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Background Aims: Hu8F4 is a T-cell receptor-like antibody with high affinity for the leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is composed of the Hu8F4 single-chain variable fragment, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from patients with acute myeloid leukemia in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor-expressing cells., Methods: We generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with Fc gamma receptor-expressing cells in vivo., Results: The primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2 + PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice., Conclusions: Herein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor binding-induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo., Competing Interests: Declaration of competing interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Tools for genome-wide rapid identification of peptide-major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell-APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515., (©2024 American Association for Cancer Research.)

Despite substantial advances in the treatment of acute myeloid leukemia (AML), only 30% of patients survive more than 5 years. Therefore, new therapeutics are much needed. Here, we present a novel therapeutic strategy targeting PR1, an HLA-A2 restricted myeloid leukemia antigen. Previously, we have developed and characterized a novel T-cell receptor-like monoclonal antibody (8F4) that targets PR1/HLA-A2 and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC). To improve the potency of 8F4, we adopted a strategy to link T-cell cytotoxicity with a bi-specific T-cell-engaging antibody that binds PR1/HLA-A2 on leukemia and CD3 on neighboring T-cells. The 8F4 bi-specific antibody maintained high affinity and specific binding to PR1/HLA-A2 comparable to parent 8F4 antibody, shown by flow cytometry and Bio-Layer Interferometry. In addition, 8F4 bi-specific antibody activated donor T-cells in the presence of HLA-A2 + primary AML blasts and cell lines in a dose dependent manner. Importantly, activated T-cells lysed HLA-A2 + primary AML blasts and cell lines after addition of 8F4 bi-specific antibody. In conclusion, our studies demonstrate the therapeutic potential of a novel bi-specific antibody targeting the PR1/HLA-A2 leukemia-associated antigen, justifying further clinical development of this strategy.

Background Aims: The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T-cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells, efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4- chimeric antigen receptor (CAR) that was transduced into T cells to mediate anti-leukemia activity., Methods: Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endo-domain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28., Results: Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4-CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro. Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner., Conclusions: Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro. Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

In this issue of Blood, Zhou et al report long-term follow-up and detailed analysis of immune reconstitution associated with a different suicide gene strategy to abrogate graft-versus-host disease (GVHD).

The Wilms tumor protein, WT-1, is a widely recognized tumor antigen that is aberrantly expressed in myeloid and lymphoid leukemia and in this issue of Blood, Doubrovina et al report the most extensive catalog heretofore of HLA-restricted immunogenic peptides derived from WT-1, which are recognized by CD8 and CD4T cells.

Vitamin D deficiency is adversely associated with diseases characterized by inflammation. The combination of the high incidence of vitamin D deficiency in patients undergoing allogeneic stem cell transplants (SCT) and the potential role of vitamin D deficiency in influencing graft-versus-host disease led us to further characterize the expression of VDR on alloreactive T cells. We hypothesized that vitamin D receptor expression may directly regulate alloreactive T cell responses. To overcome existing limitations in measuring VDR in bulk cellular populations, we developed a flow cytometric assay to measure cytoplasmic VDR in human T cells. Upon stimulation, VDR was expressed extremely early and exhibited sustained upregulation with chronic stimulation. VDR expression was also coupled to cytokine production, proliferation, and ERK1/2 phosphorylation. In addition, VDR exhibited a maturation stage-specific pattern of expression, with greatest expression on cells known to mediate GVHD, naïve and early memory T cells. Alloreactive T cells upregulated VDR, whereas the nonreactive T cells did not. Finally, repletion of vitamin D in vitro was sufficient to significantly reduce alloreactive T cell responses. These data suggest that vitamin D effects on T cells may be important in reducing graft versus host disease (GVHD) in the allogeneic stem cell transplant setting., (Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematologic malignancies. However graft-versus-host disease (GVHD) is a major limiting factor for a successful patient outcome. GVHD is a result of alloimmune responses of donor T lymphocytes attacking the recipient's cells and tissues. Chemokine receptor CCR5 plays a role in solid organ allograft rejection and mediates murine GVHD pathogenesis. Herein, we report that infiltrating lymphocytes in the skin of human acute GVHD (aGVHD) samples are predominantly CCR5(+) T cells. In addition, we characterized the features of the CCR5 expression on alloreactive T lymphocytes. We found that the CCR5(+) population exhibits the characteristics of the activated effector T cell phenotype. CCR5 expression is upregulated upon allogenic stimulation, and CCR5(+) cells are proliferating with coexpression of T cell activation markers. Furthermore, the activated T cells producing inflammatory cytokine tumor necrosis factor (TNF)alpha, interleukin (IL)-2, or interferon (IFN)-gamma, are positive for CCR5. Thus, CCR5 is a marker for GVHD effector cells and CCR5(+) T cells are active participants in the pathogenesis of human aGVHD., (Copyright (c) 2010 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

The naive phenotype of cord blood (CB) T cells may reduce graft-versus-host disease after umbilical cord blood transplantation, but this naivety and their low absolute numbers also delays immune reconstitution, producing higher infection-related mortality that is predominantly related to CMV, adenovirus (Adv), and EBV. Adoptive immunotherapy with peripheral blood-derived virus-specific cytotoxic T lymphocytes (CTLs) can effectively prevent viral disease after conventional stem cell transplantation, and we now describe the generation of single cultures of CTLs from CB that are specific for multiple viruses. Using EBV-infected B cells transduced with a clinical-grade Ad5f35CMVpp65 adenoviral vector as sources of EBV, Adv, and CMV antigens, we expanded virus-specific T cells even from CB T cells with a naive phenotype. After expansion, each CTL culture contained both CD8(+) and CD4(+) T-cell subsets, predominantly of effector memory phenotype. Each CTL culture also had HLA-restricted virus-specific cytotoxic effector function against EBV, CMV, and Adv targets. The CB CTLs recognized multiple viral epitopes, including CD4-restricted Adv-hexon epitopes and immunosubdominant CD4- and CD8-restricted CMVpp65 epitopes. Notwithstanding their naive phenotype, it is therefore possible to generate trivirus-specific CTLs in a single culture of CB, which may be of value to prevent or treat viral disease in CB transplant recipients. This study is registered at www.clinicaltrials.gov as NCT00078533.

Advances in immune assessment, including the development of T-cell receptor excision circle (TREC) assays of thymopoiesis, cytokine-flow cytometry assays of T-cell function, and higher-order phenotyping of T-cell maturation subsets have improved our understanding of T-cell homeostasis. Limited data exist using these methods to characterize immune recovery in adult cord blood (CB) transplant recipients, in whom infection is a leading cause of mortality. We now report the results of a single-center prospective study of T-cell immune recovery after cord blood transplantation (CBT) in a predominantly adult population. Our primary findings include the following: (1) Prolonged T lymphopenia and compensatory expansion of B and natural killer (NK) cells was evident; (2) CB transplant recipients had impaired functional recovery, although we did observe posttransplantation de novo T-cell responses to cytomegalovirus (CMV) in a subset of patients; (3) Thymopoietic failure characterized post-CBT immune reconstitution, in marked contrast to results in other transplant recipients; and (4) Thymopoietic failure was associated with late memory T-cell skewing. Our data suggest that efforts to improve outcomes in adult CB transplant recipients should be aimed at optimizing T-cell immune recovery. Strategies that improve the engraftment of lymphoid precursors, protect the thymus during pretransplant conditioning, and/or augment the recovery of thymopoiesis may improve outcomes after CBT.

Myeloid forms of leukemia would seem to be an ideal disease for investigators wishing to develop targeted immunotherapy because the leukemia is derived from antigen-presenting cells and because clinical data have demonstrated that there is potent T-cell immunity to chronic myeloid leukemia when donor lymphocyte infusions are used to treat relapse after transplantation. However, clinical vaccine studies have had to wait for the identification of specific antigens, some of which have recently been identified, and for a more complete understanding of basic tumor immunology. Here we review relevant fundamental T-cell biology, the nature of some important leukemia-associated antigens, and the preliminary results from recent clinical vaccine trials for leukemia. Although these studies are still early, they offer evidence that effective immunity to leukemia is possible after vaccination, thus setting the stage for future combined therapies.

Human T-cell alloreactivity plays an important role in many disease processes, including the rejection of solid organ grafts and graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. To develop a better understanding of the T cells involved in alloreactivity in humans, we developed a cytokine flow cytometry (CFC) assay that enabled us to characterize the phenotypic and functional characteristic of T cells responding to allogeneic stimuli. Using this approach, we determined that most T-cell alloreactivity resided within the CD4(+) T-cell subset, as assessed by activation marker expression and the production of effector cytokines (eg, tumor necrosis factor alpha [TNF]alpha) implicated in human GVHD. Following prolonged stimulation in vitro using either allogeneic stimulator cells or viral antigens, we found that coexpression of activation markers within the CD4(+) T-cell subset occurred exclusively within a subpopulation of T cells that significantly increased their surface expression of CD4. We then developed a simple sorting strategy that exploited these phenotypic characteristics to specifically deplete alloreactive T cells while retaining broad specificity for other stimuli, including viral antigens and third-party alloantigens. This approach also was applied to specifically enrich or deplete human virus-specific T cells.

Myelodysplastic syndromes, a heterogeneous group of clonal stem cell disorders, are characterized by ineffective and dysplastic hematopoiesis and increased risk of leukemic transformation. The clonal disorder may result in cumulative cytogenetic abnormalities in a multistep process. Intrinsic and extrinsic factors contributing to ineffective hematopoiesis include genetic predisposition, environmental effects, and iatrogenic causes. The common and often overlapping features of myelodysplastic syndrome, aplastic anemia, T-cell large granular lymphocyte lymphoproliferative disorder, and paroxysmal nocturnal hemoglobinuria suggest a shared pathophysiologic mechanism of marrow failure. Recent studies suggest that dominant clonal T cells, which may be driven to expand by hematopoietic antigenic stimuli, result in the T-cell-mediated inhibition of hematopoietic progenitors, thus contributing to cytopenia.

Poor immune reconstitution after haploidentical stem cell transplantation results in a high mortality from viral infections and relapse. One approach to overcome this problem is to selectively deplete the graft of alloreactive cells using an immunotoxin directed against the activation marker CD25. However, the degree of depletion of alloreactive cells is variable following stimulation with recipient peripheral blood mononuclear cells (PBMCs), and this can result in graft versus host disease (GVHD). We have refined this approach using recipient Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as stimulators to activate donor alloreactive T cells. Our studies demonstrate that allodepletion with an anti-CD25 immunotoxin following stimulation with HLA-mismatched host LCLs more consistently depleted in vitro alloreactivity than stimulation with host PBMCs, as assessed in primary mixed lymphocyte reactions (MLRs). Allodepletion using this approach specifically abrogates cytotoxic T-cell responses against host LCLs. In interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assays, antiviral responses to adenovirus and cytomegalovirus (CMV) were preserved following allodepletion. Likewise, using HLA-A2-pp65 tetramers, we have shown that the frequency of CMV-specific T cells is unaffected by allodepletion. Moreover, the donor anti-EBV response is partially retained by recognition of EBV antigens through the nonshared haplotype. Finally, we studied whether allodepletion affects the response to candidate tumor antigens in myeloid malignancies. Using HLA-A2-PR1 tetramer analysis, we found that the frequency of T cells recognizing the PR1 epitope of proteinase 3 was not significantly different in allodepleted and unmanipulated PBMCs from patients with chronic myeloid leukemia (CML) undergoing transplantation. Based on these data, we have embarked on a phase 1 clinical trial of addback of allo-LCL-depleted donor T cells in the haplo-identical setting.

Evidence suggests that T lymphocyte-mediated inhibition of hematopoiesis in myelodysplastic syndrome (MDS) contributes to cytopenia in some patients and can be reversed by treatment with immunosuppression. We examined the T-cell repertoires of 12 patients with MDS before and after antithymocyte globulin (ATG)-based treatment by T-cell receptor Vbeta (TCR-Vbeta) spectratype analysis. The average number of TCR-Vbeta families with skewed spectratypes, representative of clonal or oligoclonal T-cell populations, was 7.6 in MDS patients before treatment and 3.2 in healthy controls (P =.02). Four patients who recovered effective hematopoiesis after treatment lost prominent, skewed peaks on their spectratypes, suggesting loss or diminution of overrepresented clonal T-cell populations. In contrast, patients who did not recover effective hematopoiesis showed persistently skewed repertoires 3 to 6 months after treatment. In 3 patients with skewed repertoires, cDNA from the complementarity-determining region 3 (CDR3) of 4 TCR-Vbeta families was cloned and repetitively sequenced, confirming clonal T-cell dominance in each family. In one nonresponder, 16 of 19 CDR3 sequences were identical, demonstrating that 9.3% of the total T-cell population was made up of a single clone. By 6 months after treatment, this clone persisted on both spectratype and DNA sequence complementarity and when analyzed by flow cytometry was shown to be CD8(+)/CD45RA(+)/HLA-DR(-). T-cell clones were not anergic because they could be expanded 4-fold in vitro. Our results demonstrate that predominant clonal T cells that appear to be antigen-driven persist in patients with MDS unresponsive to immunosuppression, but predominant clones regress in responders to immunosuppression.