Alloreactive CD4 Research Articles

Acquired immunostimulatory phenotype of migratory CD103+ DCs promotes alloimmunity following corneal transplantation.

After transplantation, Th1-mediated immune rejection is the predominant cause of graft failure. Th1 cell sensitization occurs through complex and context-dependent interaction among antigen-presenting cell subsets, particularly CD11b+ DCs (DC2) and CD103+ DCs (DC1). This interaction necessitates further investigation in the context of transplant immunity. We used well-established preclinical models of corneal transplantation and identified distinct roles of migratory CD103+ DC1 in influencing the outcomes of the grafted tissue. In recipients with uninflamed corneal beds, migratory CD103+ DC1 demonstrate a tolerogenic phenotype that modulates the immunogenic capacity of CD11b+ DC2 primarily mediated by IL-10, suppressing alloreactive CD4+ Th1 cells via the PD-L1/PD-1 pathway and enhancing Treg-mediated tolerance via αvβ8 integrin-activated TGF-β1, thus facilitating graft survival. Conversely, in recipients with inflamed and vascularized corneal beds, IFN-γ produced by CD4+ Th1 cells induced migratory CD103+ DC1 to adopt an immunostimulatory phenotype, characterized by the downregulation of regulatory markers, including αvβ8 integrin and IL-10, and the upregulation of IL-12 and costimulatory molecules CD80/86, resulting in graft failure. The adoptive transfer of ex vivo induced tolerogenic CD103+ DC1 (iDC1) effectively inhibited Th1 polarization and preserved the tolerogenic phenotype of their physiological counterparts. Collectively, our findings underscore the essential role played by CD103+ DC1 in modulating host alloimmune responses.

Major histocompatibility complex and peptide specificity underpin CD8+ T cell direct alloresponse

The direct alloresponse, pivotal in transplant rejection, occurs when recipient T cells recognize intact allogeneic peptide-major histocompatibility complex (MHC) complexes. Despite extensive research, our understanding of alloreactive CD8+ T cells against an individual MHC allele in humans remains limited, especially their precursor frequency, MHC specificity, and peptide specificity. By using K562 cell–based artificial antigen-presenting cells expressing human leukocyte antigen (HLA)-A∗01:01, HLA-A∗02:01, or HLA-A∗03:01, we determined that the precursor frequency of alloreactive CD8+ T cells against a single MHC allele ranges from 0.1% to 0.5%. Further, these cells exhibited MHC specificity regarding proliferation, activation, interferon gamma secretion, and cytolytic ability, with limited crossreactivity toward nontargeted MHC alleles. Focusing on anti-A2 alloreactive CD8+ T cells, we developed a peptide-exchangeable artificial antigen-presenting cell that displays selected peptides on HLA-A∗02:01. From a set of 95 computationally curated A2-restricted peptides most abundant in renal tubular cells, we identified 2 immunogenic kidney peptides across multiple donors. Overall, our findings significantly enhance the understanding of direct alloresponse and provide a toolkit for future mechanistic studies and reproducible patient monitoring.

What makes the kidney so tolerant?

Various organ allografts differ in their propensity to be spontaneously accepted without any immunosuppressive treatment. Understanding the mechanisms behind these differences can aid in managing alloimmune responses in general. C57BL/6 mice naturally accept DBA/2J kidney allografts, forming tertiary lymphoid organs containing regulatory T cells (rTLOs), crucial for graft acceptance. In this issue of the JCI, Yokose and colleagues revealed that rTLOs promote conversion of cytotoxic alloreactive CD8+ T cells into exhausted/regulatory ones, through an IFN-γ-mediated mechanism. Their study provides insights into tolerance development that could help promote the acceptance of grafts at higher risk of rejection.

Alloreactive memory CD4 T cells promote transplant rejection by engaging DCs to induce innate inflammation and CD8 T cell priming

Alloreactive memory T cells have been implicated as central drivers of transplant rejection. Perplexingly, innate cytokines, such as IL-6, IL-1β, and IL-12, are also associated with rejection of organ transplants. However, the pathways of innate immune activation in allogeneic transplantation are unclear. While the role of microbial and cell death products has been previously described, we identified alloreactive memory CD4 T cells as the primary triggers of innate inflammation. Memory CD4 T cells engaged MHC II-mismatched dendritic cells (DCs), leading to the production of innate inflammatory cytokines. This innate inflammation was independent of several pattern recognition receptors and was primarily driven by TNF superfamily ligands expressed by alloreactive memory CD4 T cells. Blocking of CD40L and TNFα resulted in dampened inflammation, and mice genetically deficient in these molecules exhibited prolonged survival of cardiac allografts. Furthermore, myeloid cell and CD8 T cell infiltration into cardiac transplants was compromised in both CD40L- and TNFα-deficient recipients. Strikingly, we found that priming of naive alloreactive CD8 T cells was dependent on licensing of DCs by memory CD4 T cells. This study unravels the key mechanisms by which alloreactive memory CD4 T cells contribute to destructive pathology and transplant rejection.

Older age is associated with a distinct and marked reduction of functionality of both alloreactive CD4+ and CD8+ T cells.

Older recipient age is associated with a significant decreased risk for rejection after kidney transplantation which is incompletely understood. In a longitudinal study, circulating alloreactive T cells were assessed of young (≤45 years) and older (≥55 years) stable kidney transplant recipients. Alloreactive T-cells were identified by CD137-expression and phenotype, cytokine producing and proliferative capacity, were evaluated using multiparameter flowcytometry. The results show that before transplantation frequencies of alloreactive CD4+ and CD8+ T-cells in older KT-recipients are significantly higher and shifted towards an effector memory-phenotype. However, the frequency of polyfunctional (≥2 pro-inflammatory cytokines) CD4+ T-cells was significantly lower and less IL2 was produced. The frequency of polyfunctional alloreactive CD4+ T-cells and proliferation of alloreactive T-cells donor-specifically declined after transplantation reaching a nadir at 12 months after transplantation, irrespective of age. A striking difference was observed for the proliferative response of alloreactive CD8+ T-cells. This was not only lower in older compared to younger recipients but could also not be restored by exogenous IL2 or IL15 in the majority of older recipients while the response to polyclonal stimulation was unaffected. In conclusion, older age is associated with a distinct and marked reduction of functionality of both alloreactive CD4+ and CD8+ T-cells.

Calcineurin inhibition rescues alloantigen-specific central memory T cell subsets that promote chronic GVHD.

Calcineurin inhibitors (CNIs) constitute the backbone of modern acute graft-versus-host disease (aGVHD) prophylaxis regimens but have limited efficacy in the prevention and treatment of chronic GVHD (cGVHD). We investigated the effect of CNIs on immune tolerance after stem cell transplantation with discovery-based single-cell gene expression and T cell receptor (TCR) assays of clonal immunity in tandem with traditional protein-based approaches and preclinical modeling. While cyclosporin and tacrolimus suppressed the clonal expansion of CD8+ T cells during GVHD, alloreactive CD4+ T cell clusters were preferentially expanded. Moreover, CNIs mediated reversible dose-dependent suppression of T cell activation and all stages of donor T cell exhaustion. Critically, CNIs promoted the expansion of both polyclonal and TCR-specific alloreactive central memory CD4+ T cells (TCM) with high self-renewal capacity that mediated cGVHD following drug withdrawal. In contrast to posttransplant cyclophosphamide (PT-Cy), CSA was ineffective in eliminating IL-17A-secreting alloreactive T cell clones that play an important role in the pathogenesis of cGVHD. Collectively, we have shown that, although CNIs attenuate aGVHD, they paradoxically rescue alloantigen-specific TCM, especially within the CD4+ compartment in lymphoid and GVHD target tissues, thus predisposing patients to cGVHD. These data provide further evidence to caution against CNI-based immune suppression without concurrent approaches that eliminate alloreactive T cell clones.

(596) - NKG2D - A Potential Target for Alloreactive CD8 T Cells

(596) - NKG2D - A Potential Target for Alloreactive CD8 T Cells

Cord Blood-Derived Multipotent Stem Cells Ameliorate in Vitro/in Vivo Alloreactive Responses, and This Effect Is Associated with Exosomal Microvesicles in Vitro

Human cord blood derived-multipotent stem cells (CB-SCs) have been found to have immunomodulatory capabilities that can result in inhibition of immune activation. Clinically, when used to interact with apheresed peripheral blood mononuclear cells (PBMCs) before reinfusion, they can counteract inflammation and restore immune balance in patients with autoimmune diseases, including alopecia areata and type 1 diabetes. The present study aimed to explore the potential application of CB-SCs to control donor alloreactive responses involved in allogeneic hematopoietic cell transplantation, which often results in acute graft-versus-host disease (GVHD). Phenotypically, we demonstrated that CB-SCs express CD45, CD11b, and CD9 markers on the cell surface; express Oct3/4, a transcription factor for embryonic stem cells; are negative for CD3, CD14, and CD34 expression; and have low expression of HLA-DR. In an allogeneic mixed lymphocyte culture (MLC) using human CD4 T cell enriched PBMCs and allogeneic myeloid derived dendritic cells, direct coculture with CB-SCs decreased CD4 T cell proliferation and activation, as evidenced by a marked decrease in the expression of the late activation markers CD25 and HLA-DR and a reduction of the PKH26 cell proliferation membrane lipophilic marker. Cytokine profiling of MLC supernatants revealed decreased concentrations of inflammatory proteins, including IFN-γ, IL-17, IL-13, IL-2, IL-6, and MIP1-α, along with marked increases in IL-1RA, IP-10, and MCP-1 concentrations in the presence of CB-SCs. Furthermore, transwell MLC experiments revealed that a soluble component was partially responsible for the immunomodulatory effects of CB-SCs. In this regard, exosomal microvesicles (EVs) positive for CD9, CD63, and CD81 were found in CB-SC-derived, ultrafiltered, and ultracentrifuged culture supernatants. CB-SC-EVs inhibited T cell proliferation in allogeneic MLC, suggesting a potential mode of action in allogeneic responses. Finally, CB-SCs were evaluated for their cellular therapy potential in vivo and found to ameliorate the development of GVHD responses in a xenogeneic human PBMC-induced NSG mouse model. Taken together, our results indicate that CB-SCs can directly and indirectly attenuate alloreactive CD4 T cell activation and proliferation in vitro with a potentially related EV mode of action and may have potential as a cellular therapy to control donor T cell-mediated GVHD responses in vivo.

Dual Functions of Mitochondrial Calcium Uniporter in T Cell Alloimmunity

Despite pharmacological prophylaxis using calcineurin inhibitors, graft-versus-host disease (GVHD) still occurs in patients undergoing allogeneic hematopoietic stem cell transplantation. However, the mechanism that underlies the breakthrough of alloreactive T cell responses following calcium signaling inhibition remains poorly defined. The mitochondrial calcium uniporter (MCU), which is located in the inner mitochondrial membrane, controls Ca2+ uptake into the mitochondria matrix. Past dogma suggested that lack of mitochondria calcium uptake would compromise T cell immune response due to suboptimal NFAT activation upon TCR ligation. However, the exact role of MCU in the regulation of alloreactive T cell responses has not been previously determined. Here, we demonstrate dual functions of MCU in alloreactive T cell response and GVHD maintenance, highlighting MCU as a potential novel target to manipulate T cell alloreactivity. Using T cell-specific Mcu conditional knockout C57/BL6 (B6) mice (Mcu-cKO), we found that compared to WT T cells, B6 Mcu-cKO T cells induced less severe GVHD in Balb/c mice, with prolonged survival and significantly lower number of donor effector cells in the spleen and liver early after transplantation. Surprisingly, these Mcu-cKO T cell recipients developed severe liver GVHD by 60 days after transplantation, as evidenced by extensive lymphocytic infiltration in the liver, while to a lesser extent in the skin and intestine in these GVHD mice. Flow cytometric analysis of donor T cells, which were harvested between 5 and 8 weeks after transplantation, showed that Mcu-cKO T cells retained the IFN-g- and GM-CSF-producing capacity, but acquired stronger capacity of producing the cytotoxic molecule granzyme B. To comprehensively delineate the role of MCU in allogeneic T cell response, we used MHC-identical but minor histocompatibility antigen (miHA)-mismatched B6-to-Balb/b GVHD model, in which alloreactive CD8+ T cells can be identified with H60 peptide/MHC class-I dimer staining. Similarly, loss of Mcu in T cells caused less severe GVHD, attributed to the decreased number of H60+CD8+ effector T cells that showed impaired proliferation capacity. RNA-seq analysis of liver-infiltrating H60+CD8+ T cells revealed that Mcu ablation resulted in decreased expression of cell proliferation genes (Pclaf, Cdc45, Top2a, Cdca8, etc), increased expression of genes (Cd7, Bcl2, CD96, Eomes, Ezh1, etc) associated with memory T cells. These results demonstrated that Mcu is required for the expansion of alloreactive T cells, but restricts their memory-associated transcriptional programs during GVHD maintenance. Further characterization showed these alloreactive Mcu-cKO T cells produced higher levels of tissue migration molecules (CCR9, CCR5, etc) and lower death molecule (Lpin1) than their WT counterparts. Using gain-of-function strategy of retroviral Mcu overexpression in T cells (McuOE), we found McuOE CD8+ T cells displayed enhanced effector differentiation represented by higher expression of effector molecules (Ifng), decreased expression of stemness and memory associated molecules (Tcf7, Id3), and reduced expression of migration molecules (CCR9, CXCR3, etc). The cultured McuOE CD8+ T cells contracted earlier than WT. These results were further validated in vivo using the B6-to-Balb/c GVHD model. These data demonstrate a negative regulatory role of MCU in the tissue migration and persistence of alloreactive T cells. Notably, human T cells from patients with severe GVHD produced lower levels of MCU protein compared to that of non-GVHD patients. Our findings identified for the first time the dual functions of MCU in the regulation of T cell alloimmunity. MCU-mediated mitochondria calcium uptake promotes early expansion of alloreactive T cells but restricts the expression of memory-associated gene programs and tissue migration molecules. Strategies of enhancing MCU functionality in alloreactive T cells could potentially reduce their memory potential and tissue infiltration capacity, thereby reducing GVHD.

TCF-1is Required By CD8 + T Cells for the Maintenance of Alloimmune Responses in Graft-Vs-Host Disease

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.

Cellular Mechanisms Affecting Allogeneic CAR T Cell Expansion and Rejection in Large B-Cell Lymphoma

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold promise to improve CAR T access. However, limited expansion and persistence due to rejection in some recipients may impact efficacy. Elucidation of cellular mechanisms governing allogeneic CAR T expansion and persistence may inform patient management and technology development. We hypothesized that cell-extrinsic and intrinsic mechanisms differentially regulate rejection and expansion of allogeneic CAR T cells in patients with large B-cell lymphoma. We identified 11 patients with relapsed/refractory large B-cell lymphoma treated on the ALPHA-2 phase 1 trial (NCT04416984) with ALLO-501A, a healthy donor-derived CD19 CAR T with T cell receptor (TCR) knockout. ALLO-501A uses Cellectis technologies. Despite treatment with a single ALLO-501A lot, we observed heterogeneity in CAR T expansion and clinical outcomes. Six patients had robust expansion (expanders), and five patients had relatively poor expansion (non-expanders) [median maximum CAR vector copy number 21,571 (range 10,296-104,784) versus 141 (range 55-502) copies/µg DNA, p < 0.01]. As with autologous therapies, expanders exhibited longer CAR T persistence and a higher frequency of clinical responses than non-expanders. To elucidate cell-extrinsic mechanisms involved in allogeneic CAR T rejection, we hypothesized that recipient-derived alloreactive T cells limit expansion in non-expanders. We adapted a mixed lymphocyte reaction assay to identify alloreactive CD4 + and CD8 + T cell clones from pre-lymphodepletion PBMC samples. Briefly, labeled recipient PBMCs were incubated with irradiated ALLO-501A cells from the treatment lot. Proliferating recipient CD4 + and CD8 + T cells were isolated and underwent TCR sequencing. Alloreactive clones were defined as those enriched in this sample compared to an unstimulated sample from the same patient (p < 0.05 by Fisher's test) and were tracked post-treatment. Two expanders and two non-expanders were selected for analysis. Non-expanders exhibited more robust in vitro T cell proliferation in response to ALLO-501A than expanders (85 vs. 40% proliferating; p = 0.03). Thus, more alloreactive clones were identified in non-expanders (median 762 CD4 + clones, 314 CD8 + clones) than expanders (median 313 CD4 + clones, 90 CD8 + clones). Non-expanders also had higher median alloreactive CD8 + T cell frequencies than expanders at days 7 and 14 following allogeneic CAR T infusion ( Figure A). A similar pattern was not observed for alloreactive CD4 + clones or non-alloreactive T cell clones, highlighting the potential role of recipient alloreactive CD8 + T cells in the rejection process and the need to control this process for the success of allogeneic CAR T. Next, to define the role of cell-intrinsic mechanisms in allogeneic CAR T expansion, we tracked clonal evolution of the CAR T repertoire through TCR sequencing of the infusion product (IP) and post-treatment PBMC samples in all patients. At day 14 (peak allogeneic CAR T expansion), all expanders had numerous IP-derived clones and substantial clonotype overlap with each other. In contrast, no clonotype overlap was observed among non-expanders ( Figure B). Furthermore, we identified the same high-frequency IP-derived clones in multiple expanders and their relative abundance reflected the original clonal hierarchy of the IP. This pattern differs from autologous therapies, where clonal hierarchy is not maintained after infusion (Sheih, et al. Nat. Comm. 2020), and suggests that cell-intrinsic mechanisms may influence allogeneic CAR T expansion potential. Single-cell RNA and proteomic studies to characterize these expanding clones are ongoing. In conclusion, we compared expansion kinetics across 11 allogeneic CAR T recipients to elucidate the cellular mechanisms governing allogeneic CAR T expansion and rejection. We revealed the impact of recipient alloreactive CD8 + T cells in allogeneic CAR T rejection and identified expansion of identical allogeneic CAR T clonotypes with retained clonal hierarchy in multiple patients. While patient factors such as tumor burden and target antigen expression likely play a role as well, our results suggest that strategies to eliminate alloreactive recipient cells and enhance allogeneic CAR T fitness through improved product design, manufacturing and lymphodepletion could improve allogeneic CAR T expansion, persistence and efficacy.

Blockade of PD-L1/CD80 Interactions Augments Function of Activated Donor T Cells Results in Augmenting Gvhd Induced By Donor Naïve CD8 + t Cells and GVL Effect Mediated By Donor Memory CD8 + T Cells

Preventing graft-versus-host disease (GVHD) while preserving graft-versus-leukemia/lymphoma (GVL) activity remains an elusive goal for allogeneic hematopoietic cell transplantation (HCT). It was reported by others that while donor naïve CD8 + T cells mediate both GVHD and GVL activity, donor memory CD8 + T cells could mediate GVL activity without GVHD. We recently reported that in vivo specific blockade of PD-L1/CD80 interactions augment tumor immunity mediated by memory CD8 + T cells (Zhang et al: PNAS 2023). In the current studies, we evaluated the impact of blockade of PD-L1/CD80 on GVHD induced by donor naïve CD8 + T cells and GVL activity-mediated by donor memory CD8 + T cells. Sorted naïve CD8 + T or memory CD8 + T cells and TCD-BM cells from C57BL/6 donors were transplanted into lethal TBI-conditioned BALB/c recipients with or without bearing ALL cancer cells. 4 days after HCT, the recipients were injected I.P. with anti-PD-L1 mAb (43H12) that specifically blocks PD-L1/CD80 interactions without inferring PD-L1/PD-1 interactions or control IgG. We observed that naïve but not memory CD8 + T cells severely infiltrated GVHD target tissues, and the memory CD8 + T cells expanded mainly in the lymphoid tissues. In vivo blockade of PD-L1/CD80 interactions augmented GVHD induced by the naïve T cells and GVL activity mediated by the memory CD8 + T cells. In addition, blockade of PD-L1/CD80 interactions increased production of granzyme B and inflammatory cytokines (IFN-g and TNF-α) of activated donor CD8 + T cells. Since cytolytic activity of effector CD8 + T cell is reported to be associated with its metabolic fitness, we evaluated the effect of blockade PD-L1/CD80 interactions on metabolism of alloreactive donor CD8 + T cells. And blockade of the interactions enhanced the fitness of mitochondria, as indicated by electronic microscopy analysis. The blockade also increased ECAR, OCR, and ATP production of the activated CD8 + T cells, with increased influx of glucose and fatty acids; and the blockade augmented mitophagy of the activated CD8 + T cells, as indicated by reducing mitochondria mass and dysfunctional mitochondria in the CD8 + T cells. The results indicate that 1) PD-L1/CD80 interactions play an important role in regulating the mitochondria function of activated alloreactive CD8 + T cells; 2) Blockade of PD-L1/CD80 interactions can augment GVL activity mediated by donor memory T cells without augmenting GVHD. This is different from blockade of PD-L1/PD-1 interactions that resulted in lethal GVHD. *Zhang and Song are co-first authors of this abstract

Development of TCR-T cell therapy targeting mismatched HLA-DPB1 for relapsed leukemia after allogeneic transplantation.

Relapsed leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a significant challenge, with the re-emergence of the primary disease being the most frequent cause of death. Human leukocyte antigen (HLA)-DPB1 mismatch occurs in approximately 70% of unrelated allo-HSCT cases, and targeting mismatched HLA-DPB1 is considered reasonable for treating relapsed leukemia following allo-HSCT if performed under proper conditions. In this study, we established several clones restricted to HLA-DPB1*02:01, -DPB1*04:02, and -DPB1*09:01 from three patients who underwent HLA-DPB1 mismatched allo-HSCT using donor-derived alloreactive T cells primed to mismatched HLA-DPB1 in the recipient's body after transplantation. A detailed analysis of the DPB1*09:01-restricted clone 2A9 showed reactivity against various leukemia cell lines and primary myeloid leukemia blasts, even with low HLA-DP expression. T cell receptor (TCR)-T cells derived from clone 2A9 retained the ability to trigger HLA-DPB1*09:01-restricted recognition and lysis of various leukemia cell lines in vitro. Our study demonstrated that the induction of mismatched HLA-DPB1 specific T cell clones from physiologically primed post-allo-HSCT alloreactive CD4+ T cells and the redirection of T cells with cloned TCR cDNA by gene transfer are feasible as techniques for future adoptive immunotherapy.

Hedgehog signalling in allograft vasculopathy: a new therapeutic target?

Allograft vasculopathy (AV) leads to chronic rejection of organ transplants, but its causes are obscure. New research from the Jane-Wit laboratory showed that Sonic Hedgehog (SHH) signalling from damaged graft endothelium drives vasculopathy by promoting proinflammatory cytokine production and NLRP3-inflammasome activation in alloreactive CD4+PTCH1hiPD-1hiT memory cells, offering new diagnostic and therapeutic strategies.

Deleting Runx3 in CD4+ T Cells Disrupts Effector Functions and Facilitates Tolerance Induction in Solid Organ Transplantation

Abstract Long-term transplantation tolerance depends on the control of graft-reactive T cells. In particular, the alloreactivity of CD4+ T cells needs to be suppressed as these cells are both necessary and sufficient for cardiac allograft rejection. Although experimental protocols such as anti-CD154-mediated co-stimulation blockade can stably induce donor-specific tolerance in MHC-mismatched cardiac allografts, other more immunogenic organs such as skin are less susceptible to tolerogenic treatments, in part due to additional reactivity of the host against donor commensals that accompany the graft. The transcriptional mechanisms that enable CD4+ T cells to drive rejection and whose targeting can promote tolerance are not well understood. RNAseq comparison of the transcriptome in graft-reactive T cells from rejecting versus tolerant heart allograft recipients revealed several transcription factors differentially expressed between rejected and tolerant mice in both spleen and graft. Here, we report that the transcription factor Runx3 in alloreactive CD4+ T cells promotes Th1 and cytotoxic response against the allograft and resistance to tolerance induction. Conversely, ablating Runx3 in alloreactive CD4+ T cells augments the efficacy of anti-CD154 blockade treatment enabling skin graft acceptance.

Clonal dynamics of alloreactive T cells in kidney allograft rejection after anti-PD-1 therapy

Kidney transplant recipients are at particular risk for developing tumors, many of which are now routinely treated with immune checkpoint inhibitors (ICIs); however, ICI therapy can precipitate transplant rejection. Here, we use TCR sequencing to identify and track alloreactive T cells in a patient with melanoma who experienced kidney transplant rejection following PD-1 inhibition. The treatment was associated with a sharp increase in circulating alloreactive CD8+ T cell clones, which display a unique transcriptomic signature and were also detected in the rejected kidney but not at tumor sites. Longitudinal and cross-tissue TCR analyses indicate unintended expansion of alloreactive CD8+ T cells induced by ICI therapy for cancer, coinciding with ICI-associated organ rejection.

Conventional type I migratory CD103+ dendritic cells are required for corneal allograft survival

Corneal transplant rejection primarily occurs because of the T helper 1 (Th1) effector cell-mediated immune response of the host towards allogeneic tissue. The evidence suggests that type 1 migratory conventional CD103+ dendritic cells (CD103+DC1) acquire an immunosuppressive phenotype in the tumor environment; however, the involvement of CD103+DC1 in allograft survival continues to be an elusive question of great clinical significance in tissue transplantation. In this study, we assess the role of CD103+DC1 in suppressing Th1 alloreactivity against transplanted corneal allografts. The immunosuppressive function of CD103+DC1 has been extensively studied in non-transplantation settings. We found that host CD103+DC1 infiltrates the corneal graft and migrates to the draining lymph nodes to suppress alloreactive CD4+ Th1 cells via the programmed death-ligand 1 axis. The systemic depletion of CD103+ DC1 in allograft recipients leads to amplified Th1 activation, impaired Treg function, and increased rate of allograft rejection. Although allograft recipient Rag1 null mice reconstituted with naïve CD4+CD25- T cells efficiently generated peripheral Treg cells (pTreg), the CD103+DC1-depleted mice failed to generate pTreg. Furthermore, adoptive transfer of pTreg failed to rescue allografts in CD103+DC1-depleted recipients from rejection. These data demonstrate the critical role of CD103+DC1 in regulating host alloimmune responses.

Fibroblastic reticular cells mitigate acute GvHD via MHCII-dependent maintenance of regulatory T cells.

Acute graft versus host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) inflicted by alloreactive T cells primed in secondary lymphoid organs (SLOs) and subsequent damage to aGvHD target tissues. In recent years, Treg transfer and/or expansion has emerged as a promising therapy to modulate aGvHD. However, cellular niches essential for fostering Tregs to prevent aGvHD have not been explored. Here, we tested whether and to what extent MHC class II (MHCII) expressed on Ccl19+ fibroblastic reticular cells (FRCs) shape the donor CD4+ T cell response during aGvHD. Animals lacking MHCII expression on Ccl19-Cre-expressing FRCs (MHCIIΔCcl19) showed aberrant CD4+ T cell activation in the effector phase, resulting in exacerbated aGvHD that was associated with significantly reduced expansion of Foxp3+ Tregs and invariant NK T (iNKT) cells. Skewed Treg maintenance in MHCIIΔCcl19 mice resulted in loss of protection from aGvHD provided by adoptively transferred donor Tregs. In contrast, although FRCs upregulated costimulatory surface receptors, and although they degraded and processed exogenous antigens after myeloablative irradiation, FRCs were dispensable to activate alloreactive CD4+ T cells in 2 mouse models of aGvHD. In summary, these data reveal an immunoprotective, MHCII-mediated function of FRC niches in secondary lymphoid organs (SLOs) after allo-HCT and highlight a framework of cellular and molecular interactions that regulate CD4+ T cell alloimmunity.

Endothelial Cells Activate Alloreactive CD4+ T Cells to Initiate Acute Graft-Versus-Host Disease

Allogeneic donor T cells can exert an anti-leukemic response (GvL effect) after hematopoietic cell transplantation (allo-HCT). However, allogeneic donor T cells can also cause life-threatening acute graft-versus-host disease (aGvHD). Therefore, donor T cell priming is considered as a crucial event determining allo-HCT outcome. In aGvHD, donor CD4+ T cells are activated by antigen presenting cells (APCs), proliferate, differentiate and migrate to cause tissue damage. However, non-hematopoietic APCs have been shown to drive aGvHD pathology, making hematopoietic APCs dispensable in the initiation of aGvHD (Toubai et al., Blood 2012, Koyama et al., Nat Med 2012). During the aGvHD initiation phase allogenic T cells exclusively migrate to secondary lymphoid organs (SLOs) before they enter target tissues in the aGvHD effector phase (Beilhack et al., Blood 2005, Beilhack et al., Blood 2008). These observations led us to investigate the role of MHC class II mediated antigen presentation by non-hematopoietic lymph node (LN) stromal (SCs) and endothelial cells (ECs) in aGvHD. Recently we learned that alloreactive CD4+ T cells are activated in SLOs by non-hematopoietic APCs independently from Ccl19 expressing SCs, while these SCs provide protective niches to donor Tregs mitigating GvHD. Consequently, here we explored MHCII mediated antigen presentation by SLO blood endothelial cells (BECs) and lymph endothelial cells (LECs). BECs and LECs expressed endogenous MHCII, costimulatory receptors CD80/CD86 and acquired MHCII from dendritic cells via trogocytosis. Upon myeloablative irradiation, BECs and LECs upregulated CD80 and CD86 whereas MHCII expression decreased upon loss of radiosensitive host DCs. scRNA-seq revealed, BECs and LECs subsets expressing genes involved in MHCII antigen presentation (e.g. Cd74, Ctss, Ctsl, Tap2, H2-Aa, H2-Ab1, H2-Eb1) leading to exogenous antigen presentation (DQ-OVA) by EC subsets. To elucidate MHCII antigen presentation in ECs we crossed Cdh5-Cre to H2-Ab1fl mice to conditionally knockout MHCII on ECs (MHCIIΔCdh5). Allo-HCT in MHCIIΔCdh5 recipients utilizing an MHC major mismatched model (FVB→B6) did not alter allogeneic CD4+ T cell activation during aGvHD initiation. However, MHCIIΔCdh5 showed marginally reduced aGvHD and better survival. Notably, MHCIIΔCdh5 recipients retained functional hematopoietic APCs presenting alloantigens via MHCII. Therefore, we generated bone marrow (BM) chimeras by syngeneic (syn) HCT from MHCII-deficient (MHCIIΔ) donors into B6.WT mice. After allo-HCT of these BM chimeras, donor CD4+ T cells got activated in SLOs during the aGvHD initiation phase. However, to our surprise also syngeneic BM chimeric animals spontaneously developed GvHD-like symptoms making them unsuitable for studying MHCII mediated non-hematopoietic antigen presentation after allo-HCT. This striking phenotype could be explained by a defective CD44hiCD62Llow CD4+ T cell memory development in MHCIIΔ BM chimeras (Huang et al., J Immunol. 2014). Utilizing our scRNA seq dataset we identified Tie2 and Vav1 to be expressed on cells of hematopoietic lineage, however Tie2 expression was observed on ECs of the LNs. Therefore, we generated MHCIIΔVav1 mice by crossing Vav1-Cre to H2-Ab1fl. CD45- LNs cells from these animals activated CD4+ T cell in mixed lymphocyte reaction as well as in vivo. MHCIIΔVav1 mice developed less aGvHD with delayed kinetics. Subsequently, we surgically transplanted LNs from syngeneic MHCIIΔVav1 mice into complete MHCIIΔ mice. After MHCIIΔVav1 LN engraftment, these MHCIIΔ recipients underwent allo-HCT with allogenic CD4+ T cells. Alloreactive donor CD4+ T cells expressed more IFNγ and TNFα compared to controls. These experiments proved that non-hematopoietic cells in LNs can activate alloreactive CD4+ T cells to initiate aGvHD. Ultimately, we crossed MHCIIΔCdh5 to MHCIIΔVav1, resulting in mice deficient for MHCII in the entire hematopoietic and endothelial compartments. After allo-HCT MHCIIΔVav1ΔCdh5 were significantly better protected from aGvHD than littermate controls, revealing a previously unidentified role of ECs in initiating CD4+ T cell mediated aGvHD. Based on these findings we propose ECs as important non-hematopoietic APCs to activate alloreactive CD4+ T cells to trigger aGvHD suited for therapeutic interventions after allo-HCT.

Graft Versus Leukaemia (GvL): Identification & Characterisation of Alloreactive Antigens and Cognate T Cell Responses in Acute Myeloid Leukemia

Allogeneic stem cell transplantation (allo-SCT) is the most established, curative T cell cancer immunotherapy. The curative effect of allo-SCT relies on a graft-versus-leukemia (GvL) effect; but this can be difficult to dissociate from graft-versus-host disease (GvHD). For decades, the key unsolved challenge has been to identify common alloreactive peptide-human leukocyte antigen (HLA) complexes targeted by T cells that mediate GvL but not GvHD. We developed a workflow for unbiased identification of alloreactive antigens and analysis of cognate donor alloreactive T cells and their T cell receptors (TCRs; Fig 1). We studied sequential bone marrow and peripheral blood samples, collected over 15 years, from 12 high-risk AML patients who remain in continuous complete remission following allo-SCT (all >2 years). All patients had relapsed at least once pre-allo-SCT and/or were AML flow cytometry minimal residual disease (MRD)+ pre-allo-SCT. 7 had received donor lymphocyte infusions. No patients developed >1 grade GvHD. We performed whole exome sequencing of DNA from isolated patient T cells (patient germline), AML blasts and donor DNA to identify: (i) AML-specific coding variants; and (ii) germline coding single nucleotide polymorphisms (SNPs), mismatched between patient and donor. 98.7% of the mismatched variants were germline. More variants were identified in unrelated compared with sibling donors (mean: 5744 vs 3253 variants). To narrow the list of variants taken forward for functional studies, we applied two sequential filtering steps: (i) we restricted analysis to variants expressed in the patients' AML cells (determined by RNA-Seq). 28.7% of all mismatched coding variants were expressed in AML cells; and (ii) we used in silico prediction of HLA-binding affinities of peptides spanning variant amino acids to patient-specific HLA-I and -II using NetMHCpan 4.0 and NetMHCIIpan 3.0. To functionally identify alloreactive antigens, we then screened ~200-900 peptides per patient-donor pair (total 5300 peptides) for recognition by T cells present in patient peripheral blood collected 2-7 years after allo-SCT. This enriches for the identification of long-lived anti-AML responses at a time when patients were both infection- and GvHD- free. Response screening, using IFNγ ELISPOT assays, identified T cell responses to 24 germline, mismatched epitopes. In all cases, responses were only observed when the amino acid encoded by the patient SNP, but not the donor allele, was present in the peptide. The phenotype of the epitope-specific T cells was established by intracellular cytokine staining (ICS) and flow cytometry analysis, and the HLA restriction of each T cell response was determined using HLA-I, -DR, -DP and -DQ blocking antibodies. Of the 24 alloreactive peptides,19 elicited CD4+ HLA class II restricted responses and 3 were targeted by CD8+ class I restricted T cells. Two remain to be characterised (Fig 2). The population allele frequency of the germline variant SNPs targeted varies from 0.5% to 72% in the Caucasian population. Likewise, some of the restricting HLA alleles are very common (e.g. a HLA-DP allele presenting 3 epitope peptides is present in ~66% of the Caucasian population). ICS showed that epitope-specific CD4+ T cells produce Th1 cytokines (IFNg, TNFa) and degranulate (express CD107a) in response to peptide stimulation (Fig 2). Next, we performed single cell 5'RNA-seq with TCR-seq of epitope-restricted T cells (isolated via IFN-g catch assay) for all CD4+ epitope-specific T cells. Alloreactive CD4+ T cells have cytokine-producing and cytotoxic effector potential, expressing MIP1a and b, IFNg, granzyme B and FasL (Fig 2). Finally, lentiviral-based gene transfer of one of the candidate alloreactive epitope-specific TCRs into third party primary T cells confers the capacity to generate IFNg and TNFa in response to peptide stimulation, and to recognise primary AML cell-derived antigen in the presence of monocyte-derived dendritic cells (Fig 2). In vitro and in vivo killing assays are in progress. The high proportion of CD4+ T Cell responses identified by our pipeline is concordant with downregulation of HLA II expression on AML blasts at relapse post-allo-SCT, which is a common mechanism of immune evasion, found in one-third of patients. Our approach dissects the biology of alloreactive anti-AML GvL and GvHD responses and to manipulate them for therapeutic purposes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal