Alloreactive T-cell Research Articles

Suppressive Role of Pigment Epithelium-derived Factor in a Rat Model of Corneal Allograft Rejection.

Immunological rejection is the most common reason for corneal transplantation failure. The importance of T cells in corneal allograft rejection is well demonstrated. Recent studies highlight that pigment epithelium-derived factor (PEDF) plays an immunoregulatory role in ocular diseases by enhancing the suppressive phenotype of regulatory T cells besides its other functions in neurotrophy and antiangiogenesis. The effects of PEDF on immune rejection were examined in rat models of corneal transplantation using slit-lamp microscope observation, immunohistochemistry, flow cytometry, and Western blot. In vitro, we demonstrated PEDF reduced alloreactive T-cell activation using real-time polymerase chain reaction, flow cytometry, and Western blot. Topical administration of PEDF provided corneal transplantation rats with an improved graft survival rate of corneal allografts, reduced hemangiogenesis, and infiltration of immune cells in corneas, in particular, type 17 T helper cells while increased regulatory T cells. Moreover, nerve reinnervation within grafts was promoted in PEDF-treated recipient rats. In vitro, PEDF inhibited alloreactive T-cell activation via the c-Jun N-terminal kinase/c-Jun signaling pathway and upregulated the expressions of interleukin-10 and transforming growth factor-β, emphasizing the suppressive role of PEDF on immune responses. Our results underscore the feasibility of PEDF in alleviating corneal allograft rejection and further illustrate its potential in managing immune-related diseases.

IFN-γ induces acute graft-versus-host disease by promoting HMGB1-mediated nuclear-to-cytoplasm translocation and autophagic degradation of p53.

Acute graft-versus-host disease (aGVHD) poses a significant impediment to achieving a more favourable therapeutic outcome in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our prior investigations disclosed a correlation between p53 down-regulation in CD4+ T cells and the occurrence of aGVHD. Notably, the insufficiency of the CCCTC-binding factor (CTCF) emerged as a pivotal factor in repressing p53 expression. However, the existence of additional mechanisms contributing to the reduction in p53 expression remains unclear. Interferon (IFN)-γ, a pivotal proinflammatory cytokine, assumes a crucial role in regulating alloreactive T-cell responses and plays a complex part in aGVHD development. IFN-γ has the capacity to induce autophagy, a vital catabolic process facilitating protein degradation, in various cell types. Presently, whether IFN-γ participates in the development of aGVHD by instigating the autophagic degradation of p53 in CD4+ T cells remains an unresolved question. In the present study, we demonstrated that heightened levels of IFN-γ in the plasma during aGVHD promoted the activation, proliferation, and autophagic activity of CD4+ T cells. Furthermore, IFN-γ induced the nuclear-to-cytoplasm translocation and autophagy-dependent degradation of p53 in CD4+ T cells. The translocation and autophagic degradation of p53 were contingent upon HMGB1, which underwent up-regulation and translocation from the nucleus to the cytoplasm following IFN-γ stimulation. In conclusion, our data unveil a novel mechanism underlying p53 deficiency in CD4+ T cells among aGVHD patients. This deficiency is induced by IFN-γ and relies on autophagy, establishing a link between IFN-γ, HMGB1-mediated translocation, and the autophagic degradation of p53.

Chemokine receptors are required for effector T-cell trafficking to GVHD tissues but not to bone marrow

AbstractIn allogeneic hematopoietic stem cell transplantation (allo-SCT), alloreactive donor T cells mediate the graft-versus-leukemia effect but also attack nonhematopoietic tissues, causing graft-versus-host disease (GVHD). Reducing alloreactive T-cell trafficking to GVHD target tissues while allowing their access to bone marrow (BM) and spleen, major sites of malignant hematopoiesis, is a rational strategy for reducing the GVHD risk when using alloreactive T cells as a therapeutic. Here, we show that effector T-cell (Teff) entry into BM and spleen in unmanipulated mice and in mice that received transplantation without alloreactive T cells is augmented by pertussis toxin (PTX)-sensitive chemokine receptor signaling. However, unexpectedly, in the presence of a GVH response, chemokines no longer draw T cells into BM and spleen but remain critical for their recruitment to GVHD target tissues. Consistent with this, PTX-treated Teff cells were as efficacious as untreated T cells in killing leukemia cells in BM and spleen in mice with a concurrent GVHD response. These results suggest a strategy to improve the safety of alloreactive T-cell therapeutics in treating leukemias in the context of an allo-SCT.

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.

Rejection resistant CD30.CAR-modified Epstein-Barr virus-specific T cells as an off-the-shelf platform for CD30+ lymphoma

Off-the-shelf (OTS) adoptive T-cell therapies have many benefits such as immediate availability, improved access and reduced cost, but face the major challenges of graft-vs-host disease (GVHD) and graft rejection, mediated by alloreactive T-cells present in the graft and host, respectively. We have developed a platform for OTS T-cell therapies by using Epstein-Bar virus (EBV)-specific T-cells (EBVSTs) expressing a chimeric antigen receptor (CAR) targeting CD30. Allogeneic EBVSTs have not caused GVHD in several clinical trials, while the CD30.CAR, that is effective for the treatment of lymphoma, can also target alloreactive T-cells that upregulate CD30 on activation. Although EBVSTs express high levels of CD30, they were protected from fratricide in cis, by the CD30.CAR. Hence, they could proliferate extensively and maintained function both through their native EBV-specific T-cell receptor (TCR) and the CD30.CAR. The CD30.CAR enabled EBVSTs to persist in co-cultures with naïve and primed alloreactive T-cells and eliminate activated NK cells that can also be alloreactive. In conclusion, we show that CD30.CAR EBVSTs have the potential to be an effective OTS therapy against CD30+ tumors and if successful could then be used as a platform to target other tumor antigens.

HLA reduction of human T cells facilitates generation of immunologically multicompatible cellular products

AbstractAdoptive cellular therapies have shown enormous potential but are complicated by personalization. Because of HLA mismatch, rejection of transferred T cells frequently occurs, compromising the T-cell graft's functionality. This obstacle has led to the development of HLA knock-out (KO) T cells as universal donor cells. Whether such editing directly affects T-cell functionality remains poorly understood. In addition, HLA KO T cells are susceptible to missing self-recognition through natural killer (NK) cells and lack of canonical HLA class I expression may represent a safety hazard. Engineering of noncanonical HLA molecules could counteract NK-cell recognition, but further complicates the generation of cell products. Here, we show that HLA KO does not alter T-cell functionality in vitro and in vivo. Although HLA KO abrogates allogeneic T-cell responses, it elicits NK-cell recognition. To circumvent this problem, we demonstrate that selective editing of individual HLA class I molecules in primary human T cells is possible. Such HLA reduction not only inhibits T-cell alloreactivity and NK-cell recognition simultaneously, but also preserves the T-cell graft's canonical HLA class I expression. In the presence of allogeneic T cells and NK cells, T cells with remaining expression of a single, matched HLA class I allele show improved functionality in vivo in comparison with conventional allogeneic T cells. Since reduction to only a few, most frequent HLA haplotypes would already be compatible with large shares of patient populations, this approach significantly extends the toolbox to generate broadly applicable cellular products.

Human MAIT cells inhibit alloreactive T cell responses and protect against acute graft-versus-host disease.

Adoptive transfer of immunoregulatory cells can prevent or ameliorate graft-versus-host disease (GVHD), which remains the main cause of nonrelapse mortality after allogeneic hematopoietic stem cell transplantation. Mucosal-associated invariant T (MAIT) cells were recently associated with tissue repair capacities and with lower rates of GVHD in humans. Here, we analyzed the immunosuppressive effect of MAIT cells in an in vitro model of alloreactivity and explored their adoptive transfer in a preclinical xenogeneic GVHD model. We found that MAIT cells, whether freshly purified or short-term expanded, dose-dependently inhibited proliferation and activation of alloreactive T cells. In immunodeficient mice injected with human PBMCs, MAIT cells greatly delayed GVHD onset and decreased severity when transferred early after PBMC injection but could also control ongoing GVHD when transferred at delayed time points. This effect was associated with decreased proliferation and effector function of human T cells infiltrating tissues of diseased mice and was correlated with lower circulating IFN-γ and TNF-α levels and increased IL-10 levels. MAIT cells acted partly in a contact-dependent manner, which likely required direct interaction of their T cell receptor with MHC class I-related molecule (MR1) induced on host-reactive T cells. These results support the setup of clinical trials using MAIT cells as universal therapeutic tools to control severe GVHD or mucosal inflammatory disorders.

Experimental Data on PIRCHE and T-Cell Reactivity: HLA-DPB1-Derived Peptides Identified by PIRCHE-I Show Binding to HLA-A*02:01 in vitro and T-Cell Activation in vivo

Introduction: Human leukocyte antigen (HLA)-DPB1 mismatches during hematopoietic stem cell transplantation (HSCT) with an unrelated donor result in an increased risk for the development of graft-versus-host disease (GvHD). The number of CD8+ T-cell epitopes available for indirect allorecognition as predicted by the PIRCHE algorithm has been shown to be associated with GvHD development. As a proof of principle, PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro and in vivo. Methods: PIRCHE-I analysis was performed to identify HLA-DPB1-derived peptides that could theoretically bind to HLA-A*02:01. PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro by investigating binding affinities of HLA-DPB1-derived peptides to the HLA-A*02:01 in a competition-based binding assay. To investigate the capacity of HLA-DPB1-derived peptides to elicit a T-cell response in vivo, mice were immunized with these peptides. T-cell alloreactivity was subsequently evaluated using an interferon-gamma ELISpot assay. Results: The PIRCHE-I algorithm identified five HLA-DPB1-derived peptides (RMCRHNYEL, YIYNREEFV, YIYNREELV, YIYNREEYA, and YIYNRQEYA) to be presented by HLA-A*02:01. Binding of these peptides to HLA-A*02:01 was confirmed in a competition-based peptide binding assay, all showing an IC50 value of 21 μm or lower. The peptides elicited an interferon-gamma response in vivo. Conclusion: Our results indicate that the PIRCHE-I algorithm can identify potential immunogenic HLA-DPB1-derived peptides present in recipients of an HLA-DPB1-mismatched donor. These combined in vitro and in vivo observations strengthen the validity of the PIRCHE-I algorithm to identify HLA-DPB1 mismatch-related GvHD development upon HSCT.

Identification and analysis of alloreactive T lymphocytes from peripheral blood mononuclear cells.

Alloreactive T-cell responses against mismatched MHC or minor histocompatibility antigens may result in deleterious graft-versus-host disease (GVHD) and increased morbidity and mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nevertheless, these T-cell responses may be directed against residual tumor cells (the graft-versus-tumor effect, GVT), thus preventing relapse of the disease. Recent findings have shown that CD45RA+ naïve T cells, but not CD45RA- memory T cells are the major contributors to GVHD, thus leading to clinical trials where CD45RA+-depleted, memory-enriched T-cell products are adoptively transferred following allo-HSCT to prevent GVHD and enhance immune reconstitution. However, residual alloreactivity may still be present in the memory T-cell compartment, thus contributing to prevent disease relapse by GVT. Here, we describe a simple cell-based protocol to identify alloreactive naïve and memory T cells by co-culturing T-cell subsets and third-party antigen-presenting cells. The responding cells are identified following dilution of carboxyfluorescein succinimidyl ester (CFSE) and upregulation of the activation marker CD25. These CFSE-diluting cells can be further phenotyped by high-dimensional flow cytometry, or purified with a cell sorter for downstream genomic and functional assays.

A strategy to reconstitute immunity without GVHD via adoptive allogeneic Tscm therapy.

Adoption of allogeneic T cells directly supplements the number of T cells and rapidly induces T-cell immunity, which has good efficacy for treating some tumors and immunodeficiency diseases. However, poor adoptive T-cell engraftment and graft-versus-host disease (GVHD) limit the application of these methods. Alloreactive T-cell clones were eliminated from the donor T-cell repertoire, and the remaining T-cell clones were prepared as Tscm for T-cell adoptive treatment to reconstruct recipient T-cell immunity without GVHD. The subjects in this study included three different strains of mice. Lymphocytes from mice (C57BL/6) were used as the donor T-cell repertoire, from which the Tscm allo-reactive T cell clone was depleted (ATD-Tscm). This was confirmed by showing that the Tscm was not responsive to the alloantigen of the recipient (BALB/c). To prepare ATD-Tscm cells, we used recipient lymphocytes as a simulator, and coculture of mouse and recipient lymphocytes was carried out for 7 days. Sorting of non-proliferative cells ensured that the prepared Tscm cells were nonresponsive. The sorted lymphocytes underwent further expansion by treatment with TWS119 and cytokines for an additional 10 days, after which the number of ATD-Tscm cells increased. The prepared Tscm cells were transferred into recipient mice to observe immune reconstitution and GVHD incidence. Our protocol began with the use of 1×107 donor lymphocytes and resulted in 1 ×107 ATD-Tscm cells after 17 days of preparation. The prepared ATD-Tscm cells exhibited a nonresponse upon restimulation of the recipient lymphocytes. Importantly, the prepared ATD-Tscm cells were able to bind long and reconstitute other T-cell subsets in vivo, effectively recognizing and answering the "foreign" antigen without causing GVHD after they were transferred into the recipients. Our strategy was succeeded to prepare ATD-Tscm cells from the donor T-cell repertoire. The prepared ATD-Tscm cells were able to reconstitute the immune system and prevent GVHD after transferred to the recipients. This study provides a good reference for generating ATD-Tscm for T-cell adoptive immunotherapy.

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.

Interindividual HLA Evolutionary Divergence in Single HLA-Mismatched Unrelated Donor Hematopoietic Cell Transplantation for Malignant Hematological Disorders: A Report on Behalf of the Cellular Therapy and Immunobiology Working Party of the EBMT

In allogeneic hematopoietic cell transplantation (allo-HCT), alloreactive T-cell responses (graft versus leukemia - GvL and graft versus host disease - GvHD) are mediated by the interaction between recipient antigens and donor lymphocytes via human leukocyte antigen (HLA) molecules. HLA evolutionary divergence (HED) is a metric recently implemented as a functional proxy for the structural variability between two homologous HLA alleles at their peptide binding sites, mirroring quantitative differences in pathogen and cancer-related immunopeptidomes ( A). 1 Here, we hypothesize that differences in and between donor and recipient HEDs at given loci may modulate the direction and the intensity of alloreactivity, informing on post-transplant outcomes. We tested this hypothesis in onco-hematologic patients undergoing allo-HCT from a single HLA-A, -B, -C, -DRB1, or -DQB1 (9/10) mismatched unrelated donor (MMUD) at EBMT centers. We included patients with selected hematological malignancies, receiving a thymoglobulin-based T cell depletion, for whom complete clinical and HLA information was available in the EBMT database. We deployed individual locus-specific recipient/donor HED (HED-R, HED-D) metrics as previously described 1, and conceptualized two novel interindividual HED scores accounting for functional differences at the mismatched HLA loci ( A). The latter were considered as: 1) DeltaHED, quantifying the relative directional difference between HED-D and HED-R and informing on differences in immunopeptidome sizes, potentially capturing the direction and the intensity of the alloreactive response (GvH or HvG); 2) HEDmismatch, describing the HED of the mismatched locus as a proxy for the qualitative differences in immunopeptidomes between donor and recipient ( B). Using multivariable Cox regression (cause-specific) models adjusted for relevant clinical variables, we investigated the associations of HED-R, DeltaHED and HEDmismatch, analyzed as continuous variables, with relapse free survival (RFS), overall survival (OS), non-relapse mortality (NRM), relapse and grade II-IV acute and chronic GvHD. For each outcome and in each mismatched subgroup, we built separate models for these immunogenetic parameters and checked for proportional hazards assumption (PHA). To account for multiple comparisons, only p-values <0.01 were considered significant. Overall, 4639 adult patients, with available HLA data, received a 9/10 MMUD allo-HCT between 2010 and 2019 for acute leukemia (N=3853, 83%), myelodysplastic syndromes or myeloproliferative neoplasms (N=786, 17%). Median recipient age was 53.5 (IQR 40.2-61.9) with a male/female ratio of 1.28, whilst median donor age was 33.1 (IQR 24.5-40). Majority of patients (90%) received peripheral blood stem cells and were mismatched with their donors for locus A (33%), followed by C (21%), B (15%), DQB1 (14%) and DRB1 (6%). When analyzing the impact of HED-related parameters in each model, we observed that most recurrent associations with outcomes occurred with DRB1-related metrics. In the DRB1-mismatched subgroup (N=276), a time-varying association was found for HEDmismatch of DRB1 locus with higher HEDmismatch leading to worse RFS [HR:1.25 at t=0, (95% CI 1.1-1.42), p<0.001] and lower OS [HR:1.23 at t=0, (95%CI 1.08-1.41), p=0.002]. The strength of this associations declined over time for both endpoints (respectively RFS: HR:0.91, 95% CI 0.86-0.97, p=0.005 and OS: HR:0.93, 95% CI 0.88-0.98, p=0.013). The (directional) DeltaHED DRB1 measure instead did not display any impact on outcomes. In the same subgroup, higher HED-R in the locus C showed also a time-varying association with NRM at transplant [HR:1.89, (95% CI 1.29-2.85), p=0.002], progressively declining over time (HR:0.77, 95% CI 0.65-0.92, p=0.003). Furthermore, DRB1 HED-R modulated the probability of survival in B-mismatched subgroup, being higher scores associated with reduced OS [HR:1.03, (95%CI 1.01-1.06), p=0.009]. Here, we provide an immunogenetic benchmark to explore donor-recipient interactions in MMUD allo-HCT, demonstrating how different HLA configurations within the mismatched loci, in particular for DRB1 genes, can influence post-transplant outcomes. Given the peculiar associations observed, HED may have a role in driving early GvL effects, informing on future donor selection strategies in high-risk patients.

CAR-T Cells: A New Tool for Monitoring T-cell Alloreactivity?

CAR-T Cells: A New Tool for Monitoring T-cell Alloreactivity?

Joint models quantify associations between immune cell kinetics and allo-immunological events after allogeneic stem cell transplantation and subsequent donor lymphocyte infusion.

Alloreactive donor-derived T-cells play a pivotal role in alloimmune responses after allogeneic hematopoietic stem cell transplantation (alloSCT); both in the relapse-preventing Graft-versus-Leukemia (GvL) effect and the potentially lethal complication Graft-versus-Host-Disease (GvHD). The balance between GvL and GvHD can be shifted by removing T-cells via T-cell depletion (TCD) to reduce the risk of GvHD, and by introducing additional donor T-cells (donor lymphocyte infusions [DLI]) to boost the GvL effect. However, the association between T-cell kinetics and the occurrence of allo-immunological events has not been clearly demonstrated yet. Therefore, we investigated the complex associations between the T-cell kinetics and alloimmune responses in a cohort of 166 acute leukemia patients receiving alemtuzumab-based TCD alloSCT. Of these patients, 62 with an anticipated high risk of relapse were scheduled to receive a prophylactic DLI at 3 months after transplant. In this setting, we applied joint modelling which allowed us to better capture the complex interplay between DLI, T-cell kinetics, GvHD and relapse than traditional statistical methods. We demonstrate that DLI can induce detectable T-cell expansion, leading to an increase in total, CD4+ and CD8+ T-cell counts starting at 3 months after alloSCT. CD4+ T-cells showed the strongest association with the development of alloimmune responses: higher CD4 counts increased the risk of GvHD (hazard ratio 2.44, 95% confidence interval 1.45-4.12) and decreased the risk of relapse (hazard ratio 0.65, 95% confidence interval 0.45-0.92). Similar models showed that natural killer cells recovered rapidly after alloSCT and were associated with a lower risk of relapse (HR 0.62, 95%-CI 0.41-0.93). The results of this study advocate the use of joint models to further study immune cell kinetics in different settings.

TCR Sequencing in Mouse Models of Allorecognition Unveils the Features of Directly and Indirectly Activated Clonotypes.

Allorecognition is known to involve a large number of lymphocytes carrying diverse T-cell receptor repertoire. Thus, one way to understand allorecognition and rejection mechanisms is via high-throughput sequencing of T-cell receptors. In this study, in order to explore and systematize the properties of the alloreactive T-cell receptor repertoire, we modeled direct and indirect allorecognition pathways using material from inbred mice in vitro and in vivo. Decoding of the obtained T-cell receptor genes using high-throughput sequencing revealed some features of the alloreactive repertoires. Thus, alloreactive T-cell receptor repertoires were characterized by specific V-gene usage patterns, changes in CDR3 loop length, and some amino acid occurrence probabilities in the CDR3 loop. Particularly pronounced changes were observed for directly alloreactive clonotypes. We also revealed a clustering of directly and indirectly alloreactive clonotypes by their ability to bind a single antigen; amino acid patterns of the CDR3 loop of alloreactive clonotypes; and the presence in alloreactive repertoires of clonotypes also associated with infectious, autoimmune, and tumor diseases. The obtained results were determined by the modeling of the simplified allorecognition reaction in inbred mice in which stimulation was performed with a single MHCII molecule. We suppose that the decomposition of the diverse alloreactive TCR repertoire observed in humans with transplants into such simple reactions will help to find alloreactive repertoire features; e.g., a dominant clonotype or V-gene usage pattern, which may be targeted to correct the entire rejection reaction in patients. In this work, we propose several technical ways for such decomposition analysis, including separate modeling of the indirect alloreaction pathway and clustering of alloreactive clonotypes according to their ability to bind a single antigen, among others.

Perspectives on oral chronic graft-versus-host disease from immunobiology to morbid diagnoses.

Chronic Graft-versus-Host Disease (cGVHD) is a major long-term complication, associated with morbidity and mortality in patients following allogenic hematopoietic cell transplantation (HCT) for immune hematopoietic disorders. The mouth is one of the most frequently affected organs after HCT (45-83%) and oral cGVHD, which may appear as the first visible sign. Manifestations present with mucosal lichenoid lesions, salivary gland dysfunction and limited oral aperture. Diagnosis of oral cGVHD severity is based on mucosal lesions with symptoms of sensitivity and pain and reduced oral intake. However, diagnostic difficulties arise due to subjective definitions and low specificity to cover the spectrum of oral cGVHD. In recent years there have been significant improvements in our understanding of the underlying oral cGVHD disease mechanisms. Drawing upon the current knowledge on the pathophysiology and biological phases of oral cGVHD, we address oral mucosa lichenoid and Sjogren's Syndrome-like sicca syndromes. We consider the response of alloreactive T-cells and macrophages to recipient tissues to drive the pathophysiological reactions and biological phases of acute inflammation (phase 1), chronic inflammation and dysregulated immunity (phase 2), and subsequent aberrant fibrotic healing (phase 3), which in time may be associated with an increased malignant transformation rate. When formulating treatment strategies, the pathophysiological spectrum of cGVHD is patient dependent and not every patient may progress chronologically through the biological stages. As such there remains a need to address and clarify personalized diagnostics and management to improve treatment descriptions. Within this review, we highlight the current state of the art knowledge on oral cGVHD pathophysiology and biological phases. We address knowledge gaps of oral cGVHD, with a view to facilitate clinical management and improve research quality on lichenoid biology and morbid forms of oral cGVHD.

Chimeric HLA antibody receptor T cells to target HLA-specific B cells in solid organ transplantation.

HLA-sensitized patients on the transplant waiting list harbor antibodies and memory B cells directed against allogeneic HLA molecules, which decreases the chance to receive a compatible donor organ. Current desensitization strategies non-specifically target circulating antibodies and B cells, warranting the development of therapies that specifically affect HLA-directed humoral immune responses. We developed Chimeric HLA Antibody Receptor (CHAR) constructs comprising the extracellular part of HLA-A2 or HLA-A3 coupled to CD28-CD3ζ domains. CHAR-transduced cells expressing reporter constructs encoding T-cell activation markers, and CHAR-transduced CD8+ T cells from healthy donors were stimulated with HLA-specific monoclonal antibody-coated microbeads, and HLA-specific B cell hybridomas. CHAR T cell activation was measured by upregulation of T cell activation markers and IFNγ secretion, whereas CHAR T cell killing of B cell hybridomas was assessed in chromium release assays and by IgG ELISpot. HLA-A2- and HLA-A3-CHAR expressing cells were specifically activated by HLA-A2- and HLA-A3-specific monoclonal antibodies, either soluble or coated on microbeads, as shown by CHAR-induced transcription factors. HLA-A2 and HLA-A3 CHAR T cells efficiently produced IFNγ with exquisite specificity and were capable of specifically lysing hybridoma cells expressing HLA-A2- or HLA-A3-specific B-cell receptors, respectively. Finally, we mutated the α3 domain of the CHAR molecules to minimize any alloreactive T-cell reactivity against CHAR T cells, while retaining CHAR activity. These data show proof of principle for CHAR T cells to serve as precision immunotherapy to specifically desensitize (highly) sensitized solid organ transplant candidates and to treat antibody-mediated rejection after solid organ transplantation.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Off-the-shelf allogeneic T-cell therapies face the major challenges of graft-versus-host disease (GVHD) and graft rejection mediated by host and recipient alloreactive T cells, respectively. To address GVHD, we are using Epstein-Barr Virus-specific T cells (EBVST), which are virus specific rather than allo-specific and have not produced GVHD in over 300 allogeneic recipients. To prevent graft rejection, we modified the EBVST with a chimeric antigen receptor (CAR) targeting CD30, an antigen that is upregulated on allo-activated T-cells, which will consequently become targets for CD30.CAR EBVST. Hence, CD30.CAR EBVST can kill both CD30+ lymphoma cells and alloreactive T cells through their CAR, and may be boosted in vivo in EBV+ recipients via their native TCR. We are evaluating this approach in a phase 1 trial (NCT04288726) in patients with CD30+ lymphoma. We generated a bank of 7 CD30.CAR EBVST products by stimulating PBMC from healthy donors with overlapping EBV peptide mixtures followed by retroviral transduction with a 2nd generation, CD28-containing CD30.CAR. We have treated 16 patients (median age 36, range 22–53) with relapsed or refractory HL. Patients had undergone a median of 5 (range 3–7) prior lines of treatment. Escalating doses of 4 × 107 − 8 × 108 (DL1–4) CD30.CAR EBVST were infused after lymphodepletion. The frequency of CD30.CAR EBVST was measured by real time qPCR for the transgene in blood. Changes in the frequency of T-cells responding to EBV or tumor-associated antigens (epitope spreading) were measured by IFNɣ ELISpots. Clinical responses were assessed per Lugano criteria by PET/CT scans performed 4–8 wks post infusion. CD30.CAR EBVST were well tolerated, with no GVHD and only two patients having reversible grade 4 cytopenia. Five patients at DL3–4 had grade 1 cytokine release syndrome (CRS), but all resolved without treatment. Of the 16 patients treated, 6 had partial responses (PR) and 6/13 patients at DL2–4 had complete responses (CR), giving an overall objective response rate (ORR) of 75%. Despite these remarkable responses, qPCR for the CD30.CAR transgene showed near background levels by wk 1 post infusion. Although this could mean that CAR-T are quickly eliminated by alloreactive T-cells, additional infusions from the same donor product into the same patient can be associated with response (Figure 1); an alloreactive response would be anticipated to be amplified after each infusion and preclude additional responses. We are therefore analyzing tumor biopsy samples to determine the presence of CD30.CAR EBVST at tumor sites and characterize their phenotype for functionality. The research was funded by: Leukemia and Lymphoma Society SCOR (Heslop) and Tessa Therapeutics (Rooney/Ramos) through a grant to Baylor College of Medicine. Keywords: cellular therapies, Hodgkin lymphoma Conflicts of interests pertinent to the abstract C. A. Ramos Consultant or advisory role: Novartis, Genentech, CRISPR Therapeutics Research funding: Tessa Therapeutics, Athenex Therapeutics Other remuneration: Royalties: Tessa Therapeutics D. H. Quach Research funding: Tessa Therapeutics P. D. Lulla Research funding: Marker Therapeutics, Allovir R. H. Rouce Consultant or advisory role: Pfizer, Novartis Honoraria: Novartis, Medline Plus Research funding: Tessa Therapeutics B. J. Grilley Consultant or advisory role: Allovir, Marker Therapeutics, Tessa Therapeutics, Lokon Pharma, March Bio and Proxima Clinical Research Stock ownership: Allovir, QBRegulatory LLC N. Lapteva Consultant or advisory role: Tessa Therapeutics Research funding: Tessa Therapeutics M. K. Brenner Employment or leadership position: Founder Member Marker Therapeutics Consultant or advisory role: Tessa Therapeutics, Marker Therapeutics, Allogene, Walking Fish, Athenex, Turnstone Biologics, Posedia, Tscan, Bluebird Bio, Adaptimmune Therapeutics, Adintus, Onkimure, Triumvira Immunologics, Bellicum Pharmaceuticals, Memgen LLC, Brooklyn Immunotherapeutics, Coya, Astrazeneca Pharmaceuticals Stock ownership: Allovir, Marker Therapeutics, Tessa Therapeutics, Turnstone, Bluebird Bio, Allogene, Walking Fish, AlloVir, Tessa, Tscan Other remuneration: Royalties: Takeda, Bellicum, Marker Therapeutics H. E. Heslop Consultant or advisory role: Gilead Biosciences, Novartis, Tessa Therapeutics, Marker Therapeutics, Kiadis, PACT Pharma, Mesoblast, Ankyra Therapeutics Stock ownership: Allovir, Marker Therapeutics, Fresh Wind Biotechnologies, CoRegen Inc. Research funding: Tessa Therapeutics, Athenex Therapeutics C. M. Rooney Consultant or advisory role: Tessa Therapeutics, Marker Therapeutics, Cell Genix Stock ownership: Allovir, Marker Therapeutics Research funding: Tessa Therapeutics Other remuneration: Royalties: Takeda, Marker, Allovir, Bellicum

CAR NK-92 cell–mediated depletion of residual TCR+ cells for ultrapure allogeneic TCR-deleted CAR T-cell products

AbstractGraft-versus-host disease (GVHD) is a major risk of the administration of allogeneic chimeric antigen receptor (CAR)-redirected T cells to patients who are HLA unmatched. Gene editing can be used to disrupt potentially alloreactive T-cell receptors (TCRs) in CAR T cells and reduce the risk of GVHD. Despite the high knockout rates achieved with the optimized methods, a subsequent purification step is necessary to obtain a safe allogeneic product. To date, magnetic cell separation (MACS) has been the gold standard for purifying TCRα/β– CAR T cells, but product purity can still be insufficient to prevent GVHD. We developed a novel and highly efficient approach to eliminate residual TCR/CD3+ T cells after TCRα constant (TRAC) gene editing by adding a genetically modified CD3-specific CAR NK-92 cell line during ex vivo expansion. Two consecutive cocultures with irradiated, short-lived, CAR NK-92 cells allowed for the production of TCR– CAR T cells with <0.01% TCR+ T cells, marking a 45-fold reduction of TCR+ cells compared with MACS purification. Through an NK-92 cell–mediated feeder effect and circumventing MACS-associated cell loss, our approach increased the total TCR– CAR T-cell yield approximately threefold while retaining cytotoxic activity and a favorable T-cell phenotype. Scaling in a semiclosed G-Rex bioreactor device provides a proof-of-principle for large-batch manufacturing, allowing for an improved cost-per-dose ratio. Overall, this cell-mediated purification method has the potential to advance the production process of safe off-the-shelf CAR T cells for clinical applications.

Gene Expression Profiling and Signaling Pathway Analysis of Tolerant T cells Circulating in Adult Patients after PTCy Haplotransplantation

Abstract The mechanisms for the signaling pathways of T cell-dependent tolerance after haploidentical hematopoietic stem cell transplantation (haplo-HSCT), are incompletely understood. We tested 8 patients (Pt) with leukemia or myelodysplastic syndromes (median age:51-yrs old) following IRB approved consenting. All received Flu+TBI (+/− Cy) pre-conditioning and haplo-HSCT with post-transplant Cytoxan (PTCy) and FK506 as GvHD prophylaxis. The circulating T cells were hyporeactive in mixed lymphocyte reaction (MLR) assay to recipient DC in Pts at 1yr (or 2yrs in 1 Pt) post-haplo-HSCT (~6m post-ISD withdrawal), while these T cells responded vigorously to 3rd party APC. Neither depletion of Tregs, nor blockade of IL10R or PD1 could reverse T-cell hyporeactivity. Low-dose IL2 triggered mild anti-host responses. Cytokine profiling further confirmed the MLR responses. ImmunoSEQ™ revealed the disappearance of alloreactive T-cell clones from peripheral pool with time, even though a few alloreactive T clones remained. Low dose IL2 had minimal impact to reverse the diminishment. Unlike graft T-cell responses against host DC, tolerant T cells in circulation against hDC showed relative downregulation of allograft rejection, T-helper response, co-stimulation, and cell proliferation pathway, that was paired with upregulation of co-inhibition, PD1-PDL1, Ferroptosis and CREB pathways. In summary, hyporeactivity of donor-derived circulating T cells to recipient DC were evident. We found Treg, Tr1 activity to be insignificant in maintaining long-term tolerance. T-cell clonal deletion appears to be the dominant mechanism, along with systemically altered signaling pathways in tolerant T-cells.