Αβ T-cell Receptor Research Articles

CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells

AbstractAdoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-β is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αβ and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR–modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer–reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4+ and CD8+ T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies.

Advances in NKT cell Immunotherapy for Glioblastoma

Type I or invariant natural killer T cells belong to a unique lineage of innate T cells, which express markers of both T lymphocytes and NK cells, namely T cell receptor (TCR) and NK1.1 (CD161C), respectively. Thus, apart from direct killing of target cells like NK cells, and they also produce a myriad of cytokines which modulate the adaptive immune responses. Unlike traditional T cells which carry a conventional αβ TCR, NKT cells express semi-invariant TCR – Vα14-Jα18, coupled with Vβ8, Vβ7 and Vβ2 in mice. In humans, the invariant TCR is composed of Vα24-Jα18, coupled with Vβ11.

Staged development of long-lived T-cell receptor αβ TH17 resident memory T-cell population to Candida albicans after skin infection

Candida albicans is a dimorphic fungus to which human subjects are exposed early in life, and by adulthood, it is part of the mycobiome of skin and other tissues. Neonatal skin lacks resident memory T (TRM) cells, but in adults the Calbicans skin test is a surrogate for immunocompetence. Young adult mice raised under specific pathogen-free conditions are naive to C albicans and have been shown recently to have an immune system resembling that of neonatal human subjects. We studied the evolution of the adaptive cutaneous immune response to Candida species. We examined both human skin T cells and the de novo and memory immune responses in a mouse model of C albicans skin infection. In mice the initial IL-17-producing cells after C albicans infection were dermal γδ T cells, but by day 7, αβ TH17 effector T cells were predominant. By day 30, the majority of C albicans-reactive IL-17-producing T cells were CD4 TRM cells. Intravital microscopy showed that CD4 effector T cells were recruited to the site of primary infection and were highly motile 10days after infection. Between 30 and 90days after infection, these CD4 T cells became increasingly sessile, acquired expression of CD69 and CD103, and localized to the papillary dermis. These established TRM cells produced IL-17 on challenge, whereas motile migratory memory T cells did not. TRM cells rapidly clear an infectious challenge with C albicans more effectively than recirculating T cells, although both populations participate. We found that in normal human skin IL-17-producing CD4+ TRM cells that responded to C albicans in an MHC class II-restricted fashion could be identified readily. These studies demonstrate that C albicans infection of skin preferentially generates CD4+ IL-17-producing TRM cells, which mediate durable protective immunity.

The Quest for Psoriasis Autoantigens: Genetics Meets Immunology in the Melanocyte

The Quest for Psoriasis Autoantigens: Genetics Meets Immunology in the Melanocyte

Abstract P347: γδ T Cells Drive CD4 + And CD8 + T Cell Activation In Hypertension

Objective: Both innate (monocyte/macrophages) and adaptive immune cells (T lymphocytes) have been shown to play a role in the development of vascular injury in hypertension. Recently, we demonstrated that a small subset of “innate-like” T lymphocytes, expressing the γ/δ T cell receptor (TCR) rather than the αβ TCR, plays a key role in hypertension and vascular injury. We demonstrated an increased number and activation (CD69 + ) of γδ T cells during the development of hypertension caused by angiotensin (Ang) II infusion, and that deficiency in γδ T cells prevented Ang II-induced hypertension, resistance artery endothelial dysfunction and spleen T-cell activation in mice. We hypothesized that γδ T cells mediate activation of other T cells in hypertension. Method and Results: Fourteen to 15-week old male C57BL/6 wild-type (WT) mice were infused with Ang II (490 ng/kg/min, SC) for 3, 7 and 14 days (n=5-7) and spleen T cell profile was determined by flow cytometry. A correlation was demonstrated between the frequency (FREQ) and the number (#) of activated CD69 + γδ T cells and CD4 + CD69 + T cells (FREQ: r=0.41, P <0.05 and #: r=0.58, P <0.001) and CD8 + CD69 + T cells (FREQ: r=0.36, P <0.05 and #: r=0.50, P <0.01). We also demonstrated a high correlation between the # of CD69 + γδ T cells expressing CD27, a marker of interferon-γ expressing cells and a member of the T-T interaction molecules, with CD4 + CD69 + (r=0.88, P <0.001) and CD8 + CD69 + (r=0.81, P <0.01) T cells after 7 days of Ang II infusion. Conclusion: This study demonstrated an association between CD27 + CD69 + γδ T cells and activated T cells. These results suggest that γδ T cells drive activation of other T cells in Ang II-induced hypertension. Targeting γδ T cells may contribute to reduce inflammation in hypertension.

PTPN2 regulates T cell lineage commitment and αβ versus γδ specification.

In the thymus, hematopoietic progenitors commit to the T cell lineage and undergo sequential differentiation to generate diverse T cell subsets, including major histocompatibility complex (MHC)-restricted αβ T cell receptor (TCR) T cells and non-MHC-restricted γδ TCR T cells. The factors controlling precursor commitment and their subsequent maturation and specification into αβ TCR versus γδ TCR T cells remain unclear. Here, we show that the tyrosine phosphatase PTPN2 attenuates STAT5 (signal transducer and activator of transcription 5) signaling to regulate T cell lineage commitment and SRC family kinase LCK and STAT5 signaling to regulate αβ TCR versus γδ TCR T cell development. Our findings identify PTPN2 as an important regulator of critical checkpoints that dictate the commitment of multipotent precursors to the T cell lineage and their subsequent maturation into αβ TCR or γδ TCR T cells.

T-cell receptor αβ+ and CD19+ cell–depleted haploidentical and mismatched hematopoietic stem cell transplantation in primary immune deficiency

Allogeneic hematopoietic stem cell transplantation (HSCT) is used as a therapeutic approach for primary immunodeficiencies (PIDs). The best outcomes have been achieved with HLA-matched donors, but when a matched donor is not available, a haploidentical or mismatched unrelated donor (mMUD) can be useful. Various strategies are used to mitigate the risk of graft-versus-host disease (GvHD) and rejection associated with such transplants. We sought to evaluate the outcomes of haploidentical or mMUD HSCT after depleting GvHD-causing T-cell receptor (TCR) αβ CD3+ cells from the graft. CD3+TCRαβ+/CD19+ depleted grafts were given in conditioned (except 3) children with PIDs. Treosulfan (busulfan in 1 patient), fludarabine, thiotepa, and anti-thymocyte globulin or alemtuzumab conditioning were used in 77% of cases, and all but 4 received GvHD prophylaxis. Twenty-five patients with 12 types of PIDs received 26 HSCTs. Three underwent transplantation for refractory GvHD that developed after the first cord transplantation. At a median follow-up of 20.8months (range, 5 month-3.3years), 21 of 25 patients survived and were cured of underlying immunodeficiency. Overall and event-free survival at 3years were 83.9% and 80.4%, respectively. Cumulative incidence of grade II to IV acute GvHD was 22%±8.7%. No case of visceral or chronic GvHD was seen. Cumulative incidences of graft failure, cytomegalovirus, and/or adenoviral infections and transplant-related mortality at 1year were 4.2%±4.1%, 58.8%±9.8%, and 16.1%±7.4%, respectively. Patients undergoing transplantation with systemic viral infections had poor survival in comparison with those with absent or resolved infections (33.3% vs 100%). CD3+TCRαβ+ and CD19+ cell-depleted haploidentical or mMUD HSCT is a practical and viable alternative for children with a range of PIDs.

Abstract 3745: Dual-switch TCR: A two-ligand system to control PRAME TCR-modified T cell proliferation and death using inducible MyD88/CD40 and caspase-9

Abstract T cells engineered to express the α and β chains of antigen-specific T cell receptors (TCRs) have shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor persistence of gene-modified T cells. Additionally, severe toxicities, including patient deaths, have occurred upon infusion of large numbers of TCR-modified T cells. To enhance T cell persistence while providing a safeguard against life-threatening toxicity, we developed a dual-switch αβ TCR platform that uses a rapamycin (Rap)-induced caspase-9 (iRC9) together with a rimiducid (Rim)-controlled activation switch, inducible MyD88/CD40 (iMC). The αβ TCR sequence derived from an HLA-A2-restricted, PRAME-specific T cell clone was synthesized and placed in-frame with iMC, comprising signaling domains from MyD88 and CD40 fused to tandem Rim-binding mutant FKBP12v36 domains to generate the iMC-PRAME TCR. Caspase-9 was fused to FRB and wild-type FKBP12 domains and cloned in-frame with a selectable marker, truncated CD19 (ΔCD19), to generate iRC9-ΔCD19 retrovirus. All modules were separated by 2A polypeptide, bond-skipping sequences. Activated human T cells were dual-transduced with iMC-PRAME TCR and iRC9-ΔCD19 viruses and subsequently enriched for CD19 expression using magnetic selection. iMC and iRC9 were activated by exposing transduced T cells to 10 nM Rim or Rap, respectively. Proliferation, cytokine production and cytotoxicity of TCR-modified T cells were assessed in co-culture assays with U266 (myeloma) and THP-1 (AML) cells in the presence or absence of inducible ligands. T cells transduced with iMC-PRAME TCR and iRC9-ΔCD19 showed efficient and stable expression for TCR and ΔCD19 post-CD19 selection (82±9% CD3+Vβ1+, 96±2% CD3+CD19+). In coculture assays, dual-switch PRAME TCR-modified T cells demonstrated specific lysis of HLA-A2+PRAME+ THP-1 and U266 tumor cells compared to an irrelevant TCR (CMVpp65-specific)-modified T cells with or without iMC activation. However, Rim exposure induced a 42-fold induction of IL-2 (9±0.3 versus 385±180 pg/ml IL-2) and resulted in 13-fold greater expansion of TCR-modified T cells. The expression of iRC9 did not interfere with TCR function, nor with the synergy between TCR and iMC activation. Furthermore, exposure to Rap triggered rapid apoptosis of dual-switch TCR-modified T cells (72±5% Annexin-V+ with Rap versus 14±4% without drug), indicating that the suicide switch is also functional. iMC is a potent “Go” switch that utilizes rimiducid to provide costimulation to TCR-engineered T cells. In addition, iRC9 is a novel rapamycin-inducible suicide switch that can eliminate T cells in case of severe toxicity. This iMC-enhanced iRC9-incorporating TCR is the first reported prototype of a dual switch TCR-engineered T cell therapy to increase efficacy, durability and safety of adoptive T cell therapies. Citation Format: Tsvetelina Pentcheva-Hoang, Tania Rodriguez, David Torres, Ana Korngold, Jeannette Crisostomo, MyLinh T. Duong, Matthew Collinson-Pautz, J. Henri Bayle, Mirjam HM Heemskerk, J.H. Frederik Falkenburg, Annemarie Moseley, Kevin M. Slawin, David Spencer, Aaron E. Foster. Dual-switch TCR: A two-ligand system to control PRAME TCR-modified T cell proliferation and death using inducible MyD88/CD40 and caspase-9 [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 3745. doi:10.1158/1538-7445.AM2017-3745

Immunosurveillance by human γδ T lymphocytes: the emerging role of butyrophilins.

In contrast to conventional T lymphocytes, which carry an αβ T-cell receptor and recognize antigens as peptides presented by major histocompatibility complex class I or class II molecules, human γδ T cells recognize different metabolites such as non-peptidic pyrophosphate molecules that are secreted by microbes or overproduced by tumor cells. Hence, γδ T cells play a role in immunosurveillance of infection and cellular transformation. Until recently, it has been unknown how the γδ T-cell receptor senses such pyrophosphates in the absence of known antigen-presenting molecules. Recent studies from several groups have identified a unique role of butyrophilin (BTN) protein family members in this process, notably of BTN3A1. BTNs are a large family of transmembrane proteins with diverse functions in lipid secretion and innate and adaptive immunity. Here we discuss current models of how BTN molecules regulate γδ T-cell activation. We also address the implications of these recent findings on the design of novel immunotherapeutic strategies based on the activation of γδ T cells.

An allosteric site in the T-cell receptor C\u03b2 domain plays a critical signalling role

The molecular mechanism through which the interaction of a clonotypic αβ T-cell receptor (TCR) with a peptide-loaded major histocompatibility complex (p/MHC) leads to T-cell activation is not yet fully understood. Here we exploit a high-affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd). In addition to conformational changes in complementarity-determining regions (CDRs) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR β-chain dynamics reveals significant chemical shift effects in sites removed from the MHC-binding site. Remodelling of electrostatic interactions near the Cβ H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests a possible role for an allosteric mechanism in TCR signalling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T-cell functional assays.

Primary cutaneous aggressive epidermotropic cytotoxic T-cell lymphomas: reappraisal of a provisional entity in the 2016 WHO classification of cutaneous lymphomas

Primary cutaneous CD8-positive aggressive epidermotropic T-cell lymphoma is a rare and poorly characterized variant of cutaneous lymphoma still considered a provisional entity in the latest 2016 World Health Organization Classification of Cutaneous lymphomas. We sought to better characterize and provide diagnostic and therapeutic guidance of this rare cutaneous lymphoma. Thirty-four patients with a median age of 77 years (range 19–89 years) presented primarily with extensive annular necrotic plaques or tumor lesions with frequent mucous membrane involvement. The 5-year survival was 32% with a median survival of 12 months. A subset of 17 patients had a prodrome of chronic patches prior to the development of aggressive ulcerative lesions. We identified cases with lack of CD8 or αβ T-cell receptor expression yet with similar clinical and pathological presentation. Allogeneic stem cell transplantation provided partial or complete remissions in 5/6 patients. We recommend the term primary cutaneous aggressive epidermotropic cytotoxic T-cell lymphoma as this more broad designation better describes this clinical-pathologic presentation, which allows the inclusion of cases with CD8 negative and/or αβ/γδ T-cell receptor chain double-positive or double-negative expression. We have identified early skin signs of chronic patch/plaque lesions that are often misdiagnosed as eczema, psoriasis, or mycosis fungoides. Our experience confirms the poor prognosis of this entity and highlights the inefficacy of our standard therapies with the exception of allogeneic stem cell transplantation in selected cases.

γδ T Cells Mediate Angiotensin II-Induced Hypertension and Vascular Injury.

Innate antigen-presenting cells and adaptive immune T cells have been implicated in the development of hypertension. However, the T-lymphocyte subsets involved in the pathophysiology of hypertension remain unclear. A small subset of innate-like T cells expressing the γδ T cell receptor (TCR) rather than the αβ TCR could play a role in the initiation of the immune response in hypertension. We aimed to determine whether angiotensin (Ang) II caused kinetic changes in γδ T cells; deficiency in γδ T cells blunted Ang II-induced hypertension, vascular injury, and T-cell activation; and γδ T cells are associated with human hypertension. Male C57BL/6 wild-type and Tcrδ-/- mice, which are devoid of γδ T cells, or wild-type mice injected IP with control isotype IgG or γδ T cell-depleting antibodies, were infused or not with Ang II for 3, 7, or 14 days. T-cell profiling was determined by flow cytometry, systolic blood pressure (SBP) by telemetry, and mesentery artery endothelial function by pressurized myography. TCR γ constant region gene expression levels and clinical data of a whole blood gene expression microarray study, including normotensive and hypertensive subjects, were used to demonstrate an association between γδ T cells and SBP. Seven- and 14-day Ang II infusion increased γδ T-cell numbers and activation in the spleen of wild-type mice (P<0.05). Fourteen days of Ang II infusion increased SBP (P<0.01) and decreased mesenteric artery endothelial function (P<0.01) in wild-type mice, both of which were abrogated in Tcrδ-/- mice (P<0.01). Anti-TCRγδ antibody-induced γδ T-cell depletion blunted Ang II-induced SBP rise and endothelial dysfunction (P<0.05), compared with isotype antibody-treated Ang II-infused mice. Ang II-induced T-cell activation in the spleen and perivascular adipose tissue was blunted in Tcrδ-/- mice (P<0.01). In humans, the association between SBP and γδ T cells was demonstrated by a multiple linear regression model integrating whole blood TCR γ constant region gene expression levels and age and sex (R2=0.12, P<1×10-6). γδ T cells mediate Ang II-induced SBP elevation, vascular injury, and T-cell activation in mice. γδ T cells might contribute to the development of hypertension in humans.

An Epstein-Barr virus susceptible immature T-cell line, WILL4, established from a patient with T-lymphoblastic lymphoma bearing CD21 and a clonal EBV genome

We managed a patient with an Epstein-Barr virus-associated T-cell lymphoblastic lymphoma. Mediastinal tumor cells at initial admission were positive for CD4, CD8, and TdT. Interestingly, a lymph node at necropsy was compatible for a CD4-positive peripheral T-cell lymphoma without CD8 and TdT expression, suggesting a different phenotype from the mediastinal tumor. Tumor cells in pleural effusion continued to proliferate in in vitro and were designated as WILL4. WILL4 cells were positive for CD3, CD4, CD8, CD21, T-cell receptor (TcR) αβ, and TdT, indicating a similar phenotype to thymocytes. Southern blot analyses showed that the pleural tumor and WILL4 cells shared a TcR gene rearrangement, and that both contained a clonal EBV genome in an episomal form. RT-PCR showed that EBNA1 and LMP1 were expressed in the fresh tumor and WILL4 cells. Southern blot analyses revealed that WILL4 cells were susceptible to EBV infection in vitro using B95-8 supernatant. Anti-CD21 antibody inhibited in vitro infection of EBV, suggesting that CD21 plays a role in EBV infection into WILL4 cells. In vitro infection of EBV did not affect latent gene expression in WILL4 cells. WILL4 is a useful tool for analyzing the roles of EBV in onocogenesis in immature T-lymphoid malignancies.

Dermal γδ T Cells Do Not Freely Re-Circulate Out of Skin and Produce IL-17 to Promote Neutrophil Infiltration during Primary Contact Hypersensitivity.

The role of mouse dermal γδ T cells in inflammatory skin disorders and host defense has been studied extensively. It is known that dendritic epidermal T cells (DETC) have a monomorphic γδ T cell receptor (TCR) and reside in murine epidermis from birth. We asked if dermal γδ cells freely re-circulated out of skin, or behaved more like dermal resident memory T cells (TRM) in mice. We found that, unlike epidermal γδ T cells (DETC), dermal γδ cells are not homogeneous with regard to TCR, express the tissue resident T cell markers CD69 and CD103, bear skin homing receptors, and produce IL-17 and IL-22. We created GFP+: GFP− parabiotic mice and found that dermal γδ T cells re-circulate very slowly—more rapidly than authentic αβ TCR TRM, but more slowly than the recently described dermal αβ TCR T migratory memory cells (TMM). Mice lacking the TCR δ gene (δ-/-) had a significant reduction of 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS). We created mice deficient in dermal γδ T cells but not DETC, and these mice also showed a markedly reduced CHS response after DNFB challenge. The infiltration of effector T cells during CHS was not reduced in dermal γδ T cell-deficient mice; however, infiltration of Gr-1+CD11b+ neutrophils, as well as ear swelling, was reduced significantly. We next depleted Gr-1+ neutrophils in vivo, and demonstrated that neutrophils are required for ear swelling, the accepted metric for a CHS response. Depletion of IL-17-producing dermal Vγ4+ cells and neutralization of IL-17 in vivo, respectively, also led to a significantly reduced CHS response and diminished neutrophil infiltration. Our findings here suggest that dermal γδ T cells have an intermediate phenotype of T cell residence, and play an important role in primary CHS through producing IL-17 to promote neutrophil infiltration.

CD20-Positive T-Cell Large Granular Lymphocytic Leukemia: A Case Report and Literature Review

T-cell large granular lymphocytic leukemia (T-cell LGLL) is a rare disorder characterized by the monoclonal expansion of CD3-positive cytotoxic T cells. Cell morphology and immunophenotyping are the main tools for diagnosis. Typical phenotypic findings include the expression of markers CD3+, CD5+dim, CD8+, CD16+, CD57+, and T-cell receptor (TCR) αβ (+). However, a few cases may present with the CD3+/CD4+ phenotype or the double expression of CD4 and CD8. Similarly, in 20% of cases, TCRγ/δ is positive. In this study, we aimed to describe a rare, unusual case of T-cell LGLL that exhibited expression of the B-cell antigen, CD20. Additionally, we performed a literature review to compare the clinical characteristics of our patient to those of other patients with CD20-positive T-cell LGLL and to seek new therapeutic possibilities.

HLA-Haploidentical TCR αβ/CD19-Depleted Hematopoietic Stem Cell Transplantation in Children with Fanconi Anemia

Introduction. Allogeneic HSCT currently represents the only consolidated curative approach for Fanconi anemia (FA) patients, with best results observed in the HLA-identical sibling setting. For patients lacking an HLA-matched related or unrelated donor, haploidentical HSCT virtually assures the opportunity for nearly all patients to benefit from HSCT, offering the advantage of immediate accessibility to the transplant procedure. In order to overcome the limitation of delayed immune recovery, historically associated with this type of allograft, in the last few years we developed a novel method of ex vivo graft manipulation, consisting of the negative depletion of T-cell receptor (TCR) αβ+ T-lymphocytes and CD19 B-cells from peripheral blood stem cells (PBSC) grafts (ClinicalTrial.gov identifier: NCT01810120) (Bertaina el al., Blood 2014). Here we report our analysis in a subgroup of FA patients given TCRαβ/CD19-depleted haploidentical HSCT at our Institution.Patients and methods. Ten consecutive FA patients (6 girls and 4 boys) underwent a TCRαβ/CD19-depleted HSCT from an HLA-haploidentical relative between September 2011 and July 2015. Median age at diagnosis was 6.6 (range 2.7-22.0) years and median age at time of transplantation was 8.1 (range 4.4-22.2) years. The conditioning regimen included Cyclophosphamide 300 mg/m2/day and Fludarabine 30 mg/m2/day for 4 consecutive days (days -6 to -3), with 200 cGy single-dose TBI. Pretransplantation Fresenius® ATG was administered at a dose of 4 mg/kg/day for 3 consecutive days (days -5 to -3) in order to prevent both graft failure and graft-versus-host disease (GVHD). All patients received Rituximab 200 mg/m2 to reduce the risk of Epstein-Barr virus-related post-transplant lymphoproliferative disorders. Selective removal of TCRαβ+ and B-cells was performed on G-CSF-mobilized donor PBSC through labeling with biotinylated anti-TCRαβ antibodies and anti-CD19 antibodies, followed by incubation with anti-biotin antibodies conjugated to paramagnetic beads (CliniMACS; Miltenyi Biotec, Bergisch Gladbach, Germany). No immunosuppressive therapy was administered as post-transplantation prophylaxis against GVHD.Results. The TCRαβ/CD19-depletedgrafts contained a median of 20.40 x106/kg (range 15.80-33.40) CD34+ cells, 5.60 x106/kg (range 1.78-69.60) CD3+ lymphocytes, 0.021 x106/kg (range 0.002-0.043) TCRαβ+ lymphocytes, 5.60 x106/kg (range 1.78-69.60) TCRγδ+ lymphocytes, 0.036 x106/kg (range 0.013-0.079) CD20+ lymphocytes, and 45.30 x106/kg (range 16.2-177.0) NK cells. Engraftment with sustained full donor chimerism was achieved in 9 out of 10 patients, the cumulative incidence of graft rejection being 10% (95% CI, 0-26.8). The patient who rejected his first allograft achieved a complete engraftment after a second HSCT from one-antigen mismatched unrelated donor. No secondary graft failures were observed. The median time for neutrophil and platelet engraftment was 12 days (range, 9-15) and 9 days (range, 8-12), respectively. No patient experienced acute or chronic GVHD in the follow-up period. No transplant-related deaths occurred in our cohort. With a median follow-up of 28 months (range 13.2-39.1), the Kaplan-Meier estimates of OS and DFS were both 100%, while the EFS probability was 90% (95% CI, 47.3-98.5).Discussion. These data suggest that haploidentical HSCT after removal of TCRαβ+ and CD19+ lymphocytes is able to guarantee engraftment with excellent OS and DFS in patients affected by FA. Moreover, given the very low incidence of both acute and chronic GVHD, which has been shown to contribute to the increased risk of developing late post-transplantation malignancies in FA patients, this approach can be considered a very attractive option for FA patients in need of an allograft and lacking an HLA-identical family donor. DisclosuresNo relevant conflicts of interest to declare.

Characteristics of Early Recovery of Double Negative T-Cell Subpopulations after Haploidentical Hematopoietic Stem Cell Transplantation

Background: Immune recovery is recognized to be crucial for decreasing morbidity of the subsequent complications in patients who underwent alloHSCT. We recently compared the recoveries of T-cell subpopulations between the haploHSCT recipients with and without EBV-DNAemia and found that lower CD4/CD8 double negative T-cell (DNT) counts were significantly correlated with the increased cumulative incidence of EBV reactivation after transplantation. Multivariate analysis further showed that hampered recovery of DNT cells was an independent risk factor to predict transplant-related mortality. Based on the expression of αβ or γδ T-cell receptors, or NK cell markers, several subpopulations of DNT cells have been identified, e.g. TCR-γ/δ+DNT, TCR-α/β+DNT, NKT cells, and other cells that are incompletely characterized. Early recovery levels of DNT subsets may correlate to the occurrence of HSCT complications. Notably, reconstitution kinetics of these special T-cell subpopulations post-HSCT remains unknown.Method: A prospective study was conducted to observe the comprehensive characteristics of DNT recovery. Fifteen healthy donors and 60 consecutive patients who were diagnosed as hematological malignancy and underwent haploHSCT at our institute were included. Immnophenotyping analysis was performed to determine the recovery levels of DNT and its subpopulations including α/β+DNT, γ/δ+DNT (Vδ1+ and Vδ2+), and CD56+DNT at 30, 60, and 90 days after transplantation.Result: Percentages of total DNT, α/β+DNT, and γ/δ+DNT in recipients on day 30 were comparable with those of health donors (defined as normal range). While the percentage of total DNT significantly decreased on day 60 and day 90, percentages of α/β+DNT and γ/δ+DNT were quite stable within the 3 months after haploHSCT. Interestingly, ratio of Vδ1+/ Vδ2+ on day 30 was similar with the normal control, which was significantly reversed on day 60 and day 90. In contrast, the percentage of CD56+DNT on day 30 was 3-fold higher than the normal range, which dropped down to the normal level at 60 and 90 days. In term of the absolute numbers, all detected DNT subpopulations were hampered at 30 days post-transplantation. Only the counts of Vδ1+DNT achieved the normal level at 60 and 90 days whereas numbers of other DNT subpopulations were continuously lower during the evaluation period. The pace of Vδ2+DNT recovery was the slowest compared with others.Conclusion: Our observations give a picture of the dynamic recovery of DNT subpopulations and contribute to comprehensively understand the characteristics of immune recovery after HaploHSCT. The clinical impact of DNT-subpopulations recovery will be evaluated in the ongoing prospective study. These findings may facilitate the intervention strategies to improve the general outcome of haploHSCT recipients. DisclosuresNo relevant conflicts of interest to declare.

Inducible MyD88/CD40 (iMC) Enhances Proliferation and Survival of Tumor-Specific TCR-Modified T Cells and Improves Anti-Tumor Efficacy in Myeloma

Introduction: Use of T cells engineered to express antigen-specific T cell receptors (TCRs) has shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Additionally, MHC class I downregulation on tumor cells further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a novel, small molecule dimerizer (rimiducid)-dependent T cell "activation switch", called inducible MyD88/CD40 (iMC), along with tumor antigen-specific TCRs to regulate T cell activation and expansion, while upregulating MHC class I expression on tumor cells.Methods: Human T cells were activated with anti-CD3/CD28 and transduced with g-retroviruses encoding TCR α and β chains recognizing either the cancer-testes antigen PRAME (HLA-A*201-restricted SLLQHLIGL) or the B cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*702-restricted APAPTAVVL). Parallel "GoTCR" vectors co-expressed the αβ TCR and iMC, comprising signaling domains from MyD88 and CD40 fused in-frame with tandem rimiducid-binding FKBP12v36 domains. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed EGFPluc-expressing T2 cells (PRAME only) or PRAME+/Bob1+, HLA-A2+ HLA-B7+ EGFPluc-expressing U266 myeloma cells ± rimiducid (10 nM). MHC class I upregulation on tumor cells was measured using transwell assays and flow cytometry. In vitro tumor killing and T cell proliferation were analyzed using T cell and tumor coculture assays by either measuring loss of luciferase activity overnight or by flow cytometry over a period of 4-7 days. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 5x106-1x107 transduced T cells. iMC was activated in vivo by weekly or biweekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo bioluminescence imaging and flow cytometry, respectively.Results: All vectors efficiently (~85%) transduced activated T cells and showed antigen-specific IFN-g production and cytolytic function against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 myeloma cells. However, both TCR ligation and rimiducid-dependent iMC costimulation were required for IL-2 production against PRAME peptide-pulsed T2 cells. Coculture assays against U266 cells showed that tumor elimination was optimized with concurrent rimiducid-driven iMC activation in both "GoPRAME" and "GoBob1" constructs, and this was accompanied by greatly increased IL-2 secretion and robust T cell proliferation (~ 50-fold vs PRAME or Bob1-specific TCRs alone). Further, iMC activation produced IFN-g independently of TCR ligation, which significantly increased MHC class I expression on tumor cells (~ 7-fold) relative to PRAME TCR-transduced T cells. In NSG mice engrafted with PRAME+ U266 myeloma tumors, GoPRAME TCR-modified T cells persisted for at least 81 days post-injection and prevented tumor growth, unlike any of the other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ~1000-fold on day 81 post-injection compared to T cells expressing only the PRAME TCR (p < 0.001).Summary: iMC is a novel "Go" switch that utilizes rimiducid, a small molecule dimerizer, to provide costimulation to PRAME and Bob1-specific TCR-engineered T cells while sensitizing tumors to TCR-mediated recognition via cytokine-induced MHC I upregulation. These iMC-enhanced TCRs are prototypes of novel "GoTCR" engineered T cell therapies that may increase efficacy, safety and durability of adoptive T cell therapies. DisclosuresPentcheva-Hoang:Bellicum Pharmaceuticals: Employment. Torres:Bellicum Pharmaceuticals: Employment. Rodriguez:Bellicum Pharmaceuticals: Employment. Korngold:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Foster:Bellicum Pharmaceuticals: Employment.

Domain-swapped T cell receptors improve the safety of TCR gene therapy.

T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy.

Lack of Heterologous Cross-reactivity toward HLA-A*02:01 Restricted Viral Epitopes Is Underpinned by Distinct αβT Cell Receptor Signatures

αβT cell receptor (TCR) genetic diversity is outnumbered by the quantity of pathogenic epitopes to be recognized. To provide efficient protective anti-viral immunity, a single TCR ideally needs to cross-react with a multitude of pathogenic epitopes. However, the frequency, extent, and mechanisms of TCR cross-reactivity remain unclear, with conflicting results on anti-viral T cell cross-reactivity observed in humans. Namely, both the presence and lack of T cell cross-reactivity have been reported with HLA-A*02:01-restricted epitopes from the Epstein-Barr and influenza viruses (BMLF-1 and M158, respectively) or with the hepatitis C and influenza viruses (NS31073 and NA231, respectively). Given the high sequence similarity of these paired viral epitopes (56 and 88%, respectively), the ubiquitous nature of the three viruses, and the high frequency of the HLA-A*02:01 allele, we selected these epitopes to establish the extent of T cell cross-reactivity. We combined ex vivo and in vitro functional assays, single-cell αβTCR repertoire sequencing, and structural analysis of these four epitopes in complex with HLA-A*02:01 to determine whether they could lead to heterologous T cell cross-reactivity. Our data show that sequence similarity does not translate to structural mimicry of the paired epitopes in complexes with HLA-A*02:01, resulting in induction of distinct αβTCR repertoires. The differences in epitope architecture might be an obstacle for TCR recognition, explaining the lack of T cell cross-reactivity observed. In conclusion, sequence similarity does not necessarily result in structural mimicry, and despite the need for cross-reactivity, antigen-specific TCR repertoires can remain highly specific.