MACS-purified (Miltenyi Biotec, Auburn, CA) and mixed before transplantation into irradiated BALB/b or BALB/c recipients. Clinical GVHD was scored at least weekly.
Quantitative reverse-transcription PCR
SDS-PAGE and Western blotting
Naive (CD62LHighCD44Low) and alloreactive CD4+ and CD8+ T cells were negatively selected using an anti-NK1.1/CD19/Gr-1/CD11b/CD11c mixture. T cells were incubated on ice with anti-CD3/CD28 Abs (0.2 μg/106 cells) followed by cross-linking with anti-Armenian hamster IgG (0.5 μg/106 cells; Jackson ImmunoResearch Laboratories, West Grove, PA) in RPMI 1640 at 37°C. Alternatively, T cells were activated with PMA (Sigma-Aldrich, 50 ng/ml) or DMSO at 37°C. Cells were lysed in Laemmli buffer and 2-ME. Samples were run on 4–20% MiniTGX gels (Bio-Rad) and transferred to Immobilon-P membranes (GenHunter, Nashville, TN) (semidry transfer; Bio-Rad). Membranes were blocked in 10% FBS plus TBS-Tween (25 mM TrisBase [pH 8], 125 mM NaCl, and 0.05% Tween). Abs were from Cell Signaling Technology (Danvers, MA): anti-MEK1/2 rabbit (47E6), p44/42 MAPK-Erk1/2 mouse (3A7), phospho-MEK1/2 (Ser217/221) rabbit (41G9), and phospho-p44/42 MAPK-Erk1/2 (Thr202/Tyr204) (E10). Secondary Abs were peroxidase-conjugated goat anti-rabbit IgG (H+L) or donkey anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories). Blots were developed with ECL Substrate (Thermo Scientific) and HyBlotCL (Denville Scientific, South Plainfield, NJ).
Intracellular cAMP assay
cAMP was measured using ELISA per the manufacturer’s instructions (Enzo LifeSciences, Farmingdale, NY).
In vitro and in vivo proliferation assays
Splenocytes or CD4+ and CD8+ T cells were MACS-purified according to the manufacturer’s instructions (Miltenyi Biotec) and labeled with 2.5 μM CFSE (Sigma-Aldrich). T cells were incubated for 3 d on anti-CD3/CD28 ± recombinant human IL-2 (PeproTech). For BrdU pulse-chase experiments, splenocytes were transplanted into irradiated BALB/c mice. Starting at day 4 posttransplantation, mice received three doses of BrdU (1 mg) i.p., 12 h apart. Four hours (day 5) or 3 d (day 8) after the last BrdU injection, spleens were harvested for BrdU staining (BD Biosciences).
Serum cytokine analysis
In vivo cytotoxicity
Experiments were performed as previously described (7, 26). Briefly, lethally irradiated (900 rad) BALB/c mice received TCD BM (5 × 106 cells) alone or with wild-type (WT) or DNMAML splenocytes (8 × 106). On day 13, BALB/c recipients were challenged with a 1:1 mixture of CFSEHigh BALB/c allogeneic targets and CFSELow B6 syngeneic control cells (107 each). After 18 h, elimination of BALB/c allogeneic targets was measured by flow cytometry in the spleen.
Spleen and lymph nodes were harvested on day 8 posttransplantation. WT and DNMAML CD8+ T cells were purified by MACS according to the manufacturer’s instructions (Miltenyi Biotec). A20 (H-2d) and P815 (H-2d) cells were used as allogeneic targets, with EL4 (H-2b) as syngeneic control targets. Tumor targets were labeled with 2 MBq Na251CrO4 (PerkinElmer) for 2 h. After washing three times, labeled targets were plated at 5 × 103 cells/well in U-bottom 96-well plates (Corning-Costar, Cambridge, MA). Splenocytes were added in triplicate wells at varying E:T ratios and incubated for 5 h. [51Cr] activity in supernatants was read in a LumaPlate (PerkinElmer) in an auto-γ counter (Packard Instrument, Meriden, CT). Maximal and background release were determined by the addition of 2% Triton X-100 (Sigma-Aldrich) or media alone to targets, respectively. The percentage of specific lysis was calculated as 100 × (sample count − background count)/(maximal count − background count).
Comparison of two means was performed with two-tailed unpaired Student t test. When less than five data points were available per group, we used the unpaired Mann–Whitney U test. Linear regression analysis was used for comparison of cytotoxicity curves. Survival was compared using log-rank statistics (Prism; GraphPad, La Jolla, CA).
Notch inhibition blocks acute GVHD mediated by CD4+ or CD8+ T cells
We previously reported an essential role for Notch in CD4+ T cells during acute GVHD in an MHC-mismatched allo-BMT model (B6 anti-BALB/c) (7). To assess if alloreactive CD8+ T cells were also sensitive to Notch signaling, we used ROSA26DNMAMLf × Cd4-Cre (DNMAML) mice as source of Notch-deprived CD4+ and CD8+ T cells. These mice express the DNMAML pan-Notch inhibitor in all mature CD4+ and CD8+ T cells. WT or DNMAML B6 splenocytes were transplanted into irradiated BALB/c mice. Recipients were monitored for survival and GVHD severity. Allo-BMT recipients of B6 T cells died rapidly with severe GVHD (Fig. 1A). In contrast, recipients of DNMAML CD4+ and CD8+ T cells survived as well as mice infused only with TCD BM (Fig. 1A). We then performed allo-BMT with purified CD4+ or CD8+ T cells. DNMAML expression blocked severe GVHD induced by CD4+ T cells (Fig. 1B). Purified CD8+ T cells also induced significant lethality posttransplantation with <40% long-term survival (Fig. 1C). In contrast, DNMAML CD8+ T cell recipients had >94% survival by day 100, similar to mice receiving no T cells (Fig. 1C). These data demonstrate that Notch is an essential regulator of GVHD induced by either or both CD4+ and CD8+ T cells after MHC-mismatched allo-BMT.
To further investigate the role of Notch in CD4+ and CD8+ alloreactive T cells, we used the B6 anti-BALB/c model as well as a minor histocompatibility Ag–mismatched model (B6 anti-BALB/b) in which lethal GVHD is mediated by CD4-dependent CD8+ T cells (27). Together, WT CD4+ and CD8+ T cells induced significant lethality, with <20% long-term survival in both models (Supplemental Fig. 1A, 1B). DNMAML CD4+ and CD8+ T cell recipients achieved ∼80% survival (Supplemental Fig. 1A, 1B). When either CD4+ or CD8+ T cells expressed DNMAML, recipients were protected from severe GVHD with 80–90% of the mice surviving long-term (Supplemental Fig. 1A, 1B). These data indicate that Notch is required in both CD4+ and CD8+ T cells to fully induce GVHD in MHC- and minor histocompatibility Ag–mismatched allo-BMT models.
Alloreactive DNMAML CD8+ T cells display intrinsic and CD4+ T cell–dependent defects in IFNγ production
Notch inhibition preserves CD4+ T cell expansion after allo-BMT, but profoundly decreases IFN-γ production (7). Because CD8+ T cells are a major source of IFN-γ during GVHD, we assessed DNMAML CD8+ T cell expansion and IFN-γ production. After transplantation in the B6 anti-BALB/c model (Fig. 2A), we recovered similar numbers of donor-derived WT and DNMAML CD4+ and CD8+ T cells from lymphoid organs of allo-BMT recipients, indicating that Notch blockade did not prevent in vivo expansion of these cells (Fig. 2B, 2C). In contrast, DNMAML expression markedly decreased IFN-γ production by both alloreactive CD4+ and CD8+ T cells (Fig. 2D), suggesting parallel effects of Notch in these two subsets. These differences in IFN-γ production could not be explained by increased apoptosis of Notch-deprived T cells during the restimulation period (data not shown). To determine if changes in IFN-γ production by Notch-deprived alloreactive T cells were biologically relevant, serum IFN-γ levels were measured on day 5 after transplantation (Fig. 2E). Serum IFN-γ levels were markedly reduced in recipients of DNMAML T cells. Because IFN-γ production by CD8+ T cells is influenced by cell-intrinsic signals and CD4+ T cell help, we studied DNMAML CD8+ T cells in the presence of WT or DNMAML alloreactive CD4+ T cells (Fig. 2F). WT CD4+ and CD8+ T cells transplanted together produced abundant IFN-γ. In the presence of WT CD4+ T cells, DNMAML expression in CD8+ T cells markedly decreased but did not abolish IFN-γ production. When both DNMAML CD4+ and CD8+ T cells were infused, CD8+ T cells had little to no IFN-γ production. Thus, Notch promotes maximal IFN-γ production by alloreactive CD8+ T cells via cell-intrinsic changes in the CD8+ compartment and effects on CD4+ T cell help.
To verify that Notch signaling was potently inhibited in both alloreactive DNMAML CD4+ and CD8+ T cells, we assessed expression of the Notch target gene Dtx1 (Fig. 2G). Dtx1 transcripts were significantly reduced in both subsets of alloreactive DNMAML T cells. Collectively, inhibition of Notch signaling in alloreactive T cells preserved their expansion but profoundly reduced T cell–dependent IFN-γ production and systemic IFN-γ levels.
Notch inhibition blocks IFN-γ production in alloreactive CD4+ and CD8+ T cells despite preserved T-bet and Eomesodermin expression
Notch was suggested to control the expression of Tbx21 (encoding T-bet) in CD4+ T cells and Eomes (encoding Eomesodermin) in CD8+ T cells (13, 18, 19). To assess if these mechanisms accounted for decreased IFN-γ production by Notch-deprived CD4+ and CD8+ T cells after allo-BMT, we measured T-bet and Eomesodermin levels at the peak of the effector response. Alloreactive DNMAML CD4+ and CD8+ T cells had preserved Tbx21 and increased Eomes transcripts as compared with WT T cells (Fig. 3A). We used flow cytometry to assess intracellular T-bet and Eomesodermin and established Ab specificity by staining alloreactive T cells from WT mice compared with Tbx21−/− and Eomesf/f × Cd4-Cre mice (Fig. 3B). Consistent with mRNA findings, alloreactive DNMAML CD4+ and CD8+ T cells had preserved intracellular T-bet and increased Eomesodermin (Fig. 3C). As expected, we observed more Eomesodermin protein in CD8+ T cells than CD4+ T cells, consistent with accurate detection of Eomesodermin (28). These data indicate that decreased IFN-γ production by DNMAML CD4+ and CD8+ T cells was not caused by decreased Th1 and effector CD8+ T cell differentiation resulting from reduced expression of these master transcription factors.
PMA (a diacylglycerol analog) and ionomycin (a calcium ionophore) are often used to elicit cytokine production to study T cell differentiation. This strategy can reveal defective IFN-γ production by Tbx21−/− T cells (29). In contrast, restimulation of alloreactive DNMAML T cells with PMA/ionomycin restored IFN-γ production by both CD4+ (Fig. 3D) and CD8+ T cells (Fig. 3E) close to levels observed in WT T cells. Partial rescue of IL-2 production was also apparent (Supplemental Fig. 2). We confirmed the effects of DNMAML-mediated Notch blockade on IFN-γ production using Rbpj−/− T cells lacking CSL/RBP-Jk, a central component of the Notch transcriptional activation complex (Fig. 3F, 3G). Altogether, our observations were consistent with the presence of functional T-bet and Eomesodermin capable of activating Ifng transcription in DNMAML or CSL/RBP-Jk–deficient alloreactive T cells, suggesting that PMA/ionomycin restored activation of other pathways regulating cytokine production in these cells.
Notch-deprived alloreactive CD4+ and CD8+ T cells develop blunted Ras/MAPK and NF-κB activation
One of the major pathways activated by PMA is Ras/MAPK signaling, a key contributor to T cell cytokine gene transcription (30). To assess if alloreactive DNMAML T cells had decreased Ras/MAPK activation, we measured phosphorylation of Erk1/2 and its upstream kinase Mek1. Notch-deprived CD4+ and CD8+ T cells were primed in vivo before sort purification and ex vivo restimulation through the TCR and CD28 coreceptor. Both alloreactive DNMAML CD4+ and CD8+ T cells had a significant reduction in Mek1 and Erk1/2 phosphorylation (Fig. 4A). Interestingly, naive CD62LHighCD44Low DNMAML CD4+ T cells only had slightly decreased Erk1/2 phosphorylation, whereas naive DNMAML CD8+ T cells activated Erk1/2 normally, suggesting that the majority of the Ras/MAPK defect was acquired in vivo in the absence of Notch signaling (Fig. 4B). Next, we assessed if PMA rescued Ras/MAPK activation in DNMAML alloreactive T cells. PMA induced similar Erk2 phosphorylation in WT and DNMAML T cells, indicating restoration of Ras/MAPK signaling in DNMAML T cells (Fig. 4C).
NF-κB is another major pathway activated downstream of diacylglycerol that can regulate Ifng transcription in T cells (31). To capture the overall NF-κB activity in Notch-deprived alloreactive T cells, we bred DNMAML mice to transgenic NGL reporter mice and used F1 progeny as donors for allo-BMT (Supplemental Fig. 3) (22). On day 5 posttransplantation, alloreactive NGL and NGL/DNMAML CD4+ and CD8+ T cells were sort-purified and restimulated with anti-CD3/CD28 Abs. DNMAML CD4+ and CD8+ T cells had significantly reduced NF-κB/luciferase activity. Blunted signal transduction downstream of the TCR occurred in the absence of any changes in TCRβ (Fig. 4D) or CD3ε (Fig. 4E) surface expression, although we observed a slight but significant decrease in CD28 expression (Fig. 4F). Altogether, both DNMAML CD4+ and CD8+ alloreactive T cells acquired a blunted capacity for Ras/MAPK and NF-κB activation downstream of TCR/CD28 signals after allo-BMT.
Alloreactive DNMAML CD4+ and CD8+ T cells have increased expression of multiple negative regulators of T cell activation
Decreased Ras/MAPK signaling leading to reduced cytokine production has been described in certain forms of T cell hyporesponsiveness or anergy (32, 33). Mechanistically, increased expression of diacylglycerol kinases (Dgk) such as Dgkα and Dgkζ was linked to increased degradation of diacylglycerol into phosphatidic acid, resulting in blunted Erk1/Erk2 activation and cytokine production (34, 35). Interestingly, restimulated alloreactive DNMAML CD4+ T cells had elevated levels of Dgka and Dgkz mRNA, whereas DNMAML CD8+ T cells had increased Dgka and a trend for more Dgkz transcripts (Fig. 5A). This constellation of effects was reminiscent of T cell anergy. Therefore, we studied a panel of anergy-associated genes, starting with the NFAT-dependent genes Egr2/3 (36, 37). Egr3 but not Egr2 transcripts were increased in alloreactive DNMAML CD4+ and CD8+ T cells (Fig. 5B). We also observed increased expression of Rnf128 and Cblb, encoding Grail and Cbl-b, two E3 ubiquitin ligases that function as negative regulators of T cell activation (38, 39). In contrast, Itch expression was not significantly changed (Fig. 5C). As coinhibitory receptors can also regulate alloreactive T cell activation and function, we investigated expression of Btla, Ctla4, and Pdcd1 (encoding Pd-1) in DNMAML T cells. Although Ctla4 expression was slightly decreased, alloreactive DNMAML CD4+ T cells had increased Btla, and DNMAML CD8+ T cells elevated Pdcd1 mRNA (Fig. 5D). These mRNA changes correlated well to protein expression with decreased Ctla-4 and increased Blta and Pd-1 in alloreactive DNMAML CD4+ and CD8+ T cells (Fig. 5E). Finally, DNMAML alloreactive T cells had increased intracellular cAMP, a second messenger that provides negative-feedback regulation of T cell activation (Fig. 5F) (40).
Altogether, Notch-deprived alloreactive CD4+ and CD8+ T cells acquired features of hyporesponsive T cells, including increased expression of several negative regulators of T cell activation, some of which are NFAT dependent. Importantly, naive DNMAML CD4+ and CD8+ T cells expressed normal levels of these negative regulators (except for Cblb and Itch, which were mildly elevated in naive DNMAML CD4+ T cells) (Supplemental Fig. 4). These findings suggest that Notch deprivation results in changes in naive T cells that only become apparent after allo-BMT or that Notch inhibition has minimal influence on naive T cells, but profound effects on T cells in vivo during allo-BMT.
Notch inhibition modestly reduces in vivo proliferation of alloreactive CD4+ and CD8+ T cells while enhancing expansion of natural Tregs
DNMAML alloreactive T cells acquire increased levels of negative regulators that have previously been associated with decreased proliferation, at least using in vitro models of T cell anergy (41). However, in vitro and in vivo T cell proliferation are regulated by different stimuli. To evaluate in detail the impact of Notch blockade on T cell proliferation in vivo after allo-BMT, we used Rbpjf/f × Cd4-Cre mice (Fig. 6). As DNMAML T cells, CSL/RBP-Jk–deficient T cells fail to respond to Notch signals, but lack DNMAML-GFP fluorescence, allowing use of CFSE to track proliferation. At day 3 after allo-BMT, Notch-deficient T cells had proliferated slightly less than WT T cells (Fig. 6A). However, by day 5, >99% of donor WT and CSL/RBP-Jk–deficient T cells had proliferated for more than six to eight divisions (Fig. 6B). Thus, despite modestly reduced initial proliferation in the absence of Notch signaling, Notch-deprived T cells accumulated to levels similar to WT T cells by day 5 after allo-BMT. After this initial burst, ex vivo restimulation of Notch-deprived alloreactive T cells revealed only modest cycling defects (Fig. 6C, 6D). Similar in vivo and in vitro results were observed when DNMAML T cells were labeled with eFluor670 (data not shown). To assess ongoing in vivo proliferation, we performed a BrdU pulse-chase experiment 5–8 d after allo-BMT (Fig. 6E–G). CSL/RBP-Jk–deficient T cells demonstrated slightly reduced initial BrdU incorporation during the pulse phase (Fig. 6F). During chase, decreased loss of BrdU was apparent in Notch-deprived T cells (Fig. 6G). Thus, Notch inhibition modestly reduced in vivo proliferation of alloreactive CD4+ and CD8+ T cells, while preserving their massive initial expansion.
Traditional immunosuppressants often prevent Treg expansion, which can increase GVHD severity (42). If inhibition of Notch signaling is to be used to prevent or treat GVHD, we needed to assess the impact of Notch deprivation on Treg expansion. Using FIR+ DNMAML mice, we observed increased accumulation of DNMAML as compared with WT Foxp3+ donor T cells (Fig. 7A) (21). Furthermore, depletion of Tregs from the donor DNMAML inoculum completely prevented Treg expansion (Fig. 7B–D). These data indicate that rather than increasing conversion to induced Tregs, Notch blockade enhanced expansion of pre-existing Tregs after allo-BMT.
Preserved antihost and antitumor cytotoxicity of alloreactive Notch-deprived CD8+ T cells
Our previous work showed that alloreactive Notch-deprived CD4+ T cells had preserved cytotoxicity against allogeneic host and tumor cells after transplantation (7). To test the overall in vivo cytotoxic potential of DNMAML B6 CD4+ and CD8+ T cells, BALB/c transplant recipients were challenged with a 1:1 mix of CFSEHigh BALB/c and CFSELow B6-CD45.1 splenocytes. Cytotoxicity against BALB/c targets was measured by flow cytometry (Fig. 8A). Although recipients receiving TCD BM could not elicit cytotoxicity against the BALB/c targets, mice receiving WT or DNMAML splenocytes showed high and similar cytotoxicity against CFSEHigh BALB/c targets (Fig. 8A).
To determine if alloreactive purified DNMAML CD8+ T cells could lyse allogeneic tumor cells, an in vitro cytotoxicity assay was used. T cells were primed in vivo in lethally irradiated BALB/c recipients before assessing cytotoxicity ex vivo. Incubation of alloreactive WT or DNMAML CD8+ T cells with 51Cr-labeled allogeneic A20 and P815 (H2kd) tumor cells showed efficient cytotoxicity, with preserved cytotoxicity against A20 cells and preserved or only slightly reduced cytotoxicity against P815 cells (Fig. 8B). As expected, syngeneic EL4 (H2kb) tumor cells were not killed (Fig. 8B). Collectively, these data demonstrate that Notch inhibition in alloreactive CD8+ T cells preserved a high degree of cytotoxic potential after transplantation.
Our findings highlight a new, shared mechanism of Notch action in alloreactive CD4+ and CD8+ T cells that differs from all previously reported Notch functions in T cells. We used pan-Notch inhibition specifically in T cells to study the effects of Notch signaling in CD4+ and CD8+ T cell differentiation and function in several models of acute GVHD. Notch was absolutely required in both CD4+ and CD8+ T cells to mediate lethal acute GVHD. Notch inhibition preserved in vivo T cell expansion, but led to profoundly decreased IFN-γ production by both T cell subsets. Decreased IFN-γ production was not explained by an overall defect in Th1 CD4+ or effector CD8+ T cell differentiation, as expression of the master transcription factors T-bet and Eomesodermin was preserved or even enhanced in the absence of Notch signaling. In contrast, analysis of signal transduction pathways downstream of the TCR revealed defects in Ras/MAPK and NF-κB activation in Notch-deprived T cells, in addition to increased expression of multiple negative regulators of T cell activation. These features of hyporesponsiveness were observed in vivo upon T cell activation without Notch signaling. Importantly, Notch inhibition preserved the overall expansion of alloreactive T cells and efficient cytotoxic potential, while leading to increased accumulation of pre-existing natural Foxp3+ Tregs. This constellation of effects led to beneficial immunomodulation, indicating that Notch is an attractive therapeutic target to control GVHD after allo-BMT.
Past work in other immune contexts suggested that Notch could directly regulate Tbx21 transcription in CD4+ T cells and Eomes expression in CD8+ T cells (13, 18, 19). In contrast, Notch appeared dispensable for transcriptional activation of these genes in alloreactive T cells during GVHD (Fig. 3A–C) (7). It is possible that use of different experimental systems to manipulate Notch signaling could underlie these discrepant observations. Others reported that Notch receptors could have noncanonical effects independent of CSL/RBP-Jk and MAML, although the nature of these effects is not well defined (43). However, in alloreactive T cells at least, the dominant effects of Notch are mediated by canonical signaling and do not involve decreased Tbx21 and Eomes expression. Alternatively, different signaling pathways may be required to activate Tbx21 and Eomes transcription in distinct immune responses. After allo-BMT, T cell exposure to abundant alloantigens in a highly inflammatory environment may bypass the requirement for Notch signaling to activate these genes. Moreover, an interesting feature of Notch-deprived alloreactive T cells was increased Eomes expression, a finding that was particularly apparent in CD8+ T cells but also detected in the CD4+ compartment. Although the regulation of Eomes expression during T cell differentiation is incompletely understood, decreased IL-12 signaling and increased Foxo1 activity were reported to enhance Eomes at the expense of Tbx21 expression (44, 45). The effects of Notch deprivation on Eomes could be mediated via interference with these pathways. Interestingly, increased Eomes expression was reported in memory CD8+ T cells that acquire an enhanced ability for long-term persistence, as opposed to terminal differentiation (44, 46). This mechanism could contribute to the enhanced expansion and survival of Notch-deprived alloreactive T cells after allo-BMT.
In contrast to past findings suggesting that Notch regulates independent aspects of CD4+ and CD8+ T cell biology, our results revealed similar effects of Notch in the CD4+ and CD8+ T cell compartments during GVHD. Notch-deprived CD4+ and CD8+ T cells shared key functional properties and gene expression changes after allo-BMT. During in vivo activation, DNMAML alloreactive T cells acquired elevated levels of several negative regulators of T cell activation, including genes for which activation is NFAT dependent. A prominent feature of Notch-deprived T cells was decreased activation of Ras/MAPK and NF-κB pathways. Ras/MAPK and NF-κB activation are controlled by diacylglycerol, whose levels are negatively regulated by the lipid kinases Dgkα/ζ. Elevated Dgka and Dgkz expression in DNMAML T cells is reminiscent of past observations in models of T cell hyporesponsiveness with decreased Ras/MAPK activation (34, 35). However, DNMAML T cells also acquired elevated expression of other negative regulators of T cell activation, including E3 ubiquitin ligases and coinhibitory receptors, in addition to increased intracellular cAMP. Thus, the functional properties of Notch-deprived alloreactive T cells may not be explained entirely by decreased Ras-MAPK and NF-κB activation.
Despite the profound effects of Notch inhibition on cytokine production and a slight reduction in their initial proliferative burst, the overall accumulation of DNMAML T cells was preserved in vivo. These findings contrast with many other interventions that decrease expansion of alloreactive T cells in vivo. Interestingly, past work suggested that in vivo proliferation of alloreactive T cells was independent of IL-2 and may be controlled by IL-15, at least in MHC-mismatched allo-BMT models (47). Furthermore, classical models of T cell hyporesponsiveness and anergy have often been examined in vitro, a situation in which IL-2 production may play a critical role to support proliferation that does not reflect its effects in vivo (41). Other possible mechanisms accounting for the preserved or even enhanced in vivo expansion of DNMAML alloreactive T cells include decreased activation-induced cell death, for example, as a result of reduced exposure to IFN-γ (48). In parallel to these effects on conventional alloreactive T cells, Notch inhibition markedly enhanced in vivo expansion of Foxp3+ Tregs without inhibiting effector T cell expansion, which was explained by increased expansion of pre-existing natural Tregs present in the transplant inoculum. These findings may contribute to the protective effects of Notch inhibition in GVHD and contrast with a shortcoming of many immunosuppressive strategies, including calcineurin inhibitors, which limit Treg proliferation (42).
Alloreactive Notch-deprived CD8+ T cells had largely preserved cytotoxicity after allo-BMT, similar to our previously reported effects of Notch deprivation in CD4+ T cells (7). An interesting feature of alloreactive Notch-deprived T cells is their decreased cytokine production but preserved cytotoxic potential. Dissociation of cytokine production from cytotoxicity responses could reflect differential sensitivity of these pathways to Notch inhibition. Preserved or even enhanced T-bet and Eomesodermin expression in Notch-deprived T cells could be responsible for maintaining their cytotoxic potential after allo-BMT. Prior work has shown that T-bet and Eomesodermin are important for transcription of cytotoxic molecules (49). Collectively, Notch inhibition in alloreactive CD4+ and CD8+ T cells preserved efficient cytotoxicity while minimizing GVHD.
Altogether, our findings reveal a broad effect of Notch signaling in CD4+ and CD8+ alloreactive T cells during GVHD. Notch-deprived alloreactive T cells acquired features previously associated with hyporesponsiveness or anergy. However, this had differential effects on T cell effector functions in vivo in the posttransplantation environment, with profound inhibition of cytokine production but preserved T cell expansion, cytotoxic potential, and natural Treg accumulation. Notch-deprived T cells maintained potent cytotoxic activity in vivo, suggesting a split anergy phenotype (7, 50). Overall, Notch inhibition in T cells induced a unique combination of effects that potently inhibited GVHD, highlighting the promise of this new therapeutic strategy after allo-BMT.
The authors have no financial conflicts of interest.
We thank Dr. Tasuku Honjo (Kyoto University) for Rbpjf/f mice, Dr. Steve Reiner (Columbia University) for Eomesf/f × Cd4-Cre mice, and Dr. Benjamin Segal (University of Michigan) for Tbx21−/− mice.
This work was supported by a Damon Runyon-Rachleff Innovation award (DRR-05A-09) and a Scholar Award from the American Society of Hematology and the National Institutes of Health (RO1-AI091627) (to I.M.). Individual support included National Institutes of Health T32 training Grants AI007413-17 (to A.R.S.) and GM007315 (to J.C.), a Trainee Research Award from the American Society of Hematology (to G.T.S.), and a Miller Award for innovative immunology research (to A.R.S.). Flow cytometry was partially supported by University of Michigan Cancer Center Grant P30-CA46592.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- allogeneic bone marrow transplantation
- diacylglycerol kinase
- dominant-negative form of Mastermind-like 1
- Foxp3–internal ribosome entry site–RFP
- graft-versus-host disease
- intracellular Notch
- TCD BM
- T cell–depleted bone marrow
- regulatory T cell
- Received December 18, 2012.
- Accepted March 29, 2013.
- Copyright © 2013 by The American Association of Immunologists, Inc.