scholarly journals iNKT cells coordinate immune pathways to enable engraftment in nonconditioned hosts

2021 ◽  
Vol 4 (7) ◽  
pp. e202000999
Author(s):  
Nicholas J Hess ◽  
Nikhila S Bharadwaj ◽  
Elizabeth A Bobeck ◽  
Courtney E McDougal ◽  
Shidong Ma ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Progenitor Cell ◽  
Inflammatory Responses ◽  
Cell Activity ◽  
Cellular Immunotherapy ◽  
Human Umbilical Cord ◽  
Inkt Cells ◽  
Hematopoietic Stem

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that interact with key antigen-presenting cells to modulate adaptive T-cell responses in ways that can either promote protective immunity, or limit pathological immune activation. Understanding the immunological networks engaged by iNKT cells to mediate these opposing functions is a key pre-requisite to effectively using iNKT cells for therapeutic applications. Using a human umbilical cord blood xenotransplantation model, we show here that co-transplanted allogeneic CD4+ iNKT cells interact with monocytes and T cells in the graft to coordinate pro-hematopoietic and immunoregulatory pathways. The nexus of iNKT cells, monocytes, and cord blood T cells led to the release of cytokines (IL-3, GM-CSF) that enhance hematopoietic stem and progenitor cell activity, and concurrently induced PGE2-mediated suppression of T-cell inflammatory responses that limit hematopoietic stem and progenitor cell engraftment. This resulted in successful long-term hematopoietic engraftment without pretransplant conditioning, including multi-lineage human chimerism and colonization of the spleen by antibody-producing human B cells. These results highlight the potential for using iNKT cellular immunotherapy to improve rates of hematopoietic engraftment independently of pretransplant conditioning.

scholarly journals Acellular Wharton’s Jelly, Potentials in T-Cell Subtypes Differentiation, Activation and Proliferation

2020 ◽  
Vol 10 (4) ◽  
pp. 617-622
Author(s):  
Mehdi Talebi ◽  
Hojjatollah Nozad Charoudeh ◽  
Ali Akbar Movassaghpour Akbari ◽  
Behzad Baradaran ◽  
Tohid Kazemi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Culture Media ◽  
Cell Activity ◽  
Cell Subset ◽  
Wharton’S Jelly ◽  
Cellular Immunotherapy ◽  
Lag Phase ◽  
Wharton's Jelly ◽  
Study Results

Purpose : Because of different potentials of T-cell subtypes in T-cell based cellular immunotherapy approaches such as CAR-T cell therapies; Regarding the high cost of the serum-free specific culture media, having distinct control on T-cell subset activation, expansion and differentiation seem crucial in T-cell expansion step of cell preparation methods. By the way, there was no clear data about the effect of acellular Wharton’s Jelly (AWJ) on T-cells expansion, activation or differentiation status. So, we have launched to study the effect of AWJ on T-cell’s immunobiological properties. Methods: CD3+ T-cells were isolated from healthy bone marrow allogeneic donors, sorted by FACS method and cultured on either routine phyto-hemagglutinin complemented and different concentrations of AWJ, lag phase and doubling time of the cells calculated from cell growth curve. After 3, 7 and 14-days T-cell subtypes cell markers and cell activity related genes expression rate have been evaluated by flow cytometry and real-time polymerase chain reaction (PCR) methods respectively. Results: AWJ in a 1:1 ratio compared with contemporary lymphocyte culture media showed significant activating and proliferative capacities. The introduced condition has not affected the frequency of CD4+ subpopulation of T-cells, but significantly increased even CD8+ cells and immune-activator genes in T-cells. The regulatory and memory subsets of T-cells in this study have not affected significantly. Conclusion: the study results revealed that AWJ can be utilized as a supportive substance to increase the memory properties of the T-cells, gives control to design a selective medium for expanding and differentiating memory T-cells, relatively.

A modified CD34+ hematopoietic stem and progenitor cell isolation strategy from cryopreserved human umbilical cord blood

Transfusion ◽  
2019 ◽  
Vol 59 (12) ◽  
pp. 3560-3569
Author(s):  
Marcia F. Mata ◽  
Diana Hernandez ◽  
Evangelia Rologi ◽  
Davide Grandolfo ◽  
Enas Hassan ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Progenitor Cell ◽  
Cell Isolation ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Stem

Ex Vivo Expansion of Umbilical Cord Blood T Cells for Adoptive Immunotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 35-35
Author(s):  
Melissa A. Mazur ◽  
Young Ah Lee ◽  
Kurtzberg Joanne ◽  
Szabolcs Paul
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Adoptive Immunotherapy ◽  
Ex Vivo ◽  
Cell Activity ◽  
Ex Vivo Expansion ◽  
Hla Dr

Abstract Background: Viral infections cause significant morbidity & mortality in patients undergoing unrelated allogeneic BMT transplantation before immune reconstitution is completed. It poses a greater risk for recipients of unrelated umbilical cord blood (uUCB) transplants as there is no established antiviral immunity in naïve UCB lymphocytes available for adoptive transfer. UCB T cells also lack Th1/Tc1 cytokines, Granzymes & Perforin which are prerequisites to control viral pathogens. Another major limitation of uUCBT is the lack of donor cells available for post-transplant donor leukocyte infusions (DLI) to boost immunity or induce GVL. However, a fraction of the uCB graft could be available for T cell expansion. In this study we evaluated the feasibility of ex vivo expansion of UCB T cells. We postulated that following expansion naïve T cells may mature & acquire a phenotype compatible with effector function as assessed by expression of essential cytokines & de novo expression of members of the granzyme-perforin pathway. Methods: Thawed UBC research samples with a leukocyte content <5% of an average UCB graft are processed. T cells are enriched with “EasySep” (StemCell Tech) to deplete CD14, CD16, CD19, CD56, & glycophorin A + cells. 5–7.5*105 T cells/ml are incubated with “CD3/28 T cell Expander” artificial APC beads (Dynal) in X Vivo-15 (BioWhittacker) + 200u/ml IL2 & 10% human serum in gas permeable bags. The initial purity of the T cells is 77–92%. The starting absolute T cell numbers ranged from 0.75 to 2*106 cells. Media & cytokines are added every other day to maintain a concentration of <2*106 cells/ml. Results: At the end of 14 days UCB T Cells expanded 67 fold +/− 36, n=6. There are significant alterations in phenotype over the 2 weeks (Table 1) with up to 40% of T cells in cell cycle. Compared to the starting resting UCB T cells the majority of expanded cells have acquired the phenotype of activated (HLA-DR+, CD25+ T cells) memory cells, at the expense of naive/recent thymic immigrants (CD45RA+/CD62+). There is an inverted CD4/CD8 ratio due to the higher expansion rate of CD8 T cells (p=0.0035) while there is no difference in apoptosis (p=0.57). However, they all retain expression of CD28 (96% ±8%) along with CD27. Although some T cells have acquired the capacity to secrete granzymes A and B these are still almost a log below normal adult peripheral blood (PB) values & perforin has not been detected. Similarly, while post expansion significantly more T cells secrete cytokines upon PMA + ionomycin stimulation (Table 1) they are below levels of adult PB. Conclusions: From our preliminary results we can demonstrate effective expansion & partial maturation of UCB T Cells. For example, if one starts with 2*106 total T cells & expands them 67 fold this could provide for DLI ~5*106 T cells/kg for an average pediatric patient (25kg). We are further optimizing & characterizing this model for T cell activity & repertoire. In sum, ex vivo expansion with CD3/CD28 co-stimulation may provide clinically relevant numbers T cells available for adoptive immunotherapy that have also undergone partial maturation. Characterization of Expanded T Cells as % of all Lymphocytes Variable Median SD CD3+ 99.8 0.1 CD4+ 35 11 CD4+/CD8+ 2.3 2.8 CD45RA+/RO− 13 11 CD45RO+/RA− 55 22 CD25+ 42 21 CD45RA+/CD62+ 38 20 CD45RA+/CD27+/CD8+ 52 15 CD45RA−/CD27+/CD8+ 46 15 KI67/CD8+ 42 9 Ki67/CD4+ 32 7 HLA DR+ 40 13 Granzyme A/CD8+ 54 18 Granzyme B/CD8+ 2 2 Perforin/CD8+ 0 0

Regulation of Hematopoietic Colony Forming Cells by CD4+CD25+ T Cells: A Role for T Regulatory Cells in Hematopoiesis?.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3181-3181
Author(s):  
Maite Urbieta ◽  
Isabel Barao ◽  
Monica Jones ◽  
William J. Murphy ◽  
Robert B. Levy
Keyword(s):  
T Cells ◽  
Growth Factors ◽  
T Cell ◽  
Treg Cell ◽  
Mhc Class Ii ◽  
Progenitor Cell ◽  
Cell Activity ◽  
Class Ii ◽  
Treg Cells

Abstract CD4+CD25+ T cells (Treg) comprise a small population within the normal peripheral CD4 T cell compartment. Their primary physiological role appears to be the regulation of autoimmune responses, however, in recent years it has been established that they can modulate anti-tumor as well as transplantation responses. Treg cells have been found to exert their affects on multiple types of immunologically relevant cells including CD4, CD8 and NK populations. Although model dependent, cytokines including TGFβ and IL-10 have been identified as mediators of this population’s regulatory activity and ex-vivo, the inhibition effected is generally contact dependent. Based upon the expanding application of Treg cells in stem cell transplants for the control of GVHD, rejection (HVG) and GVL responses, we hypothesized that following T cell receptor engagement and activation in recipients, CD4+CD25+ cells may modulate hematopoietic responses via production of effector cytokines. To address this question, various populations of CD4+CD25+ T cells were initially co-cultured with unfractionated syngeneic bone marrow cells (BMC) for 24–48 hours in medium supplemented with growth factors to maintain progenitor cell (i.e. CFU) function. Following co-culture, cells were collected and replated in triplicate in methylcellulose containing medium together with hematopoietic growth factors and five-seven days later, colonies were counted. CD4+CD25+ T cells were purified from BALB/c or B6–CD8−/− mice which were then activated for 3–8 days with anti-CD3/CD28 beads (a gift of Dr. B. Blazar, U. Minn.) These cells inhibited syngeneic CFU-IL3 colony ($25 cells) formation at ratios as low as 2:1 and 0.5:1 CD4+CD25+: BMC. Notably, Tregs from B6-CD8−/− mice exhibited comparable inhibition of allogeneic (BALB/c) CFU-IL3. Non-activated CD4+CD25+ T cells co-cultured with BMC did not exhibit this inhibitory activity nor did CD4+CD25− cells which contaminated (<10%) CD4+CD25+ populations. Activated Treg cells were also found to inhibit the production of CFU-HPP, a multi-potential marrow progenitor cell population. Contact dependency was found to be required for this effect as separation of activated CD4+CD25+ T cells from BMC “targets” in trans-well cultures abrogated inhibition. Prior depletion of CD25+ cells in vivo resulted in increases in CFU-GM 7–9 days after syngeneic BMT in mice suggesting that Tregs can inhibit hematopoietic reconstitution in vivo. To examine a potential contribution of TGFβ in this model, neutralizing anti-TGFβ mab was added during CD4+CD25+ T cell + BMC co-culture. The inhibition of CFU activity was abrogated in the presence of this antibody. To begin investigating the role of MHC class II molecules in this Treg cell activity, c-kit+ enriched (>85%) BMC from B6-MHC class II KO and B6-wt mice were co-cultured with B6 Treg cells from CD8−/− mice. In contrast to B6-wt c-kit enriched populations, CFU inhibition was not detected against the MHC class II deficient c-kit enriched BMC population. Antibody experiments are in progress to determine if cognate interaction is required between c-kit enriched cells and CD4+CD25+ T cells. In summary, this is the first report demonstrating that CD4+CD25+ T cells can alter hematopoietic progenitor cell activity. We hypothesize that membrane bound TGFβ may participate in effecting such regulation via direct Treg cell interactions with progenitor cell populations.

scholarly journals Differentiation of naive cord-blood T cells into CD19-specific cytolytic effectors for posttransplantation adoptive immunotherapy

Blood ◽  
2006 ◽  
Vol 107 (7) ◽  
pp. 2643-2652 ◽  
Author(s):  
Lisa Marie Serrano ◽  
Timothy Pfeiffer ◽  
Simon Olivares ◽  
Tontanai Numbenjapon ◽  
Jennifer Bennitt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Adoptive Immunotherapy ◽  
Lymphoblastic Leukemia ◽  
Cord Blood Transplantation ◽  
Hematopoietic Stem ◽  
B Lineage

AbstractDisease relapse is a barrier to achieving therapeutic success after unrelated umbilical cord-blood transplantation (UCBT) for B-lineage acute lymphoblastic leukemia (B-ALL). While adoptive transfer of donor-derived tumor-specific T cells is a conceptually attractive approach to eliminating residual disease after allogeneic hematopoietic stem cell transplantation, adoptive immunotherapy after UCBT is constrained by the difficulty of generating antigen-specific T cells from functionally naive umbilical cord-blood (UCB)–derived T cells. Therefore, to generate T cells that recognize B-ALL, we have developed a chimeric immunoreceptor to redirect the specificity of T cells for CD19, a B-lineage antigen, and expressed this transgene in UCB-derived T cells. An ex vivo process, which is compliant with current good manufacturing practice for T-cell trials, has been developed to genetically modify and numerically expand UCB-derived T cells into CD19-specific effector cells. These are capable of CD19-restricted cytokine production and cytolysis in vitro, as well as mediating regression of CD19+ tumor and being selectively eliminated in vivo. Moreover, time-lapse microscopy of the genetically modified T-cell clones revealed an ability to lyse CD19+ tumor cells specifically and repetitively. These data provide the rationale for infusing UCB-derived CD19-specific T cells after UCBT to reduce the incidence of CD19+ B-ALL relapse.

scholarly journals The role of the thymus in T-cell immune reconstitution after umbilical cord blood transplantation

Blood ◽  
2014 ◽  
Vol 124 (22) ◽  
pp. 3201-3211 ◽  
Author(s):  
Ioannis Politikos ◽  
Vassiliki A. Boussiotis
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
T Cell Receptor ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Cord Blood Transplantation ◽  
Cell Receptor ◽  
Hematopoietic Stem ◽  
T Cell Reconstitution

Abstract Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells for patients without HLA-matched adult donors. UCB contains a low number of nucleated cells and mostly naive T cells, resulting in prolonged time to engraftment and lack of transferred T-cell memory. Although the first phase of T-cell reconstitution after UCB transplantation (UCBT) depends on peripheral expansion of transferred T cells, permanent T-cell reconstitution is mediated via a central mechanism, which depends on de novo production of naive T lymphocytes by the recipient’s thymus from donor-derived lymphoid-myeloid progenitors (LMPs). Thymopoiesis can be assessed by quantification of recent thymic emigrants, T-cell receptor excision circle levels, and T-cell receptor repertoire diversity. These assays are valuable tools for monitoring posttransplantation thymic recovery, but more importantly they have shown the significant prognostic value of thymic reconstitution for clinical outcomes after UCBT, including opportunistic infections, disease relapse, and overall survival. Strategies to improve thymic entry and differentiation of LMPs and to accelerate recovery of the thymic stromal microenvironment may improve thymic lymphopoiesis. Here, we discuss the mechanisms and clinical implications of thymic recovery and new approaches to improve reconstitution of the T-cell repertoire after UCBT.

scholarly journals Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2770-2778 ◽  
Author(s):  
AH Galy ◽  
D Cen ◽  
M Travis ◽  
S Chen ◽  
BP Chen
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Cell Activity ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Hla Dr ◽  
Adult Bone

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin-ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA-cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.

scholarly journals Reduced NFAT1 Protein Expression in Human Umbilical Cord Blood T Lymphocytes

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3101-3107 ◽  
Author(s):  
Suzanne Kadereit ◽  
Shaden F. Mohammad ◽  
Robin E. Miller ◽  
Kathleen Daum Woods ◽  
Chad D. Listrom ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Molecular Mechanisms ◽  
Constitutive Expression ◽  
Unrelated Donor ◽  
Human Umbilical Cord ◽  
Ifn Γ

Abstract Umbilical cord blood (UCB) stem cells from related and unrelated allogeneic donors have emerged as novel treatment for patients with hematologic malignancies. The incidence and severity of acute graft-versus-host disease (GVHD) after UCB transplantation compares favorably with that observed in recipients of matched unrelated donor allogeneic grafts, but remains a major cause of morbidity and mortality. It has been shown that stimulated lymphocytes from UCB have reduced production of cytokines including interferon-γ (IFN-γ) and tumor necrosis factor- (TNF-), which play a role in GVHD pathophysiology. We investigated the molecular mechanisms underlying this reduced cytokine production by analyzing expression of nuclear factor of activated T cells-1 (NFAT1) in UCB T cells. We detected no constitutive expression of NFAT1 protein in unstimulated UCB T cells compared with adult T cells. Moreover, although NFAT1 expression in UCB T cells was upregulated after prolonged (40 hours) T-cell stimulation, it was only partially upregulated when compared with adult controls. Our observation of minimal NFAT1 expression after stimulation correlated with reduced cytoplasmic IFN-γ and TNF- production in UCB T cells studied simultaneously. Reduced NFAT1 expression may blunt amplification of donor UCB T-cell alloresponsiveness against recipient antigens, thereby potentially limiting GVHD incidence and severity after allogeneic UCB transplantation.

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