TCRαβ Cells Research Articles

Outcomes of HLA-mismatched HSCT with TCRαβ/CD19 depletion or post-HSCT cyclophosphamide for inborn errors of immunity

AbstractHLA-mismatched transplants with either in vitro depletion of CD3+ T-cell receptor (TCR)αβ/CD19 (TCRαβ) cells or in vivo T-cell depletion using posttransplant cyclophosphamide (PTCY) have been increasingly used for patients with inborn errors of immunity (IEIs). We performed a retrospective multicenter study via the EBMT registry on 306 children with IEIs undergoing their first transplant between 2010 and 2019 from an HLA-mismatched donor using TCRαβ (n = 167) or PTCY (n = 139). The median age for hematopoietic stem cell transplantation (HSCT) was 1.2 years (range, 0.03-19.6 years). The 3-year overall survival (OS) was 78% (95% confidence interval (CI), 71-84) after TCRαβ and 66% (57-74) after PTCY (P = .013). Pre-HSCT morbidity score (hazard ratio [HR], 2.27; 1.07-4.80, P = .032) and non-busulfan/treosulfan conditioning (HR, 3.12; 1.98-4.92, P < .001) were the only independent predictors of unfavorable OS. The 3-year event-free survival (EFS) was 58% (50%-66%) after TCRαβ and 57% (48%-66%) after PTCY (P = .804). The cumulative incidence of severe acute graft-versus-host disease (GvHD) was higher after PTCY (15%, 9%-21%) than TCRαβ (6%, 2%-9%, P = .007), with no difference in chronic GvHD (PTCY, 11%, 6%-17%; TCRαβ, 7%, 3%-11%, P = .173). The 3-year GvHD-free EFS was 53% (44%-61%) after TCRαβ and 41% (32%-50%) after PTCY (P = .080). PTCY had significantly higher rates of veno-occlusive disease (14.4% vs TCRαβ 4.9%, P = .009), acute kidney injury (12.7% vs 4.6%, P = .032), and pulmonary complications (38.2% vs 24.1%, P = .017). Adenoviremia (18.3% vs PTCY 8.0%, P = .015), primary graft failure (10% vs 5%, P = .048), and second HSCT (17.4% vs 7.9%, P = .023) were significantly higher in TCRαβ. In conclusion, this study demonstrates that both approaches are suitable options in patients with IEIs, although they are characterized by different advantages and outcomes.

T-cell engineering strategies for tumors with low antigen density, and T-cell survival in the immunosuppressive tumor microenvironment of relapsed/refractory diffuse large B-cell lymphoma

Refractory and/or relapsed (r/r) diffuse large B-cell lymphomas after treatment with two lines of systemic chemoimmunotherapy exhibit diversity in genetics, tissue biology, and pathology, as well as poor prognosis. Patient TCRαβ cells engineered with a CD19-specific chimeric antigen receptor (CAR) have shown promising clinical outcomes in r/r diffuse large B-cell lymphoma. The ZUMA-1 study, the JULIET study, and the TRANSCEND NHL 001 study of three prototype 19CAR-T cells have indicated an overall response rate of 52–82%, a complete response rate of 40–58%, and a 12-month progression-free survival of 33.2%–46.6%, with clinically manageable treatment related toxicity. At the 5-year follow-up, relapse was observed in approximately 57% of patients within 1 year. Understanding of the risk factors for non-response remains insufficient. In addition to intrinsic tumor resistance, such as aberrant apoptotic signaling, downregulation or loss of tumor-associated antigens (TAA), an immunosuppressive tumor microenvironment, and CAR-T cell exhaustion in vivo have been suggested to be important risk factors. Mechanisms underlying 19CAR-T cell exhaustion under chronic TAA exposure, and limited 19CAR-T cell trafficking and infiltration into the tumor mass have been reported. Moreover, tumor escape in the presence of low TAA density remains a challenge in 1928ζ CAR-T cell treatment. In this review, we provide an overview of modified modular CAR elements and their synergistic effects in controlling T-cell function. We then briefly discuss novel strategies against tumors with low TAA density, such as bispecific tandem or loop CAR recognition domains, the development of human leukocyte antigen-independent synthetic TCRαβ double-chain receptors integrated into the constant region of the TCRα chain, and armored CAR-T cells targeting the tumor microenvironment.

Results of a multicenter phase I/II trial of TCR\u03b1\u03b2 and CD19-depleted haploidentical hematopoietic stem cell transplantation for adult and pediatric patients

Hematopoietic stem cell transplantation (HSCT) from haploidentical donors is a viable option for patients lacking HLA-matched donors. Here we report the results of a prospective multicenter phase I/II trial of transplantation of TCRαβ and CD19-depleted peripheral blood stem cells from haploidentical family donors after a reduced-intensity conditioning with fludarabine, thiotepa, and melphalan. Thirty pediatric and 30 adult patients with acute leukemia (n = 43), myelodysplastic or myeloproliferative syndrome (n = 6), multiple myeloma (n = 1), solid tumors (n = 6), and non-malignant disorders (n = 4) were enrolled. TCR αβ/CD19-depleted grafts prepared decentrally at six manufacturing sites contained a median of 12.1 × 106 CD34+ cells/kg and 14.2 × 103 TCRαβ+ T-cells/kg. None of the patients developed grade lll/IV acute graft-versus-host disease (GVHD) and only six patients (10%) had grade II acute GVHD. With a median follow-up of 733 days 36/60 patients are alive. The cumulative incidence of non-relapse mortality at day 100, 1 and 2 years after HSCT was 5%, 15%, and 17% for all patients, respectively. Estimated probabilities of overall and disease-free survival at 2 years were 63% and 50%, respectively. Based on these promising results in a high-risk patient cohort, haploidentical HSCT using TCRαβ/CD19-depleted grafts represents a viable treatment option.

The Importance of Bone Marrow Lymphocyte Subtypes As Predicting Factors for Molecular Recurrence in Patients with Chronic Myeloid Leukemia after Discontinuation of Imatinib

Introduction: in recent years the feasibility of the discontinuation of tyrosine kinase treatment in chronic myeloid leukemia (CML) has been proven, and several clinical factors influencing the duration of treatment-free remission (TFR) after discontinuation have been studied.Aim: we analyzed the influence of bone marrow (BM) lymphocyte subsets on the molecular recurrence after discontinuation of Imatinib (IM) in CML.Methods: in a recent discontinuation study performed at our Institution (EDI-PIO trial) we assessed BM lymphocyte subsets before and after introduction of pioglitazone which was given 3 months before discontinuation of IM. Criterias for discontinuation were: patient in chronic phase at diagnosis, a minimum of 3 years on TKI and sustained molecular remission MR4.5 for at least 2 years. Lymphocyte populations studied: B, TCD8, TCD4, T CD4+CD8+ and T CD4- CD8- besides T naïve and memory, TCRαβ, TCRγδ, NK-t and NK cells. The influence of Sokal score at diagnosis, the duration of imatinib treatment together with the BM lymphoid subsets on the time of treatment-free remission (TFR) were examined by uni- and multivariate Cox regressions.Results: we studied 30 out of 32 patients diagnosed between 1998 and 2013 that reached criteria for discontinuation that were included in EDI-PIO trial: 13 male and 17 female. Median age at diagnosis: 41 years (22-65). Median time of IM treatment: 116.3 months (38.2-209.3); 11 patients (36%) had a molecular recurrence in a median of 5,17 months (2.4-29.4). For patients remaining in TFR the median time of follow-up was 46 months (26.3-55.9). Median overall time of IM treatment (IM-Tr) was 65 months for patients recurring and 124 months for those remaining in TFR. In the univariate Cox regression a significant value was found for IM-Tr, higher percentages of T CD4+CD8+ and lower ones of TCRγδ lymphocytes at discontinuation. In the multivariate model only the T CD4+CD8+ remained.Conclusion: discontinuation of IM is feasible in patients remaining in continuous MR4.5 for more than 2 years. A longer time of IM treatment and higher values of BM T CD4+CD8+ predict a lower risk of relapse.Key words: chronic myeloid leukemia, imatinib, discontinuation, lymphocytes, pioglitazone DisclosuresPagnano: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pintpharma: Other: Lecture; EMS: Other: Lecture; Jansenn: Other: Lecture.

Natural Killer Cell Cytotoxicity Is a Predictor of Outcome for Patients with High Risk Myelodysplastic Syndrome and Oligoblastic Acute Myeloid Leukemia Treated with Azacytidine

Introduction: Hypomethylating agents (HMA) have shown significant therapeutic efficacy in the treatment of patients with MDS and AML. Although it has been suggested, that HMA act through re-activation of tumor-suppressor genes, the exact mechanism of their action has not been completely elucidated yet. The aim of our study was to identify predictors of outcome for patients treated with azacytidine (AZA).Patients and Methods: Eighteen patients with high risk MDS (RAEB-II) and oligoblastic AML (20-30% bone marrow blasts) were included in this study. Patients were not eligible for intensive induction chemotherapy due to age, or presence of comorbidities. All patients were selected for treatment with AZA.NK-cytotoxicity against K562, Rajii, and the percentage of various immune regulatory subpopulations in the peripheral blood (PB) was measured after 3-6 cycles of AZA. A previous short exposure to AZA was required with the aim: 1) to examine the effect of AZA on these populations, and 2) to identify biomarkers with the potential to predict outcome after treatment with AZA.NK-cytotoxicity against K562 and Rajii was tested by using a flow cytometry based assay as previously described (1).Quantitative estimation of immune regulatory subpopulations in PB of patients was performed by using flow cytometry. The number of myeloid derived suppressor cells (MDSCs) (CD33+/CD11b+/CD14-/HLADRlow/-) was expressed as percentage among CD45+ PB monocytes. The number of Tregs (CD3+/CD4+/CD25+/Foxp3+) was expressed as percentage among CD3+/CD4+ PB lymphocytes. The number of double negative TCRαβ (CD3+/TCRαβ+/CD4-/CD8-) was expressed as percentage among PB CD3+ lymphocytes.Comparison between groups was performed by using Mann-Whitney test for continuous variables. Receiver operating curve analysis (ROC) was performed with the aim to determine the ability of various parameters for the prediction of 2-year survival. Kaplan-Meier and log rank test were used for estimating the probability of overall survival (OS).ResultsNK cytotoxicity: The median NK-cytotoxicity against K562 of patients and healthy donors was 21.8% (95% CI, 7.5 - 32.6) and 41.8% (95% CI, 11.4 - 56.1). Patients with MDS/AML after a short exposure to azacytidine had a significantly reduced NK-cytotoxicity against K562 as compared with NK cytotoxic activity of healthy donors (p=0.01). NK cytotoxic activity against Rajii of both patients and healthy donors was very low to absent (data not shown).Immune regulatory cells: The median MDSCs of patients and healthy donors was 0.41% (95% CI, 0.13 - 1.26) and 0.60% (95% CI, 0.30 - 1.94). There was no difference between MDSCs in the PB of patients and healthy donors. There was no difference in the number of Tregs and double negative TCRαβ cells between patients and donors (data not shown).NK cytotoxicity is a predictor of overall survival in patients treated with AZA: The ability of various parameters including the PB percentage of MDSCs, Tregs, and double negative TCRαβ, as well as of NK cytotoxic activity to predict survival at 2 years after treatment with AZA was examined by using ROC analysis. NK cytotoxic activity was the only predictor of OS for patients treated with AZA (AUC=0.875, 95% CI, 0.635 - 0.979, p<0.0001). NK-cytotoxicity above 20.85% was estimated as the best cut off with sensitivity and specificity 80% and 87% in predicting 2-year survival (Figure 1A). In multivariate analysis, NK cytotoxicity above 20.85% (high NK-activity) was the only parameter statistically associated with significantly improved OS [HR=0.10, 95% CI, 0.01 - 0.90, p=0.041] (Figure 1B).Conclusions: Our study showed that NK cytotoxicity is a predictor of the outcome for patients treated with AZA. Patients with high NK-activity after a short course of AZA had significantly improved OS as compared with patients with low NK-activity. The results of our study suggest that the therapeutic activity of AZA is at least partly mediated by an immunomodulatory effect.

In Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells

SUMMARYWe report a serum-free, 3D murine artificial thymic organoid (M-ATO) system that mimics normal murine thymopoiesis with the production of all T cell stages, from early thymic progenitors to functional single-positive (CD8SP and CD4SP) TCRαβ and TCRγδ cells. RNA sequencing aligns M-ATO-derived populations with phenotypically identical primary thymocytes. M-ATOs initiated with Rag1−/− marrow produce the same differentiation block as seen in the endogenous thymus, and Notch signaling patterns in M-ATOs mirror primary thymopoiesis. M-ATOs initiated with defined hematopoietic stem cells (HSCs) and lymphoid progenitors from marrow and thymus generate each of the downstream differentiation stages, allowing the kinetics of T cell differentiation to be tracked. Remarkably, single HSCs deposited into each M-ATO generate the complete trajectory of T cell differentiation, producing diverse TCR repertoires across clones that largely match endogenous thymus. M-ATOs represent a highly reproducible and efficient experimental platform for the interrogation of clonal thymopoiesis from HSCs.

Guided Polarization of iPSC-Derived CD4SP Helper T Cells By CRISPR/Cas9-Based Genome-Editing

Guided Polarization of iPSC-Derived CD4SP Helper T Cells By CRISPR/Cas9-Based Genome-Editing

Generation and flow cytometric quality control of clinical-scale TCRαβ/CD19-depleted grafts.

The depletion of TCRαβ+ T cells and CD19+ B cells is a graft purification method for haploidentical stem cell transplantation (HSCT) retaining stem cells, NK cells and TCRγδ+ T cells. To avoid treatment-related occurrence of severe GvHD a precise quantification of residual TCRαβ+ T cells in the graft is of essential importance. Nine stem cell grafts were purified immunomagnetically on a CliniMACS device and flow cytometric quality control (QC) was performed before and after TCRαβ/CD19-depletion. As a challenge a new 10-color QC-panel was established, which enables accurate quantification of the graft composition. The binding sites of residual TCRαβ+ T and CD19+ B cells were at least partly occupied by depletion antibodies impeding flow cytometric analysis. Based on respective controls and an assumed variation coefficient of 18%, the detection limit of residual TCRαβ T cells was 1 cell/µl. and 0.002% of CD45+ cells. Log-depletion of TCRαβ and CD19 cells was -3.9 and -3.3, respectively. The recovery was 82.1%, 67.1% and 72.7% for stem cells, NK cells and for TCRγδ+ T cells. In clinical use this method may help to improve transplantation outcome, due to the correct application of the desired stem cell and the limited T cell dose. The panel is designed for the QC following TCRαβ/CD19-depletion but is adaptable to other depletion strategies as well. © 2015 International Clinical Cytometry Society.

A New Description and the Rate of Development of Inflorescence Primordia over a Full Season inVitis viniferaL. cv. Chardonnay

The crucial role of Notch signaling in cell fate decisions in hematopoietic lineage and T lymphocyte development has been well established in mice. Overexpression of the intracellular domain of Notch mediates signal transduction of the protein. By retroviral transduction of this constitutively active truncated intracellular domain in human CD34⁺ umbilical cord blood progenitor cells, we were able to show that, in coculture with the stromal MS-5 cell line, depending on the cytokines added, the differentiation toward CD19⁺ B lymphocytes was blocked, the differentiation toward CD14⁺ monocytes was inhibited, and the differentiation toward CD56⁺ NK cells was favored. The number of CD7⁺cyCD3⁺ cells, a phenotype similar to T/NK progenitor cells, was also markedly increased. In fetal thymus organ culture, transduced CD34⁺ progenitor cells from umbilical cord blood cells or from thymus consistently generated more TCR-γδ T cells, whereas the other T cell subpopulations were largely unaffected. Interestingly, when injected in vivo in SCID-nonobese diabetic mice, the transduced cells generated ectopically human CD4⁺CD8⁺ TCR-αβ cells in the bone marrow, cells that are normally only present in the thymus, and lacked B cell differentiation potential. Our results show unequivocally that, in human, Notch signaling inhibits the monocyte and B cell fate, promotes the T cell fate, and alters the normal T cell differentiation pathway compatible with a pretumoral state.

Lymphocyte subpopulations of bone marrow of breast cancer patients

We have studied bone marrow lymphocytes subpopulations in 67 patients with breast cancer and 13 patients with benign processes in the mammary gland. Morphological study of the cellular composition of the bone marrow and immunological analysis of lymphocyte subpopulations have been done. Comparative characterization of lymphocyte subpopulations in the bone marrow of patients with breast cancer and benign breast diseases was done by flow cytometry. The gate of Mature lymphocytes (CD45++) studied subpopulations of T cells (CD3), b cells (CD19), NK cells (CD56+CD3–). Among T lymphocytes (CD3+) was studied TCRγδ and TCRαβ cells, a subpopulation of CD4, CD8, CD4+, CD25+ T-regulatory cells. Within В-lymphocytes (CD19+) was studied subpopulations CD10, CD38, CD5 lymphocytes. We revealed a significant increase of CD5+, CD38+ B-cells in patients with breast cancer compared to benign tumors. Index of maturation of erythrocytes was significantly higher in BC 0.96 in comparison with benign processes 0.92. When comparing immunological data with histological structure of the tumor in the group of breast cancer patients, it was noted that the percent of CD10+ B-lymphocytes was significantly higher in infiltrative ductal cancer in comparison to infiltrative lobular carcinoma (19.3 and 8.0 % accordingly; p = 0.024).

Unconventional Intraepithelial Gut T Cells: The TCR Says It All

The intestinal epithelium harbors a large number of Tcells, including TCRαβ cells that lack expression of CD4 and CD8αβ coreceptors. In this issue of Immunity, Mayans etal. (2014) and McDonald etal. (2014) shed light on the specificity and development of this enigmatic Tcell population.

Elevated double negative T cells in pediatric autoimmunity.

Autoimmune diseases are thought to be caused by a loss of self-tolerance of the immune system. One candidate marker of immune dysregulation in autoimmune disease is the presence of increased double negative T cells (DNTs) in the periphery. DNTs are characteristically elevated in autoimmune lymphoproliferative syndrome, a systemic autoimmune disease caused by defective lymphocyte apoptosis due to Fas pathway defects. DNTs have also been found in the peripheral blood of adult patients with systemic lupus erythematosus (SLE), where they may be pathogenic. DNTs in children with autoimmune disease have not been investigated. We evaluated DNTs in pediatric patients with SLE, mixed connective tissue disease (MCTD), juvenile idiopathic arthritis (JIA), or elevated antinuclear antibody (ANA) but no systemic disease. DNTs (CD3(+)CD56(-)TCRαβ(+)CD4(-)CD8(-)) from peripheral blood mononuclear cells were analyzed by flow cytometry from 54 pediatric patients including: 23 SLE, 15 JIA, 11 ANA and 5 MCTD compared to 28 healthy controls. Sixteen cases (29.6%) had elevated DNTs (≥2.5% of CD3(+)CD56(-)TCRαβ(+) cells) compared to 1 (3.6%) control. Medication usage including cytotoxic drugs and absolute lymphocyte count were not associated with DNT levels, and percentages of DNTs were stable over time. Analysis of multiple phenotypic and activation markers showed increased CD45RA expression on DNTs from patients with autoimmune disease compared to controls. DNTs are elevated in a subset of pediatric patients with autoimmune disease and additional investigations are required to determine their precise role in autoimmunity.

Modeling Growth Of Pediatric T-ALL In Vivo and In Vitro: Clinical Meaning and Activation Of The NFkB Pathway

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by the proliferation of T-cell precursors in various sites, such as thymus, bone marrow, blood, lymph nodes or central nervous system. As T-ALL cells alone do not successfully grow in vitro, xenografts of T-ALL cells into NOD/scid/IL-2R null (NSG) mice and long-term co-cultures of T-ALL cells with stromal cells have been developed to study the biology of T-ALL cells (Armstrong et al, Blood, 2009). However, the growth of T-ALL cells in these two systems is highly variable across T-ALL samples. Moreover, the clinical relevance of both assays and, except for NOTCH pathway activation, the molecular pathways involved in successful in vivo and in vitro growths are still elusive.The aim of this work was to determine the relationships between clinical, biological and molecular characteristics of human T-ALL at diagnosis and the growth of T-ALL in these two systems.Human T-ALL blood samples were collected at diagnosis from pediatric or young adult patients with T-ALL. 50,000 T-ALL cells were intravenously injected into NSG mice. Mouse bone marrow samples were collected every 3-4 weeks from day 35 to day 210 post-transplant. Leukemic engraftment was monitored using flow cytometry measuring the % of human CD45+CD7+ leukemic cells. Time to leukemic engraftment (TTL) was defined as the time between T-ALL injection and the detection of ≥20% leukemic cells in at least one mouse. In vitro co-culture growth assay consisted in plating 200,000 cells on MS5 or MS5-DL1 (Armstrong, Blood, 2009) and count every 7 days up to 28 days.A total of 36 samples were tested of which 22 (61%) engrafted into mice. Global median TTL was 82 days (range, 36-121) defining short (TTL<82 days) and long or no engraftment (TTL>82 days) TTL groups. Patient gender, age, mediastinal involvement or abnormal karyotype had no significant impact on TTL. A trend for a shorter TTL was observed for T-ALL samples with a white blood cell count (WBC) > median WBC = 146 G/L (p =0.06). Samples containing more than 20% of TCRαβ or CD8 positive cells exhibited increased incidence of engraftment (p = 0.049 and p=0.04 respectively) whereas CD34, CD1a, CD4 or sCD3 markers were not significantly correlated with TTL. Unlike samples with TLX1, TLX3 overexpression or NOTCH/FBXW7 mutations, samples with SIL-TAL1 deletion exhibited a shorter TTL (p = 0.0004). The 2-year progression free survival of “short TTL” patients was 72% vs 70% for patients with “longer TTL” or no engraftment (p=0.38). T-ALL samples for which growth could be achieved on MS5 cells also displayed a shorter TTL.To unravel molecular mechanisms involved in the growth of leukemic cells in these two systems, micro-arrays were performed for 8 “short TTL” T-ALL versus 8 “long TTL or no engraftment” T-ALL. 346 genes were differentially express in short TTL samples compared to long/no TTL samples (P<0.05, fold change: 1.5). As expected, most of genes up-regulated in short TTL group were implicated in cell cycle function enhancing the commitment of cells to S/M phases. Analysis of regulated networks revealed that several indirect modulators of NFkB (MAL, AhR and CYLD) were significantly up/down regulated in short TTL patient samples resulting in NFkB activation.Overall, T-ALL with SIL-TAL1 deletion display an increased ability to engraft into NSG mice, in accordance with increased WBC in T-ALL patients. Contrary to B-ALL, shorter TTL is not associated with poor prognosis in T-ALL. Moreover, NSG engraftment and co-culture on stromal cells are well correlated. A shorter TTL seems to be associated with an increased leukemic proliferation through NFkB activation. Disclosures:No relevant conflicts of interest to declare.

Induction of T-cell development by Delta-like 4-expressing fibroblasts

The thymus provides a unique environment for the induction of T-cell lineage commitment and differentiation, which is predicted by specific Notch ligand-receptor interactions on epithelial cells and lymphoid progenitors, respectively. Accordingly, a bone marrow-derived stromal cell line (OP9) ectopically expressing the Notch ligand Delta-like 1 (Dll1) or Dll4 (OP9-DL1 and OP9-DL4, respectively) gains the ability to recapitulate thymus-like function, supporting T-cell differentiation of both mouse and human progenitors. In this study, we extend these findings by demonstrating that, unlike the NIH3T3 cell line, mouse primary fibroblasts made to express Dll4 (mFibro-DL4) acquire the capacity to promote and support T-cell development from hematopoietic stem cells (HSCs) into TCRαβ(+), CD4(+) and CD8(+) T-lineage cells. However, mFibro-DL4 cells showed a lower efficiency of T-cell generation than OP9-DL4 cells did. Nevertheless, progenitor T-cells (CD117(+) CD44(+) CD25(+)) generated in HSC/mFibro-DL4 co-cultures, when intravenously transferred into immunodeficient (Rag2(-/-) γc(-/-)) mice, home to the thymus, undergo differentiation, and give rise to mature T-cells that go on to populate the periphery. Surprisingly, primary human fibroblast cells expressing Dll4 showed very low efficiency in supporting human T-lineage differentiation, which could not be improved by blocking myelopoiesis. Nevertheless, mFibro-DL4 cells could support human T-cell lineage differentiation. Our results provide a functional framework for the induction of T-cell development using easily accessible fibroblasts made to express Dll4. These cells, which are amenable for in vitro applications, can be further utilized in the design of individualized systems for T-cell production, with implications for the treatment of immunodeficiencies.

Mechanisms underlying T cell guided innate immunity after inhalation of Staphylococcus enterotoxin A (SEA) (173.1)

Abstract Inhalation of Staphylococcus aureus enterotoxin A (SEA) in mice induces strong pulmonary inflammation marked by vascular permeability and alveolitis, thereby modeling aspects of lung injury in humans. After SEA inhalation CD8+Vβ3+ T cells robustly expand but the early events of this response in the lung are unclear. Our new data show that Vβ3+ T cells are activated quickly after SEA inhalation since CD69 and the high affinity IL-2 receptor α-chain (CD25) were upregulated by 3 hours. This coincided with several changes in the innate immune system including recruitment of neutrophils, monocytes and NK cells into the lung, and imprinting an activation phenotype on macrophages. Importantly, in the absence of T cells all of these effects were abrogated. As a way to understand how T cells condition innate immunity, we analyzed cytokine production and found an unexpected role for TCRαβ T cells promoting IL-17 secretion by TCRγδ T cells. We demonstrated that TCRγδ cells did not produce IL-17 when tested in TCRβ-/- mice. Currently, we are defining how TCRγδ cells are induced to synthesize IL-17 by the TCRαβ cells. In sum, TCRαβ T cells mediate pro-inflammatory responses, not only as a direct source of cytokines, but also by guiding the recruitment and activation of innate cells. Thus, our data show role reversal between the adaptive immune and innate immune system.

Hydroa Vacciniforme Is Associated with Increased Numbers of Epstein–Barr Virus–Infected γδT Cells

Hydroa vacciniforme (HV) is a rare photosensitivity disorder of childhood associated with Epstein-Barr virus (EBV)(+) T-cell infiltration. We have summarized clinical manifestations of HV, and analyzed EBV(+) T-cell subsets as well as EBV DNA load in lymphocyte fractions, in comparison with hypersensitivity to mosquito bites (HMB), an EBV-associated T/natural killer (NK) lymphoproliferative disorder. We found that 31 of 33 (93.9%) HV lesions were composed of EBV(+) T cells and reactive EBV(-) cytotoxic T cells, without significant CD56(+) cell infiltration, whereas many CD56(+) cells were present in 8 of 9 (88.9%) HMB lesions. Of 13 (20.6%) HMB patients with or without HV, 12 (92.3%) showed increased percentages (>32%) of NK cells in the peripheral blood, whereas in the 16 patients with HV alone, 14 (87.5%) showed no increase. Of the 11 HV patients, 10 (90.9%) had increased percentages (>5%) of circulating γδT cells, with a mean value of 15.7 ± 2.9%, and the γδT-cell fractions contained larger amounts of EBV DNA than non-γδT-cell fractions. A triple-labeling method revealed that all three HV patients examined had increased percentages of EBER(+), T-cell receptor (TCR)γδ(+), and TCRαβ(-) cells. Our observations indicate that HV is associated with increased numbers of EBV(+) γδT cells, whereas HMB is associated with EBV(+) NK cells.

Cellular basis for clearance of the protozoan parasite Babesia microti in immunocompromised hosts (43.13)

Abstract Babesia microti is the primary etiologic agent of human babesiosis, an emerging infectious disease in the US. In healthy individuals, babesiosis typically causes a mild flu-like illness or is asymptomatic. In immunocompromised patients, particularly those with B cell lymphoma and treated with rituximab, babesiosis is severe, persistent, and sometimes fatal. The mechanisms by which the host controls this obligate parasite of erythrocytes are poorly understood. In C57Bl/6 mice, parasitemia is marginal and transient. Lack of mature B cells due to igh6 deficiency does not hinder resistance. In rag1-/- mice, however, parasitemia is intense and persists for at least three months. This pattern also is observed in athymic (nude) mice, in mice lacking TCRαβ cells and in MHCii-/- mice. Surprisingly, parasitemia is intense but transient in cd4-/- mice. In the spleens of these mice, CD8+ T cells and CD4-CD8- T cells greatly expand during the course of infection. Parasitemia resolves in cd4-/- cd8-/- mice, but persists in cd4-/- igh6-/- mice. Likewise, parasitemia persists in cd4-/- mice depleted of B cells by repeated administration of anti-CD20. Resolution of parasitemia in cd4-/- mice is concomitant with the rise in circulating Babesia specific IgG, but does not require Fc receptors as demonstrated in cd4-/- fcegr1-/- mice. Our studies establish that, in the absence of CD4, B cells are critical for resolution of babesiosis. The role of complement and cytokines will be discussed.

Notch induces human T-cell receptor γδ+ thymocytes to differentiate along a parallel, highly proliferative and bipotent CD4 CD8 double-positive pathway

In wild-type mice, T-cell receptor (TCR) γδ(+) cells differentiate along a CD4 CD8 double-negative (DN) pathway whereas TCRαβ(+) cells differentiate along the double-positive (DP) pathway. In the human postnatal thymus (PNT), DN, DP and single-positive (SP) TCRγδ(+) populations are present. Here, the precursor-progeny relationship of the various PNT TCRγδ(+) populations was studied and the role of the DP TCRγδ(+) population during T-cell differentiation was elucidated. We demonstrate that human TCRγδ(+) cells differentiate along two pathways downstream from an immature CD1(+) DN TCRγδ(+) precursor: a Notch-independent DN pathway generating mature DN and CD8αα SP TCRγδ(+) cells, and a Notch-dependent, highly proliferative DP pathway generating immature CD4 SP and subsequently DP TCRγδ(+) populations. DP TCRγδ(+) cells are actively rearranging the TCRα locus, and differentiate to TCR(-) DP cells, to CD8αβ SP TCRγδ(+) cells and to TCRαβ(+) cells. Finally, we show that the γδ subset of T-cell acute lymphoblastic leukemias (T-ALL) consists mainly of CD4 SP or DP phenotypes carrying significantly more activating Notch mutations than DN T-ALL. The latter suggests that activating Notch mutations in TCRγδ(+) thymocytes induce proliferation and differentiation along the DP pathway in vivo.

Apple polyphenols require T cells to ameliorate dextran sulfate sodium-induced colitis and dampen proinflammatory cytokine expression

Human IBD, including UC and Crohn's disease, is characterized by a chronic, relapsing, and remitting condition that exhibits various features of immunological inflammation and affects at least one/1000 people in Western countries. Polyphenol extracts from a variety of plants have been shown to have immunomodulatory and anti-inflammatory effects. In this study, treatment with APP was investigated to ameliorate chemically induced colitis. Oral but not peritoneal administration of APP during colitis induction significantly protected C57BL/6 mice against disease, as evidenced by the lack of weight loss, colonic inflammation, and shortening of the colon. APP administration dampened the mRNA expression of IL-1β, TNF-α, IL-6, IL-17, IL-22, CXCL9, CXCL10, CXCL11, and IFN-γ in the colons of mice with colitis. APP-mediated protection requires T cells, as protection was abated in Rag-1(-/-) or TCRα(-/-) mice but not in IL-10(-/-), IRF-1(-/-), μMT, or TCRδ(-/-) mice. Administration of APP during colitis to TCRα(-/-) mice actually enhanced proinflammatory cytokine expression, further demonstrating a requirement for TCRαβ cells in APP-mediated protection. APP treatment also inhibited CXCR3 expression by TCRαβ cells, but not B or NK cells, in the colons of mice with colitis; however, depletion of CD4(+) or CD8(+) T cells alone did not abolish APP-mediated protection. Collectively, these results show that oral administration of APP protects against experimental colitis and diminishes proinflammatory cytokine expression via T cells.

CD4 and CD8 TCRαβ Cells Are selected On MHC Expressed On Thymocyte Precursors in OP9-DL1 Cultures.

Abstract 3670Poster Board III-606Human CD34+ haematopoietic precursor cells cultured on delta-like-ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells (TEC) which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4+ and cytotoxic CD8+ TCRαβ cells is not expected.Phenotypically mature CD27+ CD1− TCRγδ as well as TCRαβ cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single positive (SP) TCRαβ cells were observed. Mature CD8 SP cells consisted of two subpopulations: one expressing mainly CD8αβ and one expressing CD8αα dimers. TCRαβ CD8αα and TCRγδ cells both expressed the IL2Rβ receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8αβ+ and CD4+ TCRαβ+ cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8αβ+ and CD4+ T cells. These T cells had the characteristics of conventional T cells: CD4+ cells expressed ThPOK, CD40L and high levels of IL-2 and IL-4; CD8+ cells expressed EOMES, RUNX3 and high levels of granzyme, perforin and IFN-γ.Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8+ cells, whereas blocking MHC class I antibodies and CD8 antibodies inhibited generation of mature CD27+ CD8+ SP cells. The presence of dendritic cells was not required for the generation of mature CD4+ or CD8+ T cells.In conclusion, we have demonstrated positive selection of TCRαβ+ cells in the absence of TEC. The data suggest that positive selection is induced by interaction between T precursor cells without requirement for other haematologic or stromal elements. Disclosures:No relevant conflicts of interest to declare.