Mixed Lymphocyte Reaction Research Articles

ECCO Grant Ablating perianal fistulizing Crohn’s disease through genetically engineered patient specific induced pluripotent stem cells (iPSC)-derived stromal cells

Abstract Background and Aims Despite the wide range of available therapies treatment of perianal fistulizing Crohn’s disease (pCD) remains challenging. Therefore, exploring new effective therapies is of crucial importance. Local injection of allogeneic mesenchymal stromal cells has been reported to be effective, however, application of these cells is hindered by their limited expandability and differences in therapeutic effect due to donor variations. To overcome these problems, induced pluripotent stem cells (iPSC) can be used to create stromal cells as iPSC can be propagated indefinitely in their undifferentiated state. Therefore, the aim of our study is to generate patient specific iPSC-derived stromal cells of patients with pCD. Furthermore, we will engineer programmed death-ligand 1 (PD-L1)-expressing cultures to enhance the immunomodulatory capacity of these stromal cells. Last, preclinical testing of these cells will be performed in experimental colitis models. Methods Ten patients with pCD will be included at the outpatient clinic in the Erasmus Medical Center (EMC). Patient specific iPSC will be generated from peripheral blood in the iPSC core facility of the EMC. iPSC cultures will be exposed to a low dose of retinoic acid creating stromal cells. The variety of stromal cells will be sorted using a FACS and a stromal specific sorting panel. In parallel, cultures will be engineered, using CRISPR-Cas technology, to express PD-L1. Mixed lymphocyte reactions will be performed to assess the ability of both transduced and non-transduced cultures to provoke the proliferation of non-MHC matched T-lymphocytes. Next, cultures will be generated to express murine PD-L1 to assess their immunomodulatory capacity in established experimental colitis models to provide essential knowledge for a future clinical trial. Anticipated Impact With this study, we are taking the first steps towards improved remission rates of perianal fistulas in the future. On the long-term, this will result in reduction of health care costs.

Early allogeneic immune modulation after establishment of donor hematopoietic cell-induced mixed chimerism in a nonhuman primate kidney transplant model.

Mixed lymphohematopoietic chimerism is a proven strategy for achieving operational transplant tolerance, though the underlying immunologic mechanisms are incompletely understood. A post-transplant, non-myeloablative, tomotherapy-based total lymphoid (TLI) irradiation protocol combined with anti-thymocyte globulin and T cell co-stimulatory blockade (belatacept) induction was applied to a 3-5 MHC antigen mismatched rhesus macaque kidney and hematopoietic cell transplant model. Mechanistic investigations of early (60 days post-transplant) allogeneic immune modulation induced by mixed chimerism were conducted. Chimeric animals demonstrated expansion of circulating and graft-infiltrating CD4+CD25+Foxp3+ regulatory T cells (Tregs), as well as increased differentiation of allo-protective CD8+ T cell phenotypes compared to naïve and non-chimeric animals. In vitro mixed lymphocyte reaction (MLR) responses and donor-specific antibody production were suppressed in animals with mixed chimerism. PD-1 upregulation was observed among CD8+ T effector memory (CD28-CD95+) subsets in chimeric hosts only. PD-1 blockade in donor-specific functional assays augmented MLR and cytotoxic responses and was associated with increased intracellular granzyme B and extracellular IFN-γ production. These studies demonstrated that donor immune cell engraftment was associated with early immunomodulation via mechanisms of homeostatic expansion of Tregs and early PD-1 upregulation among CD8+ T effector memory cells. These responses may contribute to TLI-based mixed chimerism-induced allogenic tolerance.

Characterization of CD4+ and CD8+ T cells responses in the mixed lymphocyte reaction by flow cytometry and single cell RNA sequencing.

The Mixed Lymphocyte Reaction (MLR) consists in the allogeneic co-culture of monocytes derived dendritic cells (MoDCs) with T cells from another donor. This in vitro assay is largely used for the assessment of immunotherapy compounds. Nevertheless, the phenotypic changes associated with lymphocyte responsiveness under MLR have never been thoroughly evaluated. Here, we used multiplex cytokine and chemokine assays, multiparametric flow cytometry and single cell RNA sequencing to deeply characterize T cells activation and function in the context of CD4+- and CD8+-specific MLR kinetics. We showed that CD4+ and CD8+ T cells in MLR share common classical markers of response such as polyfunctionality, increased proliferation and CD25 expression but differ in their kinetics and amplitude of activation as well as their patterns of cytokines secretion and immune checkpoints expression. The analysis of immunoreactive Ki-67+CD25+ T cells identified PBK, LRR1 and MYO1G as new potential markers of MLR response. Using cell-cell communication network inference and pathway analysis on single cell RNA sequencing data, we also highlighted key components of the immunological synapse occurring between T cells and the stimulatory MoDCs together with downstream signaling pathways involved in CD4+ and CD8+ T cells activation. These results provide a deep understanding of the kinetics of the MLR assay for CD4+ or CD8+ T cells and may allow to better characterize compounds impacting MLR and eventually identify new strategies for immunotherapy in cancer.

Nanoparticles loaded with IL-2 and TGF-β promote transplantation tolerance to alloantigen.

We have previously reported that nanoparticles (NPs) loaded with IL-2 and TGF-β and targeted to T cells induced polyclonal T regulatory cells (Tregs) that protected mice from graft-versus-host disease (GvHD). Here, we evaluated whether administration of these NPs during alloantigen immunization could prevent allograft rejection by converting immunogenic responses to tolerogenic ones. Using C57BL/6 mice and BALB/c mice as either donors or recipients of allogeneic splenocytes, we found that treatment with the tolerogenic NPs in both strains of mice resulted in a marked inhibition of mixed lymphocyte reaction (MLR) to donor cell alloantigen but not to third-party control mouse cells after transfer of the allogeneic cells. The decreased alloreactivity associated with a four- to fivefold increase in the number of CD4+ and CD8+ T regulatory cells (Tregs) and the acquisition of a tolerogenic phenotype by recipient dendritic cells (DCs) in NP-treated mice. As allogeneic cells persisted in NP-treated mice, these findings suggest that tolerogenic NPs can induce alloantigen-specific Tregs and tolerogenic DCs promoting tolerogenic responses to alloantigen. By inhibiting reactivity to allotransplant, this approach could help reduce the need for immune suppression for the maintenance of allografts.

Prostate cancer therapy using immune checkpoint molecules to target recombinant dendritic cells.

We developed immune checkpoint molecules to target recombinant dendritic cells (DCs) and verified their anti-tumor efficacy and immune response against prostate cancer. DCs were generated from mononuclear cells in the tibia and femur bone marrow of mice. We knocked down the programmed death ligand 1 (PD-L1) on monocyte-derived DCs through siRNA PD-L1. Cell surface antigens were immune fluorescently stained through flow cytometry to analyze cultured cell phenotypes. Furthermore, we evaluated the efficacy of monocyte-derived DCs and recombinant DCs in a prostate cancer mouse model with subcutaneous TRAMP-C1 cells. Lastly, DC-induced mixed lymphocyte and lymphocyte-only proliferations were compared to determine cultured DCs' function. Compared to the control group, siRNA PD-L1 therapeutic DC-treated mice exhibited significantly inhibited tumor volume and increased tumor cell apoptosis. Remarkably, this treatment substantially augmented interferon-gamma and interleukin-2 production by stimulating T-cells in an allogeneic mixed lymphocyte reaction. Moreover, we demonstrated that PD-L1 gene silencing improved cell proliferation and cytokine production. We developed monocyte-derived DCs transfected with PD-L1 siRNA from mouse bone marrow. Our study highlights that PD-L1 inhibition in DCs increases antigen-specific immune responses, corroborating previous immunotherapy methodology findings regarding castration-resistant prostate cancer.

The Potential Role of Moringa oleifera Lam. Leaf Proteins in Moringa Allergy by Functionally Activating Murine Bone Marrow-Derived Dendritic Cells and Inducing Their Differentiation toward a Th2-Polarizing Phenotype.

Moringa oleifera leaves are an inexpensive substitute for staple foods. Despite limited data, Moringa oleifera leaf protein (Mo-Pr) may be allergenic in BALB/c mice. In mouse models and allergic patients, dendritic cells (DCs) may be involved in food allergy. In addition, some allergens, including food allergens, can directly activate DCs and induce Th2 polarization. We investigated whether Mo-Pr can modulate the functional profile of murine bone marrow-derived dendritic cells (BMDCs) in vitro. BMDCs were obtained from mouse bone marrow cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days and then treated with lipopolysaccharide (LPS) or Mo-Pr. BMDC phenotypes were evaluated via flow cytometry, cytokine production was assessed using ELISA, the expression of key genes was studied using qRT-PCR, the effects on T-cell differentiation were investigated using mixed lymphocyte reaction (MLR), and transcriptional changes in BMDCs were investigated using RNA-Seq. Mo-Pr-specific IgE was investigated in recipient serum after BMDC transfer. Mo-Pr treatment significantly induced BMDC maturation, increased the expression of CD80/86 and MHC II, resulted in the production of IL-12 and TNF-α, and induced T-cell differentiation. Mo-Pr treatment stimulated BMDCs' expression of the Th2 promoters OX40L and TIM-4, induced the production of the Th2-type chemokines CCL22 and CCL17, and decreased the Th1/Th2 ratio in vitro. Healthy recipients of Mo-Pr-treated BMDCs produced Mo-Pr-specific IgE.

Soluble CD83 modulates human-monocyte-derived macrophages toward alternative phenotype, function, and metabolism.

Alterations in macrophage (Mφ) polarization, function, and metabolic signature can foster development of chronic diseases, such as autoimmunity or fibrotic tissue remodeling. Thus, identification of novel therapeutic agents that modulate human Mφ biology is crucial for treatment of such conditions. Herein, we demonstrate that the soluble CD83 (sCD83) protein induces pro-resolving features in human monocyte-derived Mφ biology. We show that sCD83 strikingly increases the expression of inhibitory molecules including ILT-2 (immunoglobulin-like transcript 2), ILT-4, ILT-5, and CD163, whereas activation markers, such as MHC-II and MSR-1, were significantly downregulated. This goes along with a decreased capacity to stimulate alloreactive T cells in mixed lymphocyte reaction (MLR) assays. Bulk RNA sequencing and pathway analyses revealed that sCD83 downregulates pathways associated with pro-inflammatory, classically activated Mφ (CAM) differentiation including HIF-1A, IL-6, and cytokine storm, whereas pathways related to alternative Mφ activation and liver X receptor were significantly induced. By using the LXR pathway antagonist GSK2033, we show that transcription of specific genes (e.g., PPARG, ABCA1, ABCG1, CD36) induced by sCD83 is dependent on LXR activation. In summary, we herein reveal for the first time mechanistic insights into the modulation of human Mφ biology by sCD83, which is a further crucial preclinical study for the establishment of sCD83 as a new therapeutical agent to treat inflammatory conditions.

Abstract A114: Naturally occurring LAG3-blocking recombinant antibodies as a novel class of checkpoint inhibitors

Abstract LAG3 is a promising cancer immunotherapeutic target due to its negative regulatory role on T cells, NK cells, and B cells. Using our proprietary technology [US patent US9810694; 2017], we have isolated, sequenced, and pre-examined the potential immunological capability of fully human, naturally occurring anti-LAG3 antibodies. The best candidate, monoclonal antibody IG2 with molecular weight of 150 kDa and affinity of 4.4 nM, a complete recombinant reproduction of the antibody isolated from the human donor, was selected from 6 candidates for future research. In vitro allogeneic mixed lymphocyte reaction (MLR) assays using human T cells were performed to evaluate lymphocyte responses to stimulation with IG2 (5 μg/ml) or regular RPMI media alone. LAG3 blockade systematically resulted in an enhancement of CD3+ T cell proliferation by 45%. The main impact was on proliferation of CD4+ T cells with 50% increase. The count of CD8+ T cells also increased by 36%. Another assay evaluated IFNγ expression in NK92 cells treated with anti-LAG3 antibody. NK92 cells were starved without IL-2 for 24 hours in complete NK cell media and then cultivated with IG2 (5 μg/ml and 10 μg/ml) or media for 24 hours. Even short-term cultivation led to an increase in IFNg expression by 50%. Increasing the dose of the antibody did not affect the level of IFNγ expression. We did not observe any antibody dependent cellular cytotoxicity (ADCC) in co-cultures of NK92 cells with 786-O (renal), H358 (lung) or Hct116 (colon) cancer cell lines. Several studies demonstrate that NK cells can stimulate the expression of pro-imflammatory/pro-tumorigenic cytokines, IL-6 and IL-8, by tumor cells. Therefore, we evaluated the effects of IG2 on those genes’ expression induced by NK cells. Clear cell RCC cells (786-O) and NSCLC (H358) cell lines were co-cultured with NK92 cells for 2 hours and overnight with IG2 (1 mcg/ml) or media only. Co-cultivation of NK cells, cancer cells, and IG2 did not result in robust up-regulation of IL-6 and IL-8 by tumor cells. In order to evaluate the direct impact of the IG2 antibody on tumor cells, we incubated 786-O renal carcinoma cells with escalating concentrations of anti-LAG3 antibodies for 72 hours followed by Cell Titer Blue analysis when compared to un-treated cells (0 ug/ml). As expected, there was no direct cytotoxic effect of the antibody on tumor cells. Finally, assuming that novel naturally occurring antibodies may lead to fewer adverse events, we evaluated the preliminary toxicity in vivo. Eighteen mice were randomized in a 1:1 ratio to IG2 antibody group (1 mg/kg, intravenously, twice a week, 6 weeks) or to the untreated control group. The use of the antibody at this dose did not lead to the development of toxicity and a change in the weight of the animals compared to the control group. The results of the first studies demonstrate the potential activity and safety of a recombinant anti-LAG3 antibody reproduced from a human donor. Citation Format: Ilya Tsimafeyeu, Petr Makhov, Gennady Bratslavsky. Naturally occurring LAG3-blocking recombinant antibodies as a novel class of checkpoint inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A114.

Abstract A077: A novel, tumor-targeted immunocytokine comprising an anti-PD-L1 Affimer® fused to IL-15 exhibits potent anti-tumor activity

Abstract Affimer® biotherapeutics are novel 15kDa scaffold proteins based on the human protease inhibitor Stefin A. Affimer® proteins lack disulfide bonds and post translational modifications including glycosylation. Large, diverse phage display libraries have been created which incorporate peptide loops into the scaffold backbone. We have previously identified high-affinity competitive binders to human programmed death-ligand 1 (PD-L1) using phage display which block interaction with programmed death 1 (PD-1). Lead anti-PD-L1 Affimer® proteins were formatted by fusing to human Fc or as in-line-fusions to a serum albumin binding Affimer® to extend pharmacokinetic half-life. Here we describe novel immunocytokines that consist of half-life extended Affimer® antagonist to PD-L1 fused to human Interleukin 15 (IL-15) receptor alpha sushi domain and human IL-15 (PDL1-IL15 Affimer® fusion proteins). PD-L1 is upregulated on tumor cells, macrophages, and dendritic cells in the tumor microenvironment. PD-L1 engagement with PD-1 on T cells inhibits T cell proliferation, transcriptional activation, and cytokine release, resulting in suppressed anti-tumor response. IL-15 signals through the IL-2 receptor beta chain and the common gamma chain (IL-2Rβ/γ) and induces differentiation and proliferation of CD8+ T cells and Natural Killer (NK) cells; it does not stimulate regulatory T cells (Tregs). Immunostimulatory cytokines have therapeutic potential but are often associated with toxicity when administered systemically. PDL1-IL15 Affimer® fusion proteins have the potential to bring the immunomodulatory cytokine directly to the tumor and reverse the immunosuppressive tumor microenvironment by enhancing T cell activation, while also introducing a checkpoint inhibitor to augment the anti-tumor immune response. We characterized binding and activity of PDL1-IL15 Affimer® fusion proteins in in vitro assays. PDL1-IL15 Affimer® fusion proteins bind both PD-L1 and IL-2Rβ/γ and exhibit PD-1:PD-L1 blocking activity and IL-2Rβ/γ activation. PDL1-IL15 Affimer® fusion proteins reversed T cell exhaustion in a human mixed lymphocyte reaction assay: PDL1-IL15 Affimer® fusion proteins showed greater activity than either the PD-L1 or the IL-15 component alone. Furthermore, PDL1-IL15 Affimer® fusion proteins enhanced immune cell killing of tumor cells in co-culture assays of human peripheral blood mononuclear cells and tumor cells. In summary, data suggests that PDL1-IL15 Affimer® fusion proteins can reverse T cell exhaustion and induce immune cell activation to elicit an anti-tumor effect. Ongoing in vivo work aims to investigate the efficacy and tolerability profile of PDL1-IL15 Affimer® fusion proteins to direct future development of this therapeutic. In addition, the data will support wider utility of the Affimer® platform to develop new ways of targeting the tumor while reducing systemic toxicity. Citation Format: Victoria Juskaite, Deepa Avisetti, David H Jones, Ahmad Sobri, Gregory Billenness, Hanna Buist, Tik Nga Tong, Norhakim Yahya, Fiona McLaughlin. A novel, tumor-targeted immunocytokine comprising an anti-PD-L1 Affimer® fused to IL-15 exhibits potent anti-tumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A077.

Pegylated Long-Acting Interferon Gamma Treatment Enhances MHC Class II Expression and the Graft-Versus-Leukemia Effect in Preclinical Models of AML Relapse

Relapse after allogeneic hematopoietic stem cell transplantation (HCT)represents the most common type of treatment failure and is thought to result in part from AML cell escape from immune pressure exerted by donor immune cells (the “Graft-versus-leukemia” effect, GvL). We and others have previously reported downregulation of MHC class II (MHC-II) expression in up to half of AML cases relapsing after transplantation, but it is unclear whether a) loss of MHC-II contributes mechanistically to loss of GvL, and b) whether reversal of MHC-II downregulation can restore GvL. We tested the role of MHC-II in mediating GvL in a mouse model of minor-antigen mismatch HCT where C57BL/6 mice (B6) bearing syngeneic AML cells (H-2b) were co-transplanted with or without CD3+ T cells from C3.SW-H2b/SnJ mice (H-2b). Since murine AML cells do not generally express MHC-II at baseline, we transduced MLL-AF9-driven B6 AML cells with a fluorescently-tagged retrovirus expressing CIITA the main transcriptional regulator of MHC-II genes. MHC-II-expressing AML cells were selectively deleted when co-transplanted with donor T cells, in contrast to animals that received no T cells (Figure 1, right) or mice that received AML cells transduced with empty vector (Figure 1, left). To extend these findings to human cells, we used both MHC-II blocking antibodies and CRISPR-mediated disruption of CIITA in human AML cell lines and primary samples. Loss or blocking of MHC-II led to diminished the ability of AML cells to stimulate MHC-mismatched donor T cells in a mixed lymphocyte reaction. Loss of MHC-II expression in AML cells can be reversed in vitro by Interferon gamma (IFNG). We tested the ability of IFNG to enhance the GvL by culturing AML cell lines and primary patient samples with or without IFNG for 48 hours. IFNG-treatment enhanced the ability of AML cells from some-but not all-samples to stimulate third-party T cells. Interestingly, enhanced stimulation was observed in both CD4 and CD8 cells, suggesting that IFNG may also potentiate GvL through MHC-II-independent pathways. Since the use of IFNG in vivo may be limited by its short half-life, we tested a novel long-acting pegylated human interferon gamma (peg-IFNG, Bolder BioTechnology) in a xenograft model of AML. Immunodeficient mice were engrafted with MHC-II-low human AML cells. After engraftment, mice (n=3 each group) received six doses of IFNG, peg-IFNG, or vehicle (15mcg given s.c. M-W-F). After treatment, mice were sacrificed and MHC-II expression was quantified on human AML cells isolated from bone marrow of treated mice. IFNG treatment increased MHC-II expression modestly compared to vehicle (2.7-fold, p=0.04), while peg-IFNG much more robustly stimulated MHC-II stimulation on human AML cells in this xenograft model (12-fold, p=0.02). Finally, we tested whether peg-IFNG can enhance AML cell clearance in a xenograft model. Luciferin-labelled THP1 cells (low MHC-II expression) were injected on day 0 into immundeficient mice (n=8-9 each group). On day 7, mice started treatment with peg-IFNG or vehicle (15mcg given s.c. M-W-F for two weeks). On day 21, baseline tumor burden was measured using bioluminescence, and healthy third-party donor T cells were injected. Mice were followed with weekly CBC and bioluminescence measurments, and moribund mice were sacrificed and banked. Mice treated with peg-IFNG plus T cells had decreased tumor burden during the course of the experiment compared to untreated controls as well as mice treated with T cells plus vehicle only (Figure 2, p<0.01 by ANOVA for both comparisons). In addition, mice treated with peg-IFNG had significantly improved survival compared to control groups (median survival 79 days compared to 63 days and 67 days, p<0.05 by Log-Rank test). Together, these findings suggest that long-acting IFNG analogues may effectively enhance GvL in AML patients after HCT.

Extracellular Release of Damaged Mitochondria Induced By Cytotoxic Conditioning Exacerbates Graft-Versus-Host Disease

Despite therapeutic advancements, graft versus host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HSCT). Pathogen and damage associated molecular patterns released following pre-transplant conditioning have an important role in the initiation of GVHD. However, the role of extracellular mitochondria (exMito) as an endogenous danger signal has not been explored. Herein, we report that cytotoxic conditioning with radiation leads to mitochondrial fission and impaired PINK-1/PARKIN-dependent mitophagy in the intestinal mucosal layer. This was coupled with extracellular release of damaged mitochondria in the plasma of BALB/c mice following irradiation. Sublethal irradiation of the Caco-2 epithelial cell line confirmed the in vivo expulsion of mitochondria in a cell-death independent manner. exMito released upon irradiation were damaged, and had impaired ATP production. Incubation of splenocytes with damaged exMito increased the cell surface expression of immune stimulatory molecules and induced Th1 cytokine release. Furthermore, in mixed lymphocyte reactions, exMito pre-treated BALB/c stimulator cells increased the activation and proliferation of C57BL6/J responder T cells. Finally, co-transplantation of isolated damaged mitochondria in a C57BL6/J to BALB/c mouse HSCT model aggravated GVHD. The translational relevance of these findings was studied in a cohort of pediatric patients who underwent HSCT. Fractionated irradiation given with their conditioning regimen increased the abundance of circulating exMito and correlated with the incidence of GVHD. Our results suggest that modulation of exMito release could be a novel therapeutic target to prevent GVHD after HSCT.

Durable Multiplex Epigenetic Editing for Generation of Allogeneic CAR T without Chromosomal Rearrangements

CAR T therapies have provided significant breakthroughs for patients with CD19+ malignancies and multiple myeloma, but a major limitation is the need for individualized manufacturing of autologous T cells, increasing the cost and reducing the availability of these life-saving drugs. An allogeneic CAR T product, available off-the-shelf, could greatly reduce manufacturing burden and improve access to therapy. To date, allogeneic CAR T clinical trials have shown limited durable responses and have reduced only TCR expression to avoid a graft versus host response. Further editing to reduce MHC class I and II expression may produce a cell resistant to allogeneic CD4+ and CD8+ T cell rejection to potentially improve allogeneic CAR T persistence. Conventional genome editing technologies that rely on double or single stranded DNA breaks have the potential to introduce genomic changes including translocations, especially when multiple edits are introduced. Epigenetic editing, in contrast, enables durable modulation of gene expression without cutting or nicking the DNA sequence and is extremely well-suited for simultaneous targeting of multiple genes to enable generation of persistent allogeneic CAR T cells. In this study, we evaluated the use of a dCas9-based epigenetic editor to silence TCR and MHC class I and II expression in primary T cells to produce allogeneic CAR T transduced for expression of CAR and HLA-E. Epigenetic editor mRNA and sgRNAs targeting the genes of interest were delivered via nucleofection to activated T cells. Edited T cells were then maintained in proliferative culture, with CD3/CD28 or PMA/ionomycin restimulation, and expression of the target genes was assessed by flow cytometry. Expression of TCR and MHC class I and II was durably suppressed in both single and multiple gene target silencing, demonstrating the robustness of epigenetic editing for ex vivo cell therapies. In addition, genomic integrity assessments demonstrated that epigenetic editing did not induce genomic changes as detected by either a quantitative sequencing-based assay or a fluorescence-based technique with single-cell resolution. In primary T cells, multiplex silencing did not cause genomic rearrangements, including translocations and truncations, above the level observed in controls, whereas Cas9 multiplex editing did. Mixed lymphocyte reaction (MLR), specific killing, and degranulation assays were used to demonstrate allogeneic function of silencing edits and the overexpression of an NK shield in vitro. In these assays, epigenetically-edited cells performed similarly to control gene edited cells in which the same gene targets were knocked out. TCR-silenced T cells did not proliferate or produce IFN-γ in response to allogeneic PBMCs in an MLR assay, and MHC class I- and II-silenced T cells did not evoke a proliferative response or IFN-γ production from allogeneic CD4+ or CD8+ T cells. Furthermore, overexpression of the HLA-E NK shield protected HLA-E expressing cells relative to B2M KO cells in a specific killing assay and reduced the degranulation response of NKG2A+ NK cells without a corresponding increase in NKG2C+ NK cell degranulation observed with other single chain HLA-E constructs tested. Finally, multiplex epigenetically-edited allogeneic CAR T were produced by transduction for expression of an anti-BCMA CAR, followed by simultaneous editing to silence target genes. These cells displayed potent anti-tumor activity against the BCMA+ MM.1S cell line, comparable to activity of unedited CAR T cells. These data support multiplex epigenetic editing to produce functional allogeneic CAR T cells that (1) do not exhibit a GVHD response; (2) resist CD8+ and CD4+ T cell alloresponses; and (3) resist the NK missing-self response normally consequent of reduced MHC class I expression without introduction of genotoxic events.

Coexpression of Tim-3 and PD-1 of T Lymphocytes Prevents Acute Graft-Versus-Host Disease

T lymphocytes are critical players in acute graft-versus-host disease (aGVHD). TIM3expression on the T cell surface was identified to induce immune tolerance in a mouse GVHD model; in addition, PD1 +TIM3 + double-positive T cells displayed more potent immunosuppression compared to PD1 + T cells. However, their clinical potential is greatly limited by their low cell number in peripheral blood (PB). We already introduced a novel method to obtain a high number of PD1 +TIM3 + lymphocytes that culturing CD3 + cells isolated from G-CSF mobilized peripheral blood stem cells (CD3 +G-PBSC) with human serum albumin (HSA). We also confirmed that cultured PD1 +TIM3 + lymphocytes are the most potent subset of the CD3 + cells that inhibit T cell proliferation. In this study, we investigate the immunosuppressive effects of PD1 +TIM3 + lymphocytes after a 2-day culture with HSA (D2-CD3 +G-PBSC) or after a 4-day culture with HSA (D4-CD3 +G-PBSC). Also, we evaluate the treatment efficacy of PD1 +TIM3 + lymphocytes in the xenograft mouse aGVHD model. Methods. The healthy donors were subcutaneously injected with G-CSF (10μg/kg) for five days. G-PBSCs were collected from the donors using a COBE spectra cell separator, and CD3 + cells were isolated by positive selection by magnetic-activated cell sorting (MACS) using CD3 Dynabeads™. CD3 + G-PBSC were cultured with 5% HSA for 2 days or 4 days (1x10 5 cells/mL). PD1 and TIM3 expressions were analyzed using flow cytometry (FACS). Sorted T lymphocytes were cultured with irradiated allo-MNCs for 3 days (Mixed Lymphocyte Reaction; MLR). 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was used as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure T cell proliferation. aGVHD was induced by intraperitoneal injection of human PB MNCs to the NOD-SCID IL-2Rγ null mouse after exposure of 3Gy irradiation. The treatment efficacy of PD1 +TIM3 + lymphocytes was analyzed by determining the survival rate after tail vein injection of PD1 +TIM3 + lymphocytes (1x10 7/kg). Results. Normally, very few cells (0.2±0.2%) were PD1 +TIM3 + lymphocytesin PB. The numbers of PD1 +TIM3 + lymphocytes markedly increased by 74-fold in D2-CD3 +G-PBSC and 370-fold in D4-CD3 +G-PBSC, respectively. CD3 + G-PBSC were composed of three types of cells such as PD1 -TIM3 -, PD1 +TIM3 -, and PD1 +TIM3 + lymphocytes. To check the origin of PD1 +TIM3 + lymphocytes after HSA treatment, PD1 -Tim3 - and PD1 +Tim3 - lymphocytes from CD3 +G-PBSC were sorted and cultured with HSA. It was confirmed that PD1 +TIM3 + lymphocytes originated from PD1 -TIM3 - lymphocytes. To assess the immunosuppressive effects of cultured T lymphocytes, we used CSFE-MLR. D2-CD3 +G-PBSC significantly increased the levels of unstimulated T cells compared to D4-CD3 +G-PBSC (% of unstimulated T cells in D2-CD3 +G-PBSC vs. in D4-CD3 +G-PBSC; 38.9±3.0% vs. 13.9±6.6%). Also, both PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC demonstrated significantly enhanced levels of immunosuppression (% of unstimulated T cells PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC; 31.6±5.5 and 36.6±13.3, respectively). Lastly, we tested the effects of anti-PD1 or anti-TIM3 blocking mAb on the PD1 +TIM3 + lymphocytes. The immunosuppressive effects of PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC were significantly decreased by anti-PD1 mAb (% of unstimulated T cells in PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC; 21.1±4.5 from 31.6±5.5 and 18.4±7.8 from 36.6±13.3, respectively). However, the immunosuppression of PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC was not decrease by anti-TIM3 mAb (% of unstimulated T cells in PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC; 28.7±3.3 from 31.6±5.5 and 22.7±5.6 from 36.6±13.3, respectively). These data indicated that PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC have the same phenotype as PD1 +TIM3 + lymphocytes from D4-CD3 +G-PBSC, however, their functions are different. Next, we used the xenograft mouse aGVHD model to evaluate the treatment efficacy of PD1 +TIM3 + lymphocytes for preventing aGVHD. In the control group, 14% of the mice survived at 100 days. Mice that were treated with PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC demonstrated 100% and 71% survival rate at 100 days, respectively. Conclusion. Ex vivo HSA treatment of CD3 + G-PBSCs for 2 days shows promising therapeutic potential for the treating patients with aGVHD.

API-192, an Allogeneic CD19/20 CAR-NKT Cell Product Derived from Cord Blood CD34+ HSPCs for the Treatment of B-Cell Malignancies

Autologous chimeric antigen receptor (CAR)-T cell therapies induce durable remissions and improve survival in patients with advanced B cell malignancies. However, challenges such as manufacturing variability, cost, and time to treatment highlight the need for an allogeneic, off-the-shelf approach that preserves the efficacy of autologous CAR-T. Additionally, antigen escape is one of the established mechanisms of resistance to current CAR-T therapies which target a single antigen. Classical natural killer T (NKT) cells express an invariant T cell receptor (iTCR) that is restricted to the conserved CD1d protein, making it a promising cell type for allogeneic therapies as they do not require TCR gene editing to avoid graft-vs-host (GvH) disease. Furthermore, NKT cells mediate dendritic cell cross-priming, target immunosuppressive myeloid cells/macrophages in the tumor microenvironment, and directly kill CD1d + tumor cells through the iTCR. Endogenous NKT cells are an extremely rare population in the blood (<1% of lymphocytes), posing manufacturing challenges. API-192 is a human cord blood CD34 + hematopoietic stem and progenitor cell-derived NKT cell product engineered to express dual CARs targeting CD19 and CD20 and armored with soluble IL-15 for improved expansion and persistence. The Appia Cells Utilized for Allogeneic (ACUA) platform enables the production of large numbers of product NKT cells co-expressing multiple transgenes at high purity (>90% iTCR + 19CAR + 20CAR +) with no purification step or gene editing. Phenotypically, API-192 cells are predominantly CD4 -CD8 - or CD4 -CD8 +, express major activating NK receptors, and harbor intracellular stores of cytotoxic molecules, resembling endogenous NKT cells. API-192 kills Raji and Nalm6 tumor cells in a CAR-specific and dose-dependent manner and can control multiple rounds of tumor challenge in vitro. API-192 secretes pro-inflammatory cytokines and proliferates in response to tumor antigen . In human tumor xenograft models in immunocompromised mice, API-192 robustly expands, suppresses tumor growth, and exhibits prolonged persistence, enabling control of a tumor rechallenge 70 days after initial dosing. API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less IFN-γ release by NK and T cells in mixed lymphocyte reaction (MLR) assays, indicative of a more favorable immunogenic profile. Compared to conventional CAR-T cells, API-192 also secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with the expression of a non-HLA restricted TCR and low GvH risk. These results demonstrate the feasibility, potency, and potential safety of API-192, warranting further development as a novel allogeneic therapy.

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.

The Effects of IL-23/IL-18-Polarized Neutrophils on Renal Ischemia-Reperfusion Injury and Allogeneic-Skin-Graft Rejection in Mice.

Neutrophils display heterogeneity and plasticity with different subgroups and immune-regulatory functions under various surrounding conditions. Neutrophils induced by IL-23/IL-18 (referred to N(IL-23+IL-18) neutrophils) have a unique gene-expression profile, with highly expressing IL-17, MHC-II, and costimulatory molecules. The adoptive transfer of N(IL-23+IL-18) neutrophils significantly increased the pathogenesis in a renal ischemia-reperfusion injury mouse model. N(IL-23+IL-18) neutrophils directly and efficiently induced allogeneic T cell proliferation in vitro. N(IL-23+IL-18) neutrophils enhanced the syngeneic T cell response to allogeneic antigens in mixed-lymphocyte reaction assays. The adoptive transfer of the donor or host N(IL-23+IL-18) neutrophils significantly enhanced the antidonor antibody production in an allogeneic-skin-transplanted mouse model, accompanied by increased Tfh cells in the spleens. Therefore, the neutrophil subset induced by IL-23/IL-18 promotes tissue injury and antidonor humoral response in the allogeneic transplantation mouse model.

Circular RNA MAP2K2-modified immunosuppressive dendritic cells for preventing alloimmune rejection in organ transplantation.

Long-term patient and graft survival has been achieved in organ transplantation but at the expense of toxic side effects that are associated with long-term use of nonspecific immunosuppressive drugs. Discovering new regulators of dendritic cells is the key for development of an ideal treatment to prevent immune rejection. We hypothesized that knockdown of circMAP2K2 induces immunosuppressive DCs and that treatment with circMAP2K2 silenced-DCs can prevent alloimmune rejection. DCs were cultured and transfected with siRNA for circMAP2K2. circMAP2K2 levels were measured by qRT-PCR. DC's maturation and immune function were assessed by flow cytometry and mixed lymphocyte reactions. The function of circMAP2K2 was illustrated by a series of RIP and IP. The therapeutics of engineered DCs was tested in a mouse heart transplantation model. We found that circMAP2K2 was highly expressed in mature DCs. Knockdown of circMAP2K2 reduced expression of MHCII, CD40 and CD80, attenuated the ability of DCs to activate allogeneic naïve T cells, and enhanced CD4+CD25+FOXP3+ regulatory T cells (Treg). circMAP2K2-induced immunosuppressive DCs by interacting with SENP3. Treatment with circMAP2K2-knockdown DCs attenuated alloimmune rejection and prolonged allograft survival in a murine heart transplantation model. The immune suppression induced in vivo was donor-antigen specific. In conclusion, knockdown of circMAP2K2 can induce immunosuppressive DCs which are able to inhibit overactive immune response, highlighting a new promising therapeutic approach for immune disorder diseases.

Development of dendritic cell loaded MAGE-A2 long peptide; a potential target for tumor-specific T cell-mediated prostate cancer immunotherapy

BackgroundProstate cancer (PCa) is the second leading cause of cancer-related deaths among men worldwide. Immunotherapy is an emerging treatment modality for cancers that harnesses the immune system’s ability to eliminate tumor cells. In particular, dendritic cell (DC) vaccines, have demonstrated promise in eliciting a tumor-specific immune response. In this study, we investigated the potential of using DCs loaded with the MAGE-A2 long peptide to activate T cell cytotoxicity toward PCa cell lines.MethodsHere, we generated DCs from monocytes and thoroughly characterized their phenotypic and functional properties. Then, DCs were pulsed with MAGE-A2 long peptide (LP) as an antigen source, and monitored for their transition from immature to mature DCs by assessing the expression levels of several costimulatory and maturation molecules like CD14, HLA-DR, CD40, CD11c, CD80, CD83, CD86, and CCR7. Furthermore, the ability of MAGE-A2 -LP pulsed DCs to stimulate T cell proliferation in a mixed lymphocyte reaction (MLR) setting and induction of cytotoxic T cells (CTLs) in coculture with autologous T cells were examined. Finally, CTLs were evaluated for their capacity to produce interferon-gamma (IFN-γ) and kill PCa cell lines (PC3 and LNCaP).ResultsThe results demonstrated that the antigen-pulsed DCs exhibited a strong ability to stimulate the expansion of T cells. Moreover, the induced CTLs displayed substantial cytotoxicity against the target cells and exhibited increased IFN-γ production during activation compared to the controls.ConclusionsOverall, this innovative approach proved efficacious in targeting PCa cell lines, showcasing its potential as a foundation for the development and improved PCa cancer immunotherapy.

Cyclosporine and Voclosporin Resistant Immune Effector Cells to Improve Outcomes after Stem Cell Transplantation

Introduction: Relapse of a patient's underlying malignancy, and complications related to viral reactivation are prominent causes of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Immune effector cells (IEC) are increasingly deployed to prevent and treat these complications. These include anti-CD19 CAR-T-cells used for post-HCT B-ALL relapse, as well as viral specific T cells (VSTs) targeting EBV, CMV, and others. However, the efficacy of these IECs early after HCT may be compromised by the inhibitory effects of immunosuppressive agents used to prevent graft-versus host-disease (GVHD). A commonly employed immunosuppressant used for GVHD prophylaxis is the calcineurin inhibitor (CNI) cyclosporine (CsA). A structural analog, voclosporin (VCS), is now FDA approved for lupus nephritis and in trials to prevent rejection after solid organ transplantation. Given its favorable pharmacodynamic and toxicity profile versus CsA and tacrolimus, VCS is poised to become an important option for HCT patients. Both CsA and VCS interact with calcineurin only in the presence of their immunophilin binding partner - cyclophilin A ( PPIA). Tacrolimus and sirolimus interact with a distinct immunophilin, FKBP12. This creates the opportunity to delete a single immunophilin (eg. PPIA) to create IECs resistant to one class of CNI (CsA/VCS) but sensitive to other immunosuppressive agents, an important safety feature. Here we developed CsA/VCS-resistant IECs by CRISPR/Cas9 knockout (KO) of PPIA. These cells exhibited superior proliferation and preserved effector function in the presence of CsA and VCS. Methods: We developed IECs that are simultaneously resistant to both CsA and VCS via CRISPR-mediated KO of PPIA. Proliferation, cytotoxicity, and cytokine production of PPIA KO conventional T cells (Tcon), CMV (pp65)-specific VSTs, and CD19 CAR-T cells were tested against mock-edited (MOCK) cells in functional in vitro assays with escalating doses of CsA and VCS. Results: PPIA KO Tcon demonstrated high editing efficiency (88-92%), high viability, and robust expansion in culture. Indeed, PPIA KO efficiency was maintained over several weeks in culture, suggestive of no survival disadvantage of cells bearing this deletion. Importantly, when PPIA KO cells were used as Responders in a mixed lymphocyte reaction in the presence of CsA, the edited cells demonstrated significant resistance to drug-mediated suppression of CD4+ and CD8+ T cell proliferation across the dose-response curve (p < 0.01; Fig. 1). Similar results were observed with VCS, with PPIA KO cells significantly resistant to VCS-mediated suppression across otherwise highly suppressive doses (p<0.0001; Fig. 1). Having demonstrated that edited Tcon were resistant to CsA and VCS, we next sought to make PPIA KO IECs. CRISPR-edited VSTs and CD19 CAR-T cells showed similar editing efficiencies to Tcon. Consistent with prior results, CMV-specific PPIA KO responder VSTs were significantly resistant to suppression of proliferation by CsA vs MOCK cells at all doses tested (p=0.0004, not shown). Proliferation in the presence of drug was then assessed in KO CAR-T cells using the CD19+ NALM6 line as stimulators. Division of PPIA KO responder CD19 CAR-T cells was almost completely unaffected by CsA and VCS across escalating doses ( Fig. 2). For example, at an effector-to-target (E:T) ratio of 1:0.1 and a CsA dose of 50 ng/mL, there was 40.6 ± 2.3 % suppression of division in MOCK CD8+ CAR-T cells vs. -0.2 ± 0.4 % in PPIA KO CD8+ CAR-T cells (p<0.0001). Similar findings were observed with CD4+ responder CAR-T cells (p<0.0001, not shown). PPIA KO CAR-T cells also demonstrated intact cytotoxic function with no significant differences in % killing between the MOCK and PPIA KO groups at 3 different E:T ratios. For example, at a 1:1 E:T ratio, there was 59 ± 15% killing vs. 55 ± 12% killing in MOCK vs PPIA KO groups, respectively (not shown). Conclusion: KO of PPIA is an effective strategy for engineering resistance to CsA and VCS incellular therapies. Our results demonstrate that PPIA KO conventional T cells, VSTs, and CAR-T cells were markedly resistant to the otherwise suppressive effects on T cell proliferation that are hallmarks of both CsA and VCS. Engineering CNI-resistance into IECs will enhance the ability to deploy these cells early after transplant to prevent or treat both malignant relapse and viral reactivation.

Characteristics of Alloreactive T-Cell Clones That Predict Early Development of Acute GvHD Following Allogeneic HCT

Acute Graft-versus-Host Disease (GvHD) is mediated by T-cells recognizing alloantigens. Despite advances in prophylactic regimens that broadly deplete T-cells, incidence of acute GvHD remains high, suggesting that there is a need to further characterize alloreactive T-cells specifically. Using mixed-lymphocytes reactions (MLR) and high-throughput T cell receptor (TCR) sequencing of third complementarity-determining region (CDR3) of the TCRβ chain, we identified and characterized alloreactive T-cell clones in donor-recipient pairs of allogeneic hematopoietic cell transplantation. We sequenced and identified a total of 34,903 unique donor-derived CDR3β functional sequences (FS) from 20 donor-recipient in vitro MLRs using peripheral blood mononuclear cells (PBMC) collected from donors and patients pre-transplant. We also sequenced the respective unstimulated donor PBMC samples (Donor Type: 13 HLA-Matched/7 Mismatched, GvHD prophylaxis: 11 Tacrolimus (Tac)-Methotrexate/8 Tac-MMF/1 Sirolimus, PTCy: 12 Yes/8 No). On average, our identified alloreactive FS make up a small fraction of the sampled donor repertoire in comparison to nonalloreactive FS. (Mean fraction: 0.004 vs. 0.996, p < 0.0001) Surprisingly, we found that alloreactive FS have significantly shorter CDR3β length compared to nonalloreactive FS (mean nt 43.50 vs. 44.40, p < 10E-15). We also found that alloreactive FS which can be located within the unstimulated donor sample (n= 4522) are shorter in comparison to all derived alloreactive FS (mean nt 42.43 vs. 43.50, p < 10E-15) and to nonalloreactive FS (mean nt 42.43 vs. 44.40, p < 10E-15) (Figure 1A). Publicly shared FS have, on average, shorter CDR3β length and are overrepresented among FS associated with autoreactivity and cross-reactivity (Khosravi-Maharlooei et al., J Clin Invest, 2019). Thus, we hypothesized that alloreactive FS may be also publicly shared. We derived a list of shared FS from our donor samples, where if a FS is found in more than one donor, it is considered shared. Alloreactive clones were significantly more likely to be shared compared to nonalloreactive (OR 1.94; CI 95%: 1.88 to 2.01). In addition, using a publicly available set of cross-reactive FS (https://vdjdb.cdr3.net/search), we found that alloreactive sequences are also highly cross-reactive compared to nonalloreactive sequences (OR 6.6; CI 95%: 5.18 to 8.42). FS with greater levels of sharedness appear more frequently within a repertoire (Madi et al., Genome Res, 2014). As alloreactive sequences are highly shared, we hypothesized that donor repertoires with greater proportion of shared and alloreactive FS will increase the odds of developing clinically significant GvHD (Grade 2-4) in the recipient. Indeed, within donor repertoires, we found a higher fraction of alloreactive sequences amongst the highly expressed FS (The top 50% of FS in repertoire frequency) which correlated with development of GvHD in recipients post-transplant (mean fraction: GvHD+ group 0.013 vs GvHD- group 0.004, p < 10E-7). Due to the higher portion of shared and alloreactive FS in the top half of the donor T-cell repertoire, we used R50, an index that estimates the diversity of the top 50% of a donor repertoire, to predict the development of GvHD. Utilizing an initial validation cohort of 12 donors, we found that donors whose corresponding recipients developed GvHD within the first 3 months post-transplant had significantly lower R50 compared to those that did not develop GvHD (R50 of 0.096 vs 0.345 p < 0.0001). We also conducted a time-to-event analysis regarding GvHD development in the first 90 days post-transplant. Using the average R50 of the validation cohort as the cutoff for low and high R50 (Average R50: 0.20), we found, in an expanded bi-institutional cohort (n=57), that those with lower R50 values had a significantly greater risk of GvHD compared to those with higher R50 (Figure 1B). In conclusion, our data suggests that functional alloreactive T lymphocytes are characterized by shorter TCRβ CDR3 length. These FS are highly shared and cross-reactive, suggesting a common mechanism underlying the selection of alloreactive and autoreactive clones. From there, we were able to develop and validate a diversity metric that predicts GvHD development pre-transplant based on the donor T-cell repertoire. These findings have implications for donor selection and need to be examined prospectively.