After encountering allogeneic nonself, monocytes differentiate to DCs, which in turn activate the adaptive immune system that drives transplant rejection. The downstream mechanisms of allorecognition are largely unknown. We analyzed scRNA-seq data sets to identify transcriptional changes in monocytes occurring after allostimulation. Hspa1a, which encodes HSP70, was upregulated in monocytes after allostimulation in contrast to syngeneic controls in mice lacking T, B, and NK cells. Similar findings were seen in scRNA-seq data derived from kidney biopsies of rejecting transplant patients. To validate the role of HSP70 in innate allorecognition and transplantation, we performed allogeneic bone marrow plug and kidney transplantation into WT, Hspa1a/Hspa1b–/–, and DSG-treated (HSP70 inhibitor) B6 mice and examined the graft immune infiltrate. Histology, T cell infiltration, and survival were assessed in kidney transplanted mice. In both models, DSG-treated or HSP70-KO recipients displayed significantly reduced infiltration by monocyte-derived DCs (mo-DC). Chronic rejection of Balb/c kidney grafts in WT B6 recipients was attenuated in DSG-treated and HSP70-KO recipients as indicated by a reduction in Banff score. Further experiments demonstrated that in vitro allostimulated immature HSP70-KO BMDC showed less maturation compared with WT BMDC. Targeting HSP70 in innate immune cells offers a novel approach to reduce chronic kidney graft rejection.

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation (AlloHCT). Conditioning with chemotherapy and radiation enables donor engraftment but causes tissue injury that upregulates the alarmin interleukin-33 (IL-33) in fibroblastic reticular cells (FRCs) in secondary lymphoid organs (SLO) and tissue stromal cells. While mechanisms releasing IL-33 from nuclear sequestration remain elusive, free IL-33 directly activates donor CD4+ T cells to promote Th1 differentiation and expansion. Early IL-33 blockade limits GVHD initiation, yet the role of local IL-33 in sustaining alloimmunity remains unclear. We used preclinical models to define how IL-33 signaling to donor T cells within SLO versus the gastrointestinal tract (GIT) stroma controls active GVHD and graft-versus-lymphoma (GVL) responses. BALB/c recipients received B6 T cell—depleted bone marrow, A20 lymphomas, and T cells from donors with inducible ST2 (IL-33R) deletion to control IL-33 signaling during GVHD and GVL. To identify stromal functions, B6 recipients lacking IL-33 in FRCs (CCL19-Cre×Il33fl/fl) or gut epithelium (Vil-Cre×Il33fl/fl) were transplanted with BALB/c T cells and bone marrow. T cell—dependent IL-33 release was tested using GzmB⁻/⁻, St2+/+, and St2⁻/⁻ T cells. Late ST2 deletion resolved GVHD without compromising GVL. IL-33 signaling increased Foxp3 levels and reduced T-bet expression in donor CD4+ T cells, while sustaining TCF-1. Unexpectedly, loss of FRC-derived IL-33 exacerbated GVHD, whereas gut epithelial IL-33 deletion was protective. IL-33 induced GzmB, and GzmB⁻/⁻ T cells displayed impaired activation and expansion. Early SLO-derived IL-33 establishes effector programming that influences later tissue responses. IL-33+ stroma—T-cell crosstalk programs donor CD4+ T-cell fate and shapes local alloimmunity during GVHD. IL-33—stroma feedback is a key signal to alloreactive T cells during GVHD. NIH/NIAID F30AI147437, NIH T32 CA082084, NIH/NHLBI R01HL122489, NIH/NIAID R21AI121981, NIH/NIAID R56AI13927 Transplantation Immunology (TRAN)

Background: Graft vs. host disease (GVHD) remains a major complication of allogeneic hematopoietic stem cell transplantation (alloHSCT). To create space for donor stem cells and prevent their rejection, alloHSCT protocols rely on conditioning regimens involving chemotherapy and radiation. Conditioning causes tissue damage, which increases the tissue injury signal or “alarmin” interleukin (IL)-33 in fibroblastic reticular cells (FRC) of the secondary lymphoid organs (SLO). Mechanisms releasing IL-33 from its sequestration in the nucleus remain elusive, but free IL-33 directly stimulates donor CD4 T cells to prime IL-12-independent Type 1 T helper cell (Th1) differentiation and expansion. Targeting IL-33 early after alloHSCT limits GVHD in pre-clinical models. The gastrointestinal tract (GIT) also upregulates IL-33 in response to TBI and GVHD, but a direct role for local IL-33 in sustaining pathogenic donor responses is unclear. Our goal was to manipulate the IL-33 pathway in the SLO or GIT to better understand how stromal communications with donor T cells initiate and shape GVHD and graft vs. lymphoma (GVL) responses. Methods: We compared donor T cells (plus or minus inducible deletion of the IL-33 receptor, ST2) for their ability to mediate GVHD vs. GVL (A20 lymphoma) in BALB/c recipients receiving total body irradiation (TBI) and CD45.1+ B6 T cell depleted bone marrow (TCD BM). To define the role for IL-33-derived from the SLO vs. the GIT, we assessed survival of B6 recipients deficient in IL-33 in FRCs (CCL19-CrexIl33fl/fl) vs. those deficient in IL-33 in the epithelium of the GI tract (Vil-CrexIl33fl/fl) receiving TBI and BALB/c T cells. To investigate if donor T cells mediate IL-33 release, we completed an ex vivo model using B6 St2+/+, St2-/-, and GzmB-/- CD3 T cells co-cultured for 5 days with BALB/c TCD splenocytes and LN-derived FRCs that had been irradiated at 3500 cGy alone or with the IL-33 antagonist, sST2. Similar in vivo studies were conducted where the above donor B6 St2+/+, St2-/-, and GzmB-/- CD3 T cells were transplanted into BALB/c recipients and assessed for GzmB and donor T cell expansion on day 5 post-alloHSCT. Results: Ablating ST2 at days 10-14 post-transplant (after initial GVHD development) improved clinical scores and limited mortality. Further, sustained IL-33 signaling was not required for GVL activity. Mechanistically, late ST2 deletion was associated with increased Foxp3 expression and reciprocal Tbet decrease in donor CD4+ T cells from both SLO and GVHD target tissues. Sustained IL-33 signaling also maintained donor T cell TCF1 expression in SLO. Surprisingly, isolated deletion of FRC-derived IL-33 increased GVHD mortality in the CCL19-CrexIl33fl/flrecipients. Mechanistic studies showing FRC-derived IL-33 stimulated CD4+ PD-1 expression and blunted the total number of CD4 and CD8 T effectors in the GIT at day 21 post-alloHSCT. Whereas, deletion of IL-33 in the gut epithelium in the Vil-CrexIl33fl/fl recipients was protective and prolonged survival. RNAseq analysis suggested that IL-33 stimulates T cell granzyme B (GzmB) expression. GzmB deficient (Gzmb-/-) donor T cells displaying reduced activation and expansion in vitro and in vivo, in a phenotype similar to ST2 deficient CD4 T cells. Consistent with the importance of GzmB in mediating IL-33 signals, antagonizing IL-33 had no impact on GzmB-/- T cell responses similar to ST2 deficient CD4 T cells when compared to Gzmb+/+, which failed to expand when IL-33 was sequestered. Conclusions: Our data reveals that GzmB-mediated crosstalk between donor T cells and IL-33+ stroma orchestrates donor T cell identities and tunes local alloimmune responses after alloHSCT. Delayed deletion of ST2 signaling on donor T cells promotes survival through an upregulation of regulatory mechanisms in GVHD target tissues. Similarly, targeted deletion of IL-33 in the GIT provides protection from donor driven pathology. Whereas, targeted deletion of IL-33 from SLO FRC promotes GVHD mortality by down regulating intrinsic T cell exhaustion mechanisms in the SLO, which impacts later CD4+ T cell alloimmune responses to available IL-33 in target tissues, driving GVHD pathology. These data suggest distinct temporal and tissue specific roles for IL-33-driven programing of donor CD4+ T cells. In total, these data indicate that continual feedback between donor T cells and recipient stroma is central to the development and maintenance of GVHD.

Conditioning before allogeneic hematopoietic stem cell transplantation (AlloHSCT) increases the tissue injury signal IL-33 in fibroblastic reticular cells (FRC). Released IL-33 directly stimulates donor CD4 T cells to prime IL-12-independent Type 1 T helper cell (Th1) differentiation and expansion. Tissue stroma upregulates IL-33, but a role for IL-33 in sustaining the pathogenic donor Th1 responses causing GVHD is unclear. We compared B6 mice with inducible ST2 deletion (R26-CreERT2xSt2fl/fl) to wildtype (WT) R26-CreERT2 as T cell donors in a lethal GVHD model (B6 to BALB/c). Donor ST2 deletion at days 10-14 post AlloHSCT increased CD4 T cells Foxp3 expression with reciprocal decreases in Tbet expression in both the lymphoid organs and target tissues. Sustained IL-33 signaling also maintained donor T cell TCF1 expression. Ablating ST2 after GVHD development improved clinical scores and promoted recipient weight gain. How bioactive IL-33 is released from nuclear sequestration remains undefined. RNAseq analysis suggested that IL-33 stimulates T cell granzyme B (GzmB) expression and B6 GzmB deficient (Gzmb-/-) donor T cells displayed reduced activation and expansion similar to ST2 deficient CD4 T cells. In contrast to GzmBWT, anti-IL-33 antibodies had no impact on GzmBKO T cell responses. Thus, cross-talk between donor T cells and IL-33+ stroma orchestrates the T cell identities that are critical to sustain the pathogenic CD4 T cell responses causing GVHD.

Graft-vs-host disease (GVHD) is a common complication of allogeneic stem cell transplant (alloSCT) wherein donor T cells target alloantigens on recipient tissues. It is unclear how alloimmune responses are maintained in GVHD despite abundant antigen, which causes T cell anergy, deletion and exhaustion. Previously, we identified alloreactive TCF-1 high T cells arising post-transplant that resemble exhausted progenitors (T EXP) capable of propagating immune responses in other chronic antigen models. Here, we sought to further characterize these cells in the B6→129 MHC-matched GVHD mouse model, in which 129 recipients express the immunodominant H-2K b-restricted minor histocompatibility antigen (miHA) H60. At day +7 post-transplant, alloreactive CD8 + cells specific to H60 (as determined by MHC-I-tetramer staining; Tet H60+) were nearly uniformly PD-1 hiTox hi whereas Tet H60- cells displayed a bimodal distribution into discrete PD-1 hiTox hi and PD-1 loTox lo populations, indicative of more diverse antigen experiences. Among these both Tet H60+ and Tet H60- cells were TCF-1 hi cells. TCF-1 hi Tet H60+ cells were uniformly CD39 loTox hiPD-1 hi, which is a canonical T EXP phenotype. In contrast, among activated Tet H60- cells there were TCF-1 hi cells that were CD39 loTox hiPD-1 hi and Tox loPD-1 lo. At later times in spleen and lymph node, and in GVHD target tissues, these populations of TCF-1 + Tet H60+ and Tet H60- were found. To test if these CD39 loTCF-1 hi T EXP had proliferative advantages in GVHD, we sorted congenic TCF-1 hiCD39 lo and TCF-1 loCD39 hi CD8 + cells from recipient spleens 14-days post-transplant and adoptively transferred them in competition in a 1:1 ratio (of Tet H60+ cells) into newly transplanted recipients. Among Tet H60+ cells in all tissues at day 14 post-transfer, TCF-1 hiCD39 lo-sorted progeny greatly outperformed TCF-1 loCD39 hi-sorted progeny. In line with their role as a source of GVHD effectors, progeny of TCF-1 hiCD39 lo cells were mostly TCF-1 loCD39 hi; however, a fraction remained TCF-1 hi consistent with their being able to undergo self-renewal. Conversely, we observed few if any TCF-1 + progeny of CD39 hi cells. We next tested whether TCF-1 was an important mediator of T cell fitness or whether it was only a marker for functionality. To do so we competed congenic wild-type (WT) and Tcf7 p45-/- (p45 -/-) donor CD8 cells, which lack the N-terminal β-catenin binding domain of TCF-1, in allogeneic (129) and syngeneic (B6) recipients. Strikingly, p45 -/- CD8 cells were greatly outcompeted by WT CD8 cells in 129 recipients in all tissues and at all times post-transplant, among both Tet H60+ and Tet H60- cells. In contrast, in B6 recipients, WT and p45 -/- cells remained evenly matched, suggesting that full-length TCF-1 isoforms are dispensable for lymphopenia-induced T cell expansion. Further, p45 -/- cells were also not disadvantaged when adoptively transferred into B6 mice and acutely challenged with H60 antigen by vaccination. Together these data suggest a model wherein TCF-1 hi progenitor like T cells are seeded in GVHD target organs where they may serve as a key local source for GVHD effectors, and moreover, full-length TCF-1 is itself critical for alloreactive T cell fitness in GVH responses.

Graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation is a major cause of nonrelapse morbidity and mortality. Although ruxolitinib is now approved for the treatment of steroidrefractory GVHD, to date, no agent added to corticosteroids has been shown to improve outcomes compared with corticosteroids alone. In this issue of Blood Advances, Al Malki et al presented the results of a multicenter phase 2 study that tested whether the addition of natalizumab, a humanized antibody against the α4 subunit of α4β7 integrin, would improve the outcomes of new-onset acute GVHD. The primary end point was a complete response after 28 days, defined as the clinical resolution of GVHD in the target organs.

Summary: In allogeneic hematopoietic stem cell transplantation, donor αβ T cells attack recipient tissues, causing graft-vs-host disease (GVHD), a major cause of morbidity and mortality. A central question has been how GVHD is sustained despite T cell exhaustion from chronic antigen stimulation. The current model for GVHD holds that disease is maintained through the continued recruitment of alloreactive effectors from blood into affected tissues. Here we show, using multiple approaches including parabiosis of mice with GVHD, that GVHD is instead primarily maintained locally within diseased tissues. By tracking 1203 alloreactive T cell clones we fitted a mathematical model predicting that within each tissue a small number of progenitor T cells maintain a larger effector pool. Consistent with this, we identified a tissue-resident TCF-1+ subpopulation that preferentially engrafted, expanded, and differentiated into effectors upon adoptive transfer. These results suggest that therapies targeting affected tissues and progenitor T cells within them would be effective.

Recipient effector T cells differentiate into functional tissue-resident memory T cells, causing graft rejection after kidney transplantation. Memories of rejection Long-term graft survival after organ transplantation can be hindered by immune-mediated allograft rejection; thus, understanding these immune responses is crucial to developing new transplant-supporting therapies. Tissue-resident memory T cells (TRM), a subset of memory T cells that reside in barrier tissues and do not recirculate, are detectable in transplanted organs, but it is unclear if they contribute to allograft rejection. Abou-Daya et al. created a mouse model of T cell–mediated kidney transplant rejection, showing that adoptively transferred, kidney antigen–specific effector T cells differentiated into functional, nonrecirculating antigen-specific TRM in the transplanted kidneys. These kidney antigen–specific TRM induced allograft rejection. These data suggest that TRM in transplanted allografts can contribute to rejection and that targeting alloreactive TRM might improve long-term graft survival in transplant recipients. Tissue-resident memory T cells (TRM) contained at sites of previous infection provide local protection against reinfection. Whether they form and function in organ transplants where cognate antigen persists is unclear. This is a key question in transplantation as T cells are detected long term in allografts, but it is not known whether they are exhausted or are functional memory T cells. Using a mouse model of kidney transplantation, we showed that antigen-specific and polyclonal effector T cells differentiated in the graft into TRM and subsequently caused allograft rejection. TRM identity was established by surface phenotype, transcriptional profile, and inability to recirculate in parabiosis and retransplantation experiments. Graft TRM proliferated locally, produced interferon-γ upon restimulation, and their in vivo depletion attenuated rejection. The vast majority of antigen-specific and polyclonal TRM lacked phenotypic and transcriptional exhaustion markers. Single-cell analysis of graft T cells early and late after transplantation identified a transcriptional program associated with transition to the tissue-resident state that could serve as a platform for the discovery of therapeutic targets. Thus, recipient effector T cells differentiate into functional graft TRM that maintain rejection locally. Targeting these TRM could improve renal transplant outcomes.

In hematopoietic cell transplants, alloreactive T cells mediate the graft-versus-leukemia (GVL) effect. However, leukemia relapse accounts for nearly half of deaths. Understanding GVL failure requires a system in which GVL-inducing T cells can be tracked. We used such a model wherein GVL is exclusively mediated by T cells that recognize the minor histocompatibility antigen H60. Here we report that GVL fails due to insufficient H60 presentation and T cell exhaustion. Leukemia-derived H60 is inefficiently cross-presented whereas direct T cell recognition of leukemia cells intensifies exhaustion. The anti-H60 response is augmented by H60-vaccination, an agonist αCD40 antibody (FGK45), and leukemia apoptosis. T cell exhaustion is marked by inhibitory molecule upregulation and the development of TOX+ and CD39−TCF-1+ cells. PD-1 blockade diminishes exhaustion and improves GVL, while blockade of Tim-3, TIGIT or LAG3 is ineffective. Of all interventions, FGK45 administration at the time of transplant is the most effective at improving memory and naïve T cell anti-H60 responses and GVL. Our studies define important causes of GVL failure and suggest strategies to overcome them. In hematopoietic stem cell transplants, T cells mediate graft-versus-leukemia (GVL), but GVL can fail leading to leukemia relapse. Here the authors use a mouse model in which T cells target the minor histocompatibility antigen H60 to show how this can occur, characterize the CD8+ T cell response and demonstrate how anti-CD40 antibody therapy improves GVL.