Numbers Of Αβ Research Articles

Changes of CD3+CD56+ γδ T cell number and apoptosis during hospital admission are related to mortality in septic patients

Immunoparalysis and apoptosis of T cells are serious problems for the evolution of septic patients. We aimed to relate changes in the number of αβ and γδ T cells during hospital stay to the poor evolution of sepsis. In this prospective study, we recruited a total of 92 septic patients from the Emergency and Intensive Care Departments of two Hospitals, according to the latest criteria for the definition and management of sepsis. According to the severity of the septic process, there was a progressive decrease in T cells, being much more intense in γδ T cells. This decrease recovered in surviving patients, but CD3+CD56+ γδ T cells continued to decreased during hospital stay in non-surviving patients. Apoptosis increased in sepsis. Cell death of CD3+CD56+ γδ T cells progressively increased according to the severity of sepsis, especially in non-surviving patients.

Donor T Cells Genetically Modified with a Novel Suicide Gene (inducible Caspase 9, iC9) Expand and Persist over Time after Post-Allograft Infusion in Patients Given αβ T-Cell and B-Cell Depleted HLA-Haploidentical Allogeneic Stem Cell Transplantation (αβ haplo-HSCT) Contributing to Accelerate Immune Recovery

Background: αβ haplo-HSCT is an effective option for children with non-malignant disorders (Bertaina et al., Blood 2014) or acute leukemia (Locatelli et al. Blood 2017) and in need of an urgent allograft. In patients given this type of HSCT, recovery of adaptive immunity is, however, suboptimal. With the aim of accelerating reconstitution of adaptive immunity, we evaluated an innovative approach based on post-transplant infusion of titrated numbers of donor αβ T cells, transduced with the suicide gene iC9 (BPX-501 cells). Since the transduced gene contains sequences for the CD19 marker, BPX-501 cells are CD3+/CD19+ and, thus, can be easily tracked in peripheral blood. We report data on immune recovery of children transplanted at Bambino Gesù Children's Hospital, Rome, and enrolled in a multicenter prospective phase I-II trial on BPX-501 cells (sponsored by Bellicum Pharmaceuticals®, ClinicalTrials.gov identifier: NCT02065869).Patients and study design: Included in this analysis were 108 children. Median age at transplant was 5.6 years (range, 0.3-18) and the male/female ratio was 61/47. Patients were subdivided according to the original disorder in 5 groups: group A (53 children with acute leukemia), group B (22 with primary immune-deficiency, PIDs), group C (14 with erythroid disorders), group D (7 with Fanconi anemia) and group E (12 with other diseases). No patient was given any post-transplant graft-versus-host disease (GvHD) prophylaxis. Nine children were enrolled in the phase I portion of the trial consisting of 3 cohorts, receiving escalating doses of BPX-501 cells of 2.5x105, 5x105, and 1x106 cells/kg, respectively. The remaining 99 patients included in the phase II portion received 1x106 BPX-501 cells/kg. Per protocol, BPX-501 cells were to be infused within the first month after αβ haplo-HSCT. We evaluated the absolute number of circulating CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, and NK cells at 1, 2, 3, 6, 9, 12 and 24 months after αβ haplo-HSCT. We also analyzed the number of BPX-501 cells, αβ T cells and γδ T cells at the same time points. The impact of patient gender, age (0-2, 2-6, 6-12 and >12 years), original disease, use of total body irradiation (TBI) during conditioning regimen, occurrence of cytomegalovirus (CMV) reactivation, of acute GvHD and of leukemia relapse (only in group A patients), on recovery of different lymphocyte subsets was investigated, as well. Data were expressed as mean value+SEM (standard error of mean). Six patients given Rimiducid, the agent activating iC9, were censored at time of drug infusion.Results: After infusion, BPX-501 cells expanded progressively reaching a peak at 9 months after the allograft, when their mean value was 144+36/μL. In the 16 patients with 2-year follow-up, the mean number of BPX-501 cells was still 62+23/μL. Details on expansion/persistence of BPX-501 cells, as well as on recovery of CD3+, CD4+ and CD8+ T cells, are shown in Figure 1A and 1B, respectively. A mean number of CD3+ cells greater than 1.000/μL was reached at 6 months after transplantation. Figure 1C depicts the progressive recovery of B cells. NK and γδ T cells promptly recovered after the allograft; the number of αβ T cells overcomes that of γδ T cells at 2 months after transplant. CMV reactivation promoted a statistically better expansion of BPX-501 cells (see also Figure 1D). This effect was paralleled by an improved recovery of both CD3+ and αβ T cells in patients who experienced CMV reactivation. A statistically higher number of both CD3+ and αβ T cells was observed at 6 and 12 months after αβ haplo-HSCT in patients who did not receive TBI. Neither acute GvHD nor leukemia recurrence occurrence statistically affected the number of BPX-501 cells. Children with an age comprised between 0-2 years or with PIDs had a better recovery of both CD3+ and αβ T cells at 6, 9 and 12 months after the allograft.Conclusions: Our results indicate that BPX-501 cells infused after αβ haplo-HSCT expand in vivo and persist over time, contributing to fasten adaptive immunity recovery. Their peak of expansion is reached at 9 months after the allograft, but they are consistently detected also at 2 years after infusion. CMV reactivation is the main driver of BPX-501 cell expansion, this finding suggesting that BPX-501 cells cooperate to the viral clearance. Overall, the whole pattern of immune recovery of these 108 children is improved as compared with that of patients given αβ haplo-HSCT without BPX-501 cell infusion. [Display omitted] DisclosuresO'Neill:Bellicum Pharmaceuticals: Other: contractor. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders . Foster:Bellicum Pharmaceuticals: Employment, Other: stockholders .

γδ T cells are the predominant T cell type in opossum mammaries during lactation

Milk provides mammalian neonates with nutritional support and passive immunity. This is particularly true in marsupials where young are born highly altricial and lacking many components of a fully functional adaptive immune system. Here we investigated the T cell populations in the mammaries of a lactating marsupial, the gray short-tailed opossum Monodelphis domestica. Immunohistochemistry confirmed the presence of T cells within the opossum mammaries throughout lactation. Results of quantifying transcript abundance for lymphocyte markers are consistent with γδ T cells being the most common T cell type within lactating mammaries. Numbers of γδ T cells appear to peak early during the first postnatal week, and then decline throughout lactation until weaning. In contrast, numbers of αβ T cells and γμ T cells appear to be low to non-existent in the lactating mammaries. The results support an ancient and conserved role of immune cells in the evolution and function of mammalian mammary tissue.

Transfer of Ex Vivo Expanded NK and γδT Cells from Untouched Posttransplant PBMCs to Clear Minimal Residual Disease in Acute Myeloid Leukemia

GMP-grade NK cell expansion for clinical purpose has been demonstrated feasible and safe. Here we share our pilot data on posttransplant immunotherapy with ex vivo expanded NK cells to treat minimal residual disease in a pediatric patient with posttransplant relapsed myeloid leukemia. Our patient, a 13 year old boy who underwent 2nd allogeneic stem cell transplantation (haploidentical stem cell transplantation from his mother) due to posttransplant relapsed acute myeloid leukemia. After the 2nd haploidentical stem cell transplantation (SCT) minimal residual disease (MRD) was detected by multiparameter flow cytometry and by two molecular markers CALM-AF10 fusion transcript and a NRAS-mutation. For posttransplant compassionate use immunotherapy by NK cell transfer, NK cells were expanded from untouched isolated PBMCs of the patient post 2nd haploidentical SCT. GMP-grade expansion of the NK cells was done under static conditions in our GMP-facility. Isolated PBMCs were pooled with 100Gy irradiated K562mb15 4-1BBL feeder cells (kindly provided by Dario Campana) in a proportion of 1:20 (NK to K562mb15 4-1BBL). PBMCs and K562mb15 4-1BBL were seeded in conventional cell culture flasks (175cm2) at a density of 1.1E6 cells/ml. Cell culture media contained RPMI1640 supplemented with 10% AB-human serum, 1% L-glutamine and 100IU Proleukine® IL2/ml. Cell culture was monitored daily for cell number, white blood cell differentiation, pH of the cell culture, glucose metabolism, lactate production and microbial sterility testing at the beginning and the end of the expansion period. The cell product was harvested on day 15-17. Fresh isolated PBMCs and the expanded NK cell product were characterized by flow cytometry. NK cells were expanded >1000 fold (3.1 and 3.4 log-fold) in 14-17 days. The product contained a total number of 9.8E9 and 19.9E9 cells, which was 328 and 665E6/kgBW. The expansion protocol supports NK and γδ T cell expansion whereas the number of αβ T cells stays stable. Cytotoxicity assay against various targets revealed excellent cellular cytotoxicity and antibody dependent cellular cytotoxicity. To prevent relapse in our patient with posttransplant MRD positivity, NK cells from the patient post 2nd haploidentical SCT were expanded for cellular immunotherapy. 2 weeks post 1st NK cell transfer (day +170) the patient achieved complete MRD response in the bone marrow. Unfortunately the patient showed detectable MRD one month later. Therefore another NK cell expansion and transfer was done. 2 weeks post 2nd NK cell transfer (day +232) the patient again achieved complete MRD response in the bone marrow and is in complete molecular remission ever since (day +340). The NK cell products were tolerated well. Transient coughing and temporary increase of temperature were registered. Both, in vitro and in vivo effect of the NK cell product were documented. Clinical use of expanded and activated NK cells and γδ T cells can induce molecular remission in posttransplant MRD positive acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Gammadelta (γδ) T lymphocytes do not impact the development of early atherosclerosis

ObjectiveGammadelta (γδ) T cells are a subset of pro-inflammatory innate-like T lymphocytes that serve as a bridge between innate and adaptive immunity. γδ T cells are highly enriched in cholesterol compared to αβ T cells. In this study, we aimed to identify the role of γδ T cells in atherosclerosis, a cholesterol and inflammation-driven disease. MethodsWe found that the percentages of γδ T cells are increased in ApoE−/− mice fed a Western diet. We generated TCRδ−/−ApoE−/− mice and fed them either rodent chow or a Western diet for ten weeks for the assessment of atherosclerosis. ResultsThe atherosclerotic lesion size in diet-fed TCRδ−/−ApoE−/− mice was similar to that of diet-fed ApoE−/− mice. There were no differences in cytokine production or numbers of αβ T cells in aorta of TCRδ−/−ApoE−/− mice. Plasma lipoprotein profiles were unchanged by the absence of γδ T cells. ConclusionOur data suggest that γδ T cells do not contribute to early atherosclerotic plaque development.

Rapid entry and downregulation of T cells in the central nervous system during the reinduction of experimental autoimmune encephalomyelitis

We investigated the mechanisms whereby a previous attack of experimental autoimmune encephalomyelitis (EAE) modifies a subsequent attack in the Lewis rat. Active immunization with myelin basic protein (MBP) and complete Freund’s adjuvant 28 days after the passive transfer of MBP-sensitized spleen cells induced a second episode of EAE, which occurred earlier than in naive control animals, but was less severe overall. The pattern of neurological signs was also different in rechallenged rats, which had less severe tail and hindlimb weakness but more severe forelimb weakness. In rechallenged rats, inflammation was more severe in the cervical spinal cord, cerebellum, brainstem and cerebrum, but less severe in the lumbar spinal cord, than in controls. The early onset of EAE in rechallenged rats was explained by a memory T cell response to MBP 72–89 in the draining lymph node and spleen, and by the enhanced entry of T cells into the central nervous system (CNS). However, the number of αβ T cells in the spinal cord of rechallenged rats declined faster than in controls, especially in the lumbosacral cord, where the number of Vβ8.2 + T cells and the frequency of T cells reactive to MBP 72–89 rapidly decreased, indicating rapid downregulation of the immune response in the previously inflamed spinal cord. Apoptosis of inflammatory cells in the CNS was increased in the rechallenged rats and is likely to contribute to this downregulation. Furthermore, during the disease course the generation of encephalitogenic T cells in the peripheral lymphoid organs was limited compared with controls. Thus, a previous attack of EAE modifies a subsequent attack through the interaction of the following processes: a memory T cell response to MBP; facilitated T cell entry into the CNS; downregulation of the immune response in the CNS, including increased apoptosis of inflammatory cells; and a limited generation of encephalitogenic T cells in the peripheral lymphoid organs.

Roles of chloroform-resistant variants in mouse models of experimental colitis

We have recently reported that the intestinal microflora, especially chloroform-resistant variants (CRV), plays an important role in the maturation of murine intestinal mucosal T cell developments. In this study, to elucidate the role of CRV in the development of colitis, we compared the susceptibility to the colitis of the specific-pathogen-free (SPF) flora-associated and CRV-associated ex-GF Balb:c mice by means of intra-rectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS). The association of SPF flora or CRV with GF Balb/c induced phenotypic maturation in large intestinal (LI)-intestinal intraepithelial lymphocytes (IELs) and LI-lamina propria (LP) T cells. In LI-IELs, the CD4/CD8 ratio decreased with contamination by SPF flora or CRV with GF Balb/c mice. Among LP cells, the number of αβ T cell receptor (TCR) T cells increased. The percentage of the CD4 + CD45RB high phenotype in CD4 + αβ TCR LI-LP T cells, which is a critical marker molecule of virgin T cells, decreased in both SPF flora and CRV-associated Balb/c mice compared to GF Balb/c mice. These results suggested that the CRV flora converted the LI-IEL and LI-LP T cell characteristics from the GF to the conventional type. After the induction of colitis by means of an intra-rectal enema of TNBS in 50% ethanol, the body weight decreased in the group of Balb/c mice contaminated with the fecal flora of SPF mice but not that of CRV flora mice. Moreover, the colo-rectal tissue weight increased in the SPF flora-associated Balb/c mice but not in CRV-associated ones. Flow-cytometric analysis indicated that the percentage of LI-LP αβ TCR T cells increased in both the SPF flora and CRV-associated Balb/c mice. However, CD4 + CD45RB high LI-LP αβ TCR T cells only decreased in the SPF flora-associated Balb/c mice. Semi-quantitative RT-PCR analysis indicated that mRNA in both IL-12 and IFN- γ in LI-LP cells was higher in SPF flora-associated Balb/c mice than in CRV-associated ones. Taken together, these results suggested that the CRV flora is important of the maturation for LI-IELs and LI-LP T cells from GF to the conventional type. However, this flora is not critical for the development of colitis. Keywords: Clostridia, colitis, cytokine, inflammatory bowel disease, intestinal flora, intestinal intraepithelial lymphocyte, lamina propria, TNBS.

Defective B-1 Cell Development and Impaired Immunity against Angiostrongylus cantonensis in IL-5Rα-Deficient Mice

We generated interleukin-5 receptor α chain (IL-5Rα)–deficient (IL-5Rα −/−) mice by gene targeting. The IL-5Rα −/− mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3. They showed no IL-5-induced enhancement of B cell responses to T-independent antigens. The number of αβ T cell receptor–positive thymocytes tended to decrease in 3-week-old IL-5Rα −/− mice, returning to normal by 6 weeks of age. The IL-5Rα −/− mice produced basal levels of eosinophils, while their bone marrow cells failed to form eosinophilic colonies in response to IL-5. Impaired eosinophilopoiesis in IL-5Rα −/− mice enhanced the survival of Angiostrongylus cantonensis. These results indicate that IL-5-induced eosinophils serve as potent effector cells in the killing of Angiostrongylus cantonensis in mice.

Treatment of Arthritis in Lewis Rats by a Monoclonal Antibody against αβ T Cell Receptor: Differential Sensitivity of Yersinia-Induced Arthritis versus Adjuvant Arthritis

Lewis rats experimentally infected with Yersinia enterocolitica develop sterile arthritis similar to Yersinia-associated reactive arthritis in humans. To investigate the putative role of αβ T cells in the pathogenesis of Yersinia-induced arthritis (YIA) rats were treated with the monoclonal antibody (mAb) R73 mAb directed against the rat αβ T cell receptor. In spite of reduction of αβ T cells in peripheral blood and in liver lesions of Yersinia-infected rats this serotherapy had no suppressive effect on YIA. Moreover, R73 mAb treatment had no influence on the number of αβ T cells in the inflammed synovial tissue. In contrast, R73 mAb serotherapy in Mycobaterium tuberculosis-immunized rats blocked development of adjuvant arthritis (AA) and suppressed the presence of αβ T cells in the synovial tissue. These results suggest fundamental differences between the immunopatho-mechanism of YIA caused by bacterial infection and AA induced by bacterial immunization and known to be T cell mediated. These data might have consequences for putative serotherapy of arthritis in humans.

Changes in the distribution of αβ and γδ T cells in blood and in lymph nodes from fetal and postnatal lambs

We have examined the distribution of αβ and γδ T cells, together with B cells, in a range of lymph nodes and blood before and after birth. The proportions of blood-borne αβ and γδ T cells changed markedly during development with a wave of increasing numbers of blood-borne γδ T cells occurring in the fetus during the last month of gestation and early postnatal life. γδ T cells constituted 18% of T cells in the blood of fetal lambs one month before birth and 60% of T cells in the blood of lambs one month after birth. There were also changes in the numbers of αβ T cells circulating through lymph nodes after birth. The proportion of CD4 + and CD8 + cells in mesenteric nodes increased substantially after birth, whereas that of γδ T cells decreased. Although B cells were present in much larger numbers in ileal lymph nodes in both the fetus and lamb, there was a large increase in the concentration of B cells in all lymph nodes in lambs after birth. In addition to the differences in the distribution of lymphocyte subsets circulating in fetal and postnatal lambs, markedly different growth patterns were also observed between lymph nodes before and after birth.