TCR Alpha Beta Research Articles

Investigating MAIT cell phenotype using MR1 and TCR Dextramer™ reagents

Abstract Objective Here, we demonstrate a workflow, which enables the study of mucosal-associated invariant T (MAIT) cells using MR1 reagents. The workflow covers initial detection of MAIT cells with TCR sequencing as well as cloning, production, and validation of selected MAIT TCR molecules. Methods Human PBMC samples were stained with relevant antibodies and MR1/5-OP-RU dCODE Dextramer™ reagents. Following staining, sorting, and partitioning on a BD RhapsodyTM Single-Cell Analysis System, the cells underwent sequencing for characterization of gene and surface marker expression as well as paired alpha-beta TCR sequences. Selected TCR sequences were produced as TCR molecules and subsequently used to detect MR1/5-OP-RU on the surface of antigen-presenting cells. Results The majority (70%) of the MAIT cells express typical TCRs containing TRAV1-2 and TRAJ33 gene segments. The remaining MR1/5-OP-RU-reactive MAIT cells express highly diverse, non-classical MAIT TCR sequences. The three most frequent TCR clonotypes were produced and their specificity validated using in vitro assays and staining of MR1/5-OP-RU-presenting cells. Summary Our workflow allows identification of MAIT cells and elucidation of their TCR sequences. The high sensitivity of MR1 dCODE Dextramer reagents enables detection of both weak interactions and rare cell types. Subsequent production of MAIT TCR molecules allows deeper characterization of individual MAIT cell specificity and its role in health and disease.

Abstract 2062: Identification of functional MAIT-specific TCRs using MR1 dCODE Dextramer® reagents and evaluating antigen presentation

Abstract Objectives: To support TCR discovery and enable evaluation of TCR target expression on the surface of cells, we have established a streamlined workflow allowing TCR Discovery, validation and evaluation of MHC-antigen expression on target cells. As proof of concept, MR1 dCODE Dextramer® reagents were used to identify antigen-specific MAIT cells using single-cell sequencing, TCR sequences were extracted from the resulting data and then cloned and expressed as TCR monomers. The specificity of the TCRs was subsequently validated as TCR Dextramer® reagents and used to evaluate target MHC-peptide presentation on cell surface. Methods: Mixed PBMC samples from three healthy donors were stained with antibodies targeting relevant phenotypic markers as well as MR1 dCODE Dextramer® reagents carrying the MAIT cell-specific ligand 5-OP-RU. Following staining, sorting, and partitioning on a BD Rhapsody™ Single-Cell Analysis System, the cells underwent sequencing for a characterization of gene and surface marker expression as well as paired alpha-beta TCR sequences. Single alpha and beta chains were individually expressed, refolded together and purified. Refolded TCR monomer functionality was validated using an artificial cell assay. The functional TCRs can be multimerized on a Dextramer® backbone and used to evaluate expression of target on cell surface. Results: The majority of the identified CD161+ MAIT cells (~70%) expressed typical TCRs consisting of TRAV1-2 and TRAJ33 as well as TRBV6/20, TRBD1/2, and TRBJ2 gene segments. Three alpha-beta TCR sequences were selected: Two TCRs having common TRAV+TRAJ, TRBV+TRBJ combinations and one having a rare TRAV+TRAJ one. TCR alpha and beta chains were successfully expressed for all selected TCRs but only 2 TCRs showed a correctly refolded TCR as demonstrated by proper recognition of MR1/5-OP-RU. Based on these two functional TCRs, TCR Dextramer® reagents were generated and used to evaluate expression of target antigen on cell surface. Conclusion: We demonstrate a workflow allowing: (i) Identification of MAIT cells and their corresponding TCR sequences. (ii) Generation of soluble TCR molecules based on the identified sequences and validation of their specificity. (iii) Generation of TCR Dextramer® reagents allowing identification of target expression on surface of cells. This workflow is also useful for identification of other antigen-specific T cells and creates a new tool for evaluation of antigen-expression by MHC on surface of cells. By combining the high sensitivity of dCODE Dextramer® technology with TCR sequencing and creation of TCR Dextramer® reagents, both low-affinity interactions and rare cell types can be detected and studied in unique detail. Citation Format: Kevin Lenogue, Bjarke Hansen, Kivin Jacobsen, Liselotte Brix, Thomas Holberg Blicher. Identification of functional MAIT-specific TCRs using MR1 dCODE Dextramer® reagents and evaluating antigen presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2062.

396 - TCR Alpha Beta and CD19+ Depleted Haploidentical Peripheral Stem Cell Transplantation Using Reduced Toxicity Conditioning Regimen with Pharmacokinetics Guided Busulfan, Fludarabine, Thiotepa and Thymoglobulin Offers Durable Donor Engraftment in Children with Primary Immune Deficiency Disorders

396 - TCR Alpha Beta and CD19+ Depleted Haploidentical Peripheral Stem Cell Transplantation Using Reduced Toxicity Conditioning Regimen with Pharmacokinetics Guided Busulfan, Fludarabine, Thiotepa and Thymoglobulin Offers Durable Donor Engraftment in Children with Primary Immune Deficiency Disorders

416 - Immune Profiling Following TCR Alpha Beta T and B-Cell Depleted Hematopoietic Cell Transplantation in Pediatric Patients

416 - Immune Profiling Following TCR Alpha Beta T and B-Cell Depleted Hematopoietic Cell Transplantation in Pediatric Patients

Characteristics and Outcomes of Adult Patients with T Prolymphocytic Leukemia: A Real World Study of the French Innovative Leukemia Group (FILO)

Introduction T-prolymphocytic leukemia (T-PLL) is a rare T-cell neoplasm of aggressive clinical course and poor outcome. Treatment with the monoclonal antibody to CD52-alemtuzumab (alem) has considerably improved outcomes, yet with transient responses. Bendamustine (benda) showed encouraging results, particularly in alem-refractory patients (pts). Allogeneic hematopoietic stem cells transplantation (Allo-HSCT) has been reported to be effective in T-PLL, yielding durable remissions and improved overall survival (OS). New approaches using well-tolerated therapies and drugs that target such growth and survival signals as that the JAK/STAT pathways or BCL2 have recently been tested in small series of pts ineligible to intensive therapy. Methods The present multicenter study aimed at evaluating clinical and biological characteristics, as well as outcome of a cohort of 157 T-PLL pts over 18 years old (yo) treated in any of 24 centers of the French Innovative Leukemia Organization (FILO) between 03/1997 and 05/2022. OS, relapse, progression and progression-fee survival (PFS) were evaluated. Molecular analyzes searched for JAK1, JAK3, STAT5B, IL2RG, NOCH1, NOTCH2, EZH2, TET2 and TP53 mutations/deletions in 39 pts. Results Pts characteristics are shown in Table 1. The median age at first therapy was 72 yo (range 30-94). Neuro-meningeal and extra-nodal involvement were present at diagnosis and first relapse in respectively 4.6% / 10.5% and 5.9% / 14.3% of pts. At diagnosis, 12% of pts had hemoglobin <100 g/L, 32.6% thrombocytopenia <100x109/L and 43% lymphocytosis >40x109/L. Flow cytometric immunophenotyping showed pan-T antigens expression (CD2,CD3, CD5, CD7) in nearly all pts (table). Alpha-beta TCR was present for 64/73 pts (87.7%) and CD25 for 17/42 (40.2%). The TPLL was CD4+ for 73/98 pts (74.5%), CD8+ for 7/93 (7.5%), double positive (CD4+/CD8+) for 15/93 (16.1%) and double negative (CD4-/CD8-) for 3/92 (3.2%). Cytogenetics/FISH showed recurrent changes involving chromosome 14 in 69/95 (72.6%) pts [inv(14) and t(14;14) in 47/69 (68.1%) and 7/69 (10.3%)] and complex karyotype in 67/95 (70.5%). C-MYC gain, ATM and TP53 deletions/mutations were identified in respectively 22.6%, 27.5% and 16.4% of the pts analyzed at diagnosis, while JAK1, JAK3, and STAT5B were mutated in 15.4 %, 64.3% and 21.4% of tested cases. Additional results will be presented at the meeting. Pts received 1 to 5 lines of treatment (median 1). In first line, 45/127 (35.4%) evaluable pts received alem 30 mg IVx3 per week, after ramp-up (median:6 cycles; 1-10), with for 7 of them a previous sequence of benda (90 mg/m2 on day 1 [D1]). Thirty pts (23.6%) received first line benda 90 mg/m2 on D1 then 2x6 cycles every 28 days (median 3; 1-6), 15 (11.8%) received pentostatin, 11 (8.6%) had a CHOP-like regimen. Best supportive care was provided to 21 pts (16.5%). No treatment was proposed to 3 indolent pts (2.3%). Various schedules were applied to the remaining pts. Post-CR Allo-HSCT was performed for 20 pts (15.7%). Median follow-ups are 9.5 months (0.03-217) for the whole cohort (Figure1) and 11.8 (0.03-221) for Allo-HSCT recipients. Median OS are respectively 12.5 (8.4-17.5) and 23.2 months (5.82-not reached [NR]). The median PFS of the whole cohort is 2.6 months (0-217). Median OS for patients treated with alem or benda in first line are respectively 14.1 (8.4-25.4) and 7.8 (4.3-21) months, while OS for pts who had a benda bridge is 8 months (3.22-NR). The overall response rate (ORR) after first line is 42.7 % (CR 25.6%, partial response [PR] 17.1%). ORRs after first line alem or benda are respectively 53.5% (CR 41.9%, PR 11.6%) and 35.7% (CR: 14.3%, PR 21.4%). In univariate analysis, platelets<100x109/L (HR: 1.95 [1.28-2.95]; p=0.002%) and complex karyotype (HR: 2.08 [1.19-3.63]; p=0.010) statistically significantly appear to impact survival. Conclusion This large study demonstrates that alem therapy significantly improves the survival of T-PLL pts. Consolidation by Allo-HSCT should however be performed for all eligible patients achieving CR. Other therapeutic agents should be reserved for relapse. Prospective trials are needed to define the most effective therapeutic strategy and improve clinical responses to frontline therapy. New approaches using well-tolerated targeted therapies involving growth and survival signals are needed for the majority of patients unable to receive intensive chemotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Humanized Murine Model to Identify Neoantigen-Specific T Cells in CBFA2T3-GLIS2 Positive Acute Megakaryoblastic Leukemia

CBFA2T3-GLIS2 is an oncogenic fusion gene that acts as the driver mutation in ~18% of pediatric patients with acute megakaryoblastic leukemia (AMKL) that do not have Down syndrome. CBFA2T3-GLIS2 driven leukemia carries the worst prognosis among all pediatric acute myeloid (AML) subtypes, with event free and overall survival probabilities of 8% and 14% respectively. Chemotherapy and stem cell transplantation fail to cure this disease, thus innovative treatment approaches are needed. While monoclonal antibody and chimeric antigen receptor T-cell (CAR-T) therapies have proven successful in acute lymphoblastic leukemia (ALL), similar successes haven't been realized in AML due to the lack of targetable antigens that eradicate leukemia cells while minimizing off target toxicities such as depletion of normal myeloid cells which can lead to prolonged neutropenia. An alternative is T cell receptor engineered T cell (TCR-T) immunotherapy which use heterodimers consisting of alpha and beta peptide chains to recognize polypeptide fragments presented by MHC molecules on the tumor cells. An advantage of this approach is the ability to recognize intracellular tumor specific and tumor associated antigen fragments in addition to extracellular proteins which results in a wider range of targets. To investigate the immunogenicity of CBFA2T3-GLIS2 positive AMKL cells, we established a humanized patient derived xenograft (PDX) murine model by conditioning 15 immunodeficient NSG-SGM3 mice with 200 rads 24 hours prior to transplantation of 3.5 million CBFA2T3-GLIS2 positive PDX cells derived from an individual female patient. After 20 days of engraftment, 2.5 million HLA class I matched PBMCs from an unrelated female donor were introduced. Experimental mice (engrafted with both PDX cells and PBMCs), leukemia only control mice, and PBMC only control mice were sacrificed at days 30, 35, and 40. Flow cytometry analysis at day 40 showed dramatic reduction of leukemic blasts (huCD45lo, huCD34+) when exposed to PBMCs compared to the leukemia only control (1.48% and 93.7%, respectively). Circulating human Granzyme A was elevated at day 40 in the PDX mice with PBMCs compared to the PBMC only control (9665.4 pg/ml vs. 789.7 pg/ml, p<0.001), indicating a potential leukemia-directed cytokine lymphocyte response. Single cell TCR repertoire analysis of CD8+ T cells from day 40 revealed a decrease in alpha beta TCR diversity when exposed to PDX cells compared to the PBMC only control, consistent with a clonal expansion of neoantigen-directed TCRs. Single TCR expressing stable cell lines have been generated for the top expanded clones. In ongoing experiments, we are utilizing these cell lines to assess leukemia clearance in our model and measuring specificity and functionality for the putative neoantigens through tetramer staining and peptide stimulation assays, respectively. For the latter studies, we have taken two approaches for the identification of potential neoantigens that may be directing this observed clonal expansion. First, to interrogate potential neoantigens spanning the fusion junction we used the algorithm from NetMHCcons which identified two peptides with intermediate binding affinity (defined by an IC50 of 150 - 500 nM). Second, we utilized immunopeptidomics to identify HLA class I presented peptides on the surface of the PDX cells. One of our top significant hits was a peptide that corresponds to the 5‘ untranslated region (UTR) of bone morphogenetic protein 2 (BMP2). This peptide fragment was not present in the human Uniprot database, suggesting the 5‘ UTR is not expressed in normal tissues. Although its role in leukemia is not well described, BMP2 is aberrantly upregulated in CBFA2T3-GLIS2 positive AMKL and required for serial replating in colony formation unit assays; therefore, this peptide is of great interest as a candidate leukemia-associated antigen. In summary, our novel humanized PDX model sheds light on the immunogenicity of CBFA2T3-GLIS2 mediated AMKL and the potential for TCR-T immunotherapy in the setting of minimal residual disease for this chemotherapy resistant malignancy. Studies are ongoing to identify the TCR clone(s) responsible for the anti-leukemic response seen in our model and the leukemia-associated antigens that are mediating this response. DisclosuresGruber: Kura Oncology: Consultancy.

Single-cell immunopathology of systemic contact allergy associated with corticosteroids

Rationale Allergic contact dermatitis (ACD) is a classic delayed hypersensitivity reaction commonly associated with corticosteroids that can rarely manifest as a delayed rash following systemic administration. Methods A 50-year old woman developed local edema and delayed spreading rash following interarticular injection with dexamethasone and methylprednisolone acetate (MA). 6-months following this intradermal skin testing (IDST) was positive to MA and methylprednisolone sodium succinate, and IDST and patch test negative to other corticosteroids. She tolerated dexamethasone challenge. Cells were isolated from 4-mm punch biopsies from 48-hour positive IDST and unaffected skin with liberase digestion and sorted on CD3. scTCR and scRNA-seq were performed using plate-based assays (Smart-seq-2). Data was filtered using Seurat and analysed with Visual Genomics Analysis Studio (VGAS) software. Results Histopathology showed a superficial perivascular dermatitis with epidermal spongiosis in keeping with ACD. T-cells were 18x more prevalent in affected skin and those from both affected and unaffected sites were predominantly CD8+ T-cells that expressed CD45RO and polyclonal TCR alpha beta. Differentially expressed genes (DEG) in T-cells from affected skin reflected homing (CCR7,S1PR1), T-cell activation and proliferation (TNFRS9, GRAP2, ZYG11A, ZHX1), T-cell effector differentiation (IL-21R), and stress survival (EEF1A1, IFI6) without representation of markers of T-cell residency (ITGAE) or regulation. Conclusions We demonstrate insights into the immunopathogenesis of drug-induced ACD by showing that IDST+ methylprednisolone associated ACD is corticosteroid-specific and associated with a polyclonal population of primarily CD45RO+ memory CD8+ T-cells showing upregulation of markers of homing, T-cell activation, proliferation and differentiation but lacking markers of T-cell residency and regulation.

Lymphoproliferative Syndrome With Autoimmunity: A Possible Genetic Basis for Dominant Expression of the Clinical Manifestations

Fas (CD95/Apo-1) mutations were previously reported as the genetic defect responsible for human lymphoproliferative syndrome associated with autoimmune manifestations (also known as autoimmune lymphoproliferative syndrome or Canale-Smith syndrome). We have identified 14 new heterozygous Fas mutations. Analysis of patients and families allow us to further dissect this syndrome with regards to the relationship between Fas mutations, inheritance pattern, and phenotype as observed on long-term follow-up. In vitro studies show that lymphocytes from all Fas mutant carriers exhibit a Fas-antibody-induced apoptosis defect. However, among the 8 inherited mutations, 4 of 4 Fas missense mutations were associated with high clinical penetrance, whereas 3 of 4 mutations leading to a truncated Fas product were associated with variable clinical penetrance. This suggests that a second defect, in another yet undefined factor involved in apoptosis and/or lymphoproliferation control, is necessary to induce full clinical expression of the disease. These results also indicate that the currently available antibody-mediated in vitro apoptosis assay does not necessarily reflect the in vivo ability of abnormal Fas molecules to trigger lymphocyte death. In addition, we found that lymphoproliferative manifestations resolved with age, whereas immunological disorders [ie, hypergammaglobulinemia and detection of TcR alphabeta(+) CD4(-) CD8(-) lymphocytes] persisted. This observation suggests that Fas-mediated apoptosis plays a more important role in lymphocyte homeostasis in early childhood than later on in life.

Perforins Expression by Cutaneous Gamma Delta T Cells.

Gamma delta (GD) T cells are an unconventional T cell type present in both the epidermis and the dermis of human skin. They are critical to regulating skin inflammation, wound healing, and anti-microbial defense. Similar to CD8+ cytotoxic T cells expressing an alpha beta (AB) TCR, GD T cells have cytolytic capabilities. They play an important role in elimination of cutaneous tumors and virally infected cells and have also been implicated in pathogenicity of several autoimmune diseases. T cell cytotoxicity is associated with the expression of the pore forming protein Perforin. Perforin is an innate immune protein containing a membrane attack complex perforin-like (MACPF) domain and functions by forming pores in the membranes of target cells, which allow granzymes and reactive oxygen species to enter the cells and destroy them. Perforin-2, encoded by the gene MPEG1, is a newly discovered member of this protein family that is critical for clearance of intracellular bacteria. Cutaneous GD T cells express both Perforin and Perforin-2, but many questions remain regarding the role that these proteins play in GD T cell mediated cytotoxicity against tumors and bacterial pathogens. Here, we review what is known about Perforin expression by skin GD T cells and the mechanisms that contribute to Perforin activation.

Abstract 900: Intermittent cyclophosphamide and vinorelbine reshape the immune cell environment, induce T cell clonal replacement and increase the efficacy of PD-1 inhibition in models of triple negative breast cancer

Abstract The clinical success of cyclophosphamide (Cy)-based haploidentical stem cell transplants in hematological malignancies indicates that this drug has the potential to reorchestrate the immune system against cancer cells. Along a similar way, Cy is administered before the infusion of CAR-T cells to improve their clinical efficacy. We have previously found that daily, low/medium-dose Cy (20mg/Kg), in association with vinorelbine (V) was able to improve the preclinical efficacy of checkpoint inhibitors (CIs) anti-PD-1 and anti-PD-L1 in preclinical models of breast cancer and lymphoma (Orecchioni et al, 2018). Randomized clinical trials have recently indicated that the addition of CIs to other chemotherapy drugs such as taxanes (T), doxorubicin (D), or platinum (P) might be beneficial in triple negative breast cancer (TNBC) patients. Thus, we designed the present study in two orthotopic, immunocompetent, local and metastatic models of TNBC (CI-resistant 4T1 and EMT6, moderately responsive to CIs) to investigate whether Cy and V, in different schedules and doses, were more effective than T, D, or P. In vivo studies in local and metastatic models using both 4T1 and EMT6 TNBC cells clearly indicated that intermittent (ie every 6 days), medium-dosage (140 mg/Kg) Cy, was more effective than any other combinatorial regimens including CIs plus daily low/medium-dose Cy, or T, D, or P at their regular dosages. The association of V further increased the preclinical efficacy of intermittent Cy plus CIs, and abrogated tumor growth in both 4T1 and EMT6 models. In vivo studies with neutralizing monoclonal antibodies targeting in separate experiments T, B, NK, and myeloid cells demonstrated that CD3+CD4+, CD3+CD8+ T cells as well as CD11b+ monocyte/macrophage dendritic cells were crucial to abrogate tumor growth in TNBC-bearing mice treated with intermittent Cy, V and CIs. Single-cell transcriptome analysis of more than 60,000 intratumoral immune cells and flow cytometry studies in circulating and intratumoral subsets of immune cells indicated that V promoted the generation and maturation of myeloid APC cells, and that intermittent Cy generated new clones of tumor-infiltrating CD3+CD4+ and CD3+CD8+ TCR alpha beta cells. After treatment with intermittent Cy, V and CIs, intratumoral immune cells showed a unique gene signature of 42 genes (CD3D, Ptprc, CD69, Lat, Lck, CD2, CD28, CD27, and CD3E among others) suggestive of a change in morphology and behavior resulting from exposure to a mitogen, cytokine, chemokine, cellular ligand, or an antigen for which it is specific, as well as APC-to-T cell adhesion. Taken together, our data support the hypothesis that APC priming and T cell clonal replacement can significantly improve CI efficacy in vivo in TNBC models. We are further investigating the Cy-related mechanisms inducing intratumoral T-cell clonal replacement and how these T cells cooperate with V-induced APCs. Citation Format: Paolo Falvo, Stefania Orecchioni, Roman Hillje, Alessandro Raveane, Patrizia Mancuso, Chiara Camisaschi, Lucilla Luzi, Francesco Bertolini. Intermittent cyclophosphamide and vinorelbine reshape the immune cell environment, induce T cell clonal replacement and increase the efficacy of PD-1 inhibition in models of triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 900.

THU0501 EARLY DIAGNOSIS OF THE AUTOIMMUNE LYMPHOPROLIFERATIVE SYNDROME (ALPS) IN PATIENTS WITH UNDEFINED AUTOINFLAMMATORY OR AUTOIMMUNE DISORDERS: THE PRACTICAL ROLE OF A FLOW CYTOMETRY PANEL

Background:ALPS is a rare disorder due to a defective apoptotic mechanism leading to abnormal lymphoproliferation and autoimmunity. The disease is difficult to identify in the early phase when it may be misdiagnosed. Elevated TCR alpha-beta CD4-CD8- lymphocytes (double negative T lymphocytes DNT) together with hyperIgG, high levels of IL10, Il18, vitamin B12 and soluble Fas ligand have been suggested as the main ALPS hallmarks (1). Therefore, a specific flow cytometry panel (DNT cells, ratio of CD25+CD3+ to HLA−DR+CD3+ cells, increased B220+ T-cells, and decreased CD27+ memory B cells) has been proposed to serve as a diagnostic screen for ALPS (2).Objectives:To evaluate the usefulness of a specific lymphocyte flow cytometry panel in the early identification of ALPS/ALPS-like disorders in a cohort of patients with undefined autoinflammatory or autoimmune disorders.Methods:The clinical data of patients referred to the pediatric Rheumatology Unit of the Istituto Giannina Gaslini Hospital for a suspicion of autoimmune or autoinflammatory condition from October 2015 to April 2018, were retrospectively analyzed. Data on clinical manifestations, laboratory workup, genetic analysis and treatment were collected. Flow cytometry was included among the screening panel: DNT, CD25+CD3+, HLA−DR+CD3+ cells, B220+ T-cells, and CD27+ memory B cells were included. A statistical analysis was performed: data were analyzed with an univariate logistic regression analysis, to identify the most significant variables associated with ALPS. These variables were then included in a multivariate analysis to select a set of clinical and laboratory parameters, each of them associated with a significant probability to be associated with ALPS independently from other variables.Results:475 patients were retrospectively analized. 211 patients not fulfilling the inclusion criteria were excluded. The patients were classified as follows: i) Autoimmune disease 26 pts (10 SLE; 3 MCTD; 6 jDM; 5 Behçet; 1 SjS; 1 Kawasaki) ii) Juvenile Idiopathic Arthritis 35 pts iii) Monogenic systemic autoinflammatory disease (MSAID) 27 pts (17 FMF; 3 MKD; 1 TRAPS; 4 DADA2; 2 SAVI) iv) PFAPA 100 pts v) Systemic Undefined Recurrent Fever 45 pts vi) Undetermined-SAID 15 pts vii) ALPS/ ALPS probable 16 pts. The flow cytometry panel showed, as expected, an elevation of DNT in all ALPS patients. Among the other parameters, CD3CD25+/CD3HLADR+, and B220+ T cells, were significatively altered in 75% of ALPS patients. Conversely, B CD27+ did not differentiate ALPS from the other subgroups. The multivariate analysis revealed 5 clinical/laboratory parameters that showed the higher independent association to ALPS in the cohort of patients. Splenomegaly, female gender, elevated DNT, arthralgia and elevated alfabeta+B220+ lymphocytes were positively and significantly associated to ALPS.Conclusion:The use of the specific flow cytometry panel, comprehensive of DNT, B220+, HLA-DR and CD25, in patients with undefined autoinflammatory or autoimmune disorders may identify a subgroup of patients with ALPS.

Innate-like CD8 T cells in the teleost olfactory-CNS axis express NK cell markers and mount rapid polyclonal responses to viral antigens

Abstract T lymphocytes can perform innate immune functions and display tissue specificity. Wheres innate T cells have been extensively studied in mammals, little is known about their presence and function in other vertebrates. Previous work in our laboratory identified a subset of TCRalpha beta T cells that rapidly infiltrate the olfactory organ of rainbow trout upon nasal vaccination. This rapid response suggested that these lymphocytes play important functions in mucosal innate immune responses. Here we report that CD8 T cells rapidly infiltrate the olfactory organ of rainbow trout not only in response to nasal viral immunization but also in response to other nasal microbial immunizations including killed and live bacteria. Nasal delivery of PAMPS such as LPS or poly I:C did not elicit these responses. Further phenotyping of these cells using qPCR and FISH staining indicate that these innate lymphocytes are NK T cells since they express the teleost NK cell markers NCCRP1 and NITR2. Repertoire analyses of infiltrating CD8 T cells showed a public but polyclonal response with 14 amino acid long CDR3 in response to the virus. Our findings represent the first characterization of NK T cells in early vertebrates and underscore the importance of NK T cells at defending the mucosal barriers of vertebrate animals.

Comparison of two T-cell depletion methods of grafts from alternative donors for allogeneic hematopoietic cell transplantation of patients with fanconi anemia.

Background & Aim Our program has used 2 T-cell depleted HSCT approaches for patients with FA for transplants from donors other than HLA matched siblings: 1) the Isolex 300i Magnetic Cell Selection System (Baxter) + sheep RBC depletion (E-) (TCD1) was used to achieve an approximate 5 log10 depletion of CD3+ cells, and (2) the CliniMACS device (Miltenyi; TCD2) was used as a sole TCD method with an approximate 4.5 log10 depletion of CD3+cells. Methods, Results & Conclusion We retrospectively reviewed charts of consecutive patients with FA who received a first HCT from alternative donors. We identified 42 patients with aplastic anemia, MDS or AML, 24 who received TCD1 (1999-2009) and 18 who received TCD2 (2010-present). Most patients in TCD1 received a TBI-based conditioning, while most in TCD2 received busulfan-based conditioning. Donors were matched unrelated, mismatched unrelated, or mismatched related. Because of the differences in the treatment periods, conditioning regimens, and disease breakdown, we analyzed the data specifically related to the T-cell depletion. Distributions of continuous variables were compared using a Mann-Whitney-Wilcoxon test, while binary and categorical variables were compared using a Fisher's exact test. Grafts were G-CSF mobilized peripheral blood progenitor cells. T-cell depletions were performed as described. Table 1 provides demographics, cell doses and hematologic recovery data for TCD1 and TCD2. Median CD34 cell doses of the grafts did not differ significantly while median CD3 dose did (N=0.048). All but one patient engrafted. Two patients receiving grafts from mismatched unrelated donors developed grade 2-4 acute GvHD in TCD1, none in TCD2. Neutrophil engraftment did not significantly differ while platelet engraftment was more rapid with TCD1 (N=0.0045). CD4 recovery (Abs CD4 >200) and PHA recovery (50th%ile) differences were comparable (NS). In conclusion, no difference were noted between the Isolex E- T-cell depletion and the CliniMACS CD34 selection system, except for a more rapid platelet reconstitution for the CliniMACS column. There was no difference in risks of graft rejection and GvHD, and immune reconstitution was comparable in both cohorts. Future plans include the comparison of these methods to the TCR alpha beta depletion in this patient population.

A novel method for high throughput TCR single cell VDJ-pairing with phenotypic analysis

Abstract Single cell transcriptional analysis reveals a wealth of information that is obscured when analyzing the RNA of a sample en masse. Furthermore, it is important to know the identity of the TCR chain VDJ-rearrangement associated with the phenotypic state. This information can provide direct calculations of clonal frequency in various cell subsets, tracking of specific lymphocytes with treatment, and reveal paired information for both chains of the receptor for downstream Car-T development. Here, we discuss a novel method that identifies paired VDJ-rearrangements for both TCR alpha and beta chains from single cells barcoded via the BD Rhapsody Express single cell system. The phenotype of the cell is obtained using the BD Rhapsody RNA-seq kit, while the receptor information is amplified from the same cDNA using an iRepertoire-derived method incorporating a multiplex mix of primers associated with both the TCR alpha and beta loci. This targeted-seq strategy requires less read depth when compared to template-switch approaches. In one experiment, 3,000 TCR-beta unique CDR3 (uCDR3) were identified from ~7,000 barcoded cells using 1.5 million NGS reads with a 0.83 Pearson correlation upon repeat. Approximately 1,000 TCR alpha-beta paired uCDR3 were also called from the data. Additionally, the BD phenotyping results independently verify the immune repertoire data since the TCR beta chain constant gene overlays with the TCR-beta uCDR3 clonotypes for CD4+ and CD8+ subsets but is largely absent in the CD14+ monocyte population for both analyses. Thus, the developed technology provides the ability to simultaneously pair both alpha and beta chains for thousands of T-cells while obtaining information related to the functional status of each cell.

HLA-Haploidentical Hematopoietic Cell Transplantation after TCR-Alpha Beta and CD45RA+ Depletion Following Reduced Intensity Conditioning in Adults and Children with Hematological Malignancies

▪Background: Haploidentical (Haplo) hematopoietic cell transplantation (HCT) provides an alternative option for patients without HLA-matched donor. GVHD, engraftment failure, and infectious complications continue to be the main causes of non-relapse mortality (NRM). We hypothesized that selective depletion of TCRαβ+ and CD45RA+ naïve T cells subset will permit hematopoietic engraftment, while effectively reducing GVHD, and provide improved donor immune reconstitution through adoptive transfer of donor's mature NK and γδ T cells and CD45RA- memory T cells.Methods: GCSF +/- plarixefor (N=13) mobilized PBSC apheresis product were divided into two fractions in 9:1 ratio, and depleted using CliniMACS device after labeling with TCRαβ and CD45RA reagents (Miltenyi Biotec, Bergish-Gladbach, Germany), respectively. The conditioning regimen consisted of total nodal irradiation 6 - 7.5 Gy over 3 equal fractions, 160 mg/m2 Fludarabine divided daily over 4 days, Thiotepa 10 mg/kg divided twice daily for 1 day and Melphalan 70-140 mg/m2 for 1 day. Short term GVHD prophylaxis for 30 days was given to 1 patient using MMF, 11 using tacrolimus or 2 using Sirolimus.Results: We transplanted 20 adults (median age 42 years, range 20 - 68 ) and 3 children (age range 7-14 years) with high risk AML (n=14), ALL (n=6), MDS (n=2), myeloma (N=1). The patients were infused with TCRαβ and CD45RA depleted graft containing a median of 9.94 x 106 (range, 5.58 - 20.78) CD34+ cells/kg, 0.19 x 104 (range 0 - 8.53) CD45RA+CD3+ cells/kg, and 4.21 x 106 (range, 0.15 - 11.67) CD45RO+CD3+ cells/kg. In addition, the TCRαβ depleted graft fraction contained a median of 0.19 x 104 (range 0 - 8.53) TCRαβ+ cells/kg, and 8.87 x 106 (range 1.73 - 29.74) TCRγδ + cells/kg. All patients except 1 had engraftment of ANC > 500 cells/µL at a median of 11 day (range, 8 - 19) and PLT > 20,000 cells/µL at a median of 12 day (range, 7 - 19). Complete donor chimerism was seen at the time of engraftment in all patients with primary engraftment. Only 1 patient experienced primary graft failure. There was no secondary graft failure.Eleven patients developed acute GVHD of grade II-IV, with the day 100 cumulative incidence (C.I.) of acute GVHD of Gd II-IV, and Gd III-IV of 50% (95% CI 27.3 - 69.0%) and 17.9 % (95% CI 5.3- 36.4%), respectively. One patient experienced chronic GVHD. Day 180 C.I. of NRM and relapse were 23.9 % (95% 8.3-43.9%) and 5.6 % (95% CI 0.3-23.3%), respectively. NRM was attributed to aGVHD in 3 of the 6 deaths. Viral reactivation included CMV (n=7), HHV6 (N=4), EBV (N=3) and adenovirus (N=2), with no fatal viral infection occurred within 100 days. Two patients had fatal blood stream infection within 100 days. With a median follow up of 306 days (range 18 - 551 days) in surviving patients, the 6 month and 1 year overall survival (OS) were 76.9% (95% CI 52.9-89.7%) and 71 % (95% CI 46-85.9%), respectively. (Figure 1).Conclusions: Our preliminary results suggest that RIC haplo-HCT with TCRαβ and CD45RA+ depleted grafts allows successful allograft in high-risk patients lacking a suitable matched donor. NRM due to GVHD remains the major obstacle for the successful HCT. Fine adjustment of T cell dose with short course of immunosuppressant may reduce GVHD while preserving antitumor and antimicrobial immunocompetence. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PO-391 High-throughput single-cell T-cell receptor profiling by SMART technology

IntroductionSingle-cell T-cell receptor (scTCR) clonotype analysis permits the determination of the specific TCR alpha-beta (α/β) chain pairing expressed on each cell. This pairing information allows researchers to gain insight into Tcell heterogeneity and plasticity, determine the contribution of the pairing to antigen specificity of the individual TCR, and design therapeutic antibodies. Here we employ a 5’-RACE-like approach and SMART technology, in conjunction with two novel next-generation sequencing (NGS) library preparation kits, using the same primer pairs, to capture full-length variable regions of TCR-α and -β chains.Material and methodsMethod 1, using the SMARTer Human scTCRa/b Profiling Kit, permits NGS library preparation of FACS-sorted cells in 96-well plates. Method 2 is an adaptation of the process above that scales to ~1200 single cells using the SMARTer ICELL8 Single-Cell System, which enables single-cell isolation and nanoliter PCR in a nanowell chip.Results and discussionsWe present data showing α/β pairing from Jurkat, CCRF, PBMCs, and CD4 +T cells. In addition to the sensitivity of this method, the ability to pool the cDNA from 96wells into 12 sequencing libraries adds to the ease of use. Consistent with immunology reports, unstimulated CCRF-CEM cells examined with this kit expressed a TCR-β but not a TCR-α chain.scTCR pofiling with SMARTer ICELL8 Single-Cell System as a proof of principle of scTCRα/β was performed on Jurkat cells and CCRF-CEM cells.Jurkat cells and CCRF-CEM cells were processed using an ICELL8 chip preprinted with barcoded oligos. Paired TCRα/β Jurkat clonotypes were detected in 77% and 87% cells in mixed and single cell populations, respectively.ConclusionThe ability of the core biochemistry and PCR components of these kits to be used with either FACS-sorted cells in 96-well plates or >1,000 cells in novel ICELL8 chips (in development) points to the general utility and scalability of this approach in understanding paired scTCR clonotype diversity.

Identification of paired heavy and light chains from single B-cells from immunized Malian adults with rapid functional confirmation using iPair-BCR™, NGS, and iScreen™

Abstract Information about the cognate pairing of TCR alpha-beta chains and BCR IgH and IgKL chains encoded by individual T and B cells is key to understanding adaptive immune responses and developing therapeutic applications. We have previously reported the development of a sensitive technology that allows the amplification and identification of the paired human TCR alpha and beta chains from single T cells, termed iPair-TCR. Here, we report the extension of this technology to identify paired human BCR IgH and IgKL chains from antigen-specific single B cells. In this proof of concept study, we identified paired-VDJ-receptors from antigen-specific B-cells from nine Pfs230-EPA immunized Malian adults using the iPair-BCR method. Next, we developed a method to rapidly generate Fab fragments and demonstrate the binding of several of these single cell targets to the original Pfs230 antigen. Single cells of interest were identified based upon their repeated frequency on the plate, which indicates a clonal selection. The corresponding heavy and light chains were PCR amplified from selected wells. Using overlap extension PCR, all necessary elements for in vitro transcription and translation and either the CH1 or C-kappa-domain were added to both the 5′ and 3′ ends of the single cell VDJ. After in vitro transcription and translation, four out of five tested Fab fragments demonstrated binding through a colorimetric ELISA assay. The overall process after VDJ identification can be performed in under a week indicating the utility of our technology for rapid identification of antigen-specific BCRs and functional binding characteristics.

A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies.

Adoptive T cell therapy using TCR transgenic autologous T cells has shown great potential for the treatment of tumor patients. Thorough characterization of genetically reprogrammed T cells is necessary to optimize treatment success. Here, we describe the generation of triple parameter reporter T cells based on the Jurkat 76 T cell line for the evaluation of TCR and chimeric antigen receptor functions as well as adoptive T cell strategies. This Jurkat subline is devoid of endogenous TCR alpha and TCR beta chains, thereby circumventing the problem of TCR miss-pairing and unexpected specificities. The resultant reporter cells allow simultaneous determination of the activity of the transcription factors NF-κB, NFAT and AP-1 that play key roles in T cell activation. Human TCRs directed against tumor and virus antigens were introduced and reporter responses were determined using tumor cell lines endogenously expressing the antigens of interest or via addition of antigenic peptides. Finally, we demonstrate that coexpression of adhesion molecules like CD2 and CD226 as well as CD28 chimeric receptors represents an effective strategy to augment the response of TCR-transgenic reporters to cells presenting cognate antigens.

Abstract 625: Eradication of cancer cells by T-cell receptor-engineered T cells targeting neoantigens/oncoantigens

Abstract Cytotoxic T lymphocytes (CTLs) play critical roles in cancer-immune responses, and functional characterization of CTLs and their cancer-specific antigens will facilitate cancer immunotherapies. Immunogenic peptides, which can be derived from oncogenic proteins specifically expressed in cancer cells but not expressed in normal organs except testis (oncoantigens), or from peptides with somatic nonsynonymous mutations (neoantigens), are known as good targets for CTLs to eradicate cancer cells. In this study, we aimed to establish a method to efficiently identify oncoantigen/neoantigen-specific CTLs. Firstly, we screened candidate HLA-A2402-restricted oncoantigen/neoantigen peptides by in silico prediction of their binding affinity to MHC class I molecules. We conducted an in-vitro stimulation of CD8 lymphocytes carrying HLA-A24:02 allele by each peptide, and then confirmed clonal expansion of the peptide-specific CTLs by TCR repertoire sequencing analysis, interferon-γ enzyme-linked immunospot (ELISPOT) and/or peptide-HLA multimer assays. After identification of TCR alpha-beta pairs, we conducted retroviral transduction and prepared the TCR-engineered T cells to evaluate their cytotoxic activities against cancer cells. As oncoantigens, we isolated the CTLs for FOXM1 and UBE2T from healthy donors, and found these CTLs showed strong cytotoxicity against HLA-A2402-positive cancer cells expressing target proteins, but not against HLA-unmatched cancer cells. Similarly, the TCR-engineered T cells for FOXM1 and UBE2T showed killing effects for only HLA-A2402-positive cancer cells. Neoantigen-specific TCR-engineered CTLs also exhibited the mutated peptide-specific response, but did not cross-react to the nonmutated peptide. In addition, neoantigen-specific cytotoxicity was observed against HLA-A2402-positive cancer cells expressing the proteins with target somatic mutations. In conclusion, we developed the pipeline to screen possible onconatigens/neoantigens and establish antigen-specific TCR-engineered CTLs from peripheral blood lymphocytes. Our approach provides a promising strategy to develop personalized immunotherapies using onconatigen/neoantigen-reactive TCR-engineered T cells to treat cancer. Citation Format: Tatsuo Matsuda, Taigo Kato, Yuji Ikeda, Matthias Leisegang, Sachiko Yoshimura, Tetsuro Hikichi, Makiko Harada, Makda Zewde, Jae-Hyun Park, Hans Schreiber, Kazuma Kiyotani, Yusuke Nakamura. Eradication of cancer cells by T-cell receptor-engineered T cells targeting neoantigens/oncoantigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2017-625

BPX-501 Cells (Donor T Cells Transduced with iC9 Suicide Gene) Treatment Following TCR Alpha Beta Depleted Stem Cell Transplantation in Adults and Children with Hematological Disorders

BPX-501 Cells (Donor T Cells Transduced with iC9 Suicide Gene) Treatment Following TCR Alpha Beta Depleted Stem Cell Transplantation in Adults and Children with Hematological Disorders