Natural killer (NK) cells have different roles in the host response against Plasmodium-induced malaria depending on the stage of infection. Liver NK cells have a protective role during the initial ...hepatic stage of infection by production of the TH1-type cytokines IFN-γ and TNF-α. In the subsequent erythrocytic stage of infection, NK cells also induce protection through Th1-type cytokines but, in addition, may also promote development of cerebral malaria via CXCR3-induction on CD8(+) T cells resulting in migration of these cells to the brain. We have recently shown that the regulatory Ly49E NK receptor is expressed on liver NK cells in particular. The main objective of this study was therefore to examine the role of Ly49E expression in the immune response upon Plasmodium berghei ANKA infection, for which we compared wild type (WT) to Ly49E knockout (KO) mice. We show that the parasitemia was higher at the early stage, i.e. at days 6-7 of Plasmodium berghei ANKA infection in Ly49E KO mice, which correlated with lower induction of CD69, IFN-γ and TNF-α in DX5(-) liver NK cells at day 5 post-infection. At later stages, these differences faded. There was also no difference in the kinetics and the percentage of cerebral malaria development and in lymphocyte CXCR3 expression in WT versus Ly49E KO mice. Collectively, we show that the immune response against Plasmodium berghei ANKA infection is not drastically affected in Ly49E KO mice. Although NK cells play a crucial role in Plasmodium infection and Ly49E is highly expressed on liver NK cells, the Ly49E NK receptor only has a temporarily role in the immune control of this parasite.
The NOD-LtSZ scid/scid (NOD/SCID) repopulation assay is the criterion for the study of self-renewal and multilineage differentiation of human hematopoietic stem cells. An important shortcoming of ...this model is the reported absence of T-cell development. We studied this aspect of the model and investigated how it could be optimized to support T-cell development. Occasionally, low-grade thymic engraftment was observed in NOD/SCID mice or Rag2−/−γc−/− mice. In contrast, the treatment of NOD/SCID mice with a monoclonal antibody against the murine interleukin-2Rβ, (IL-2Rβ) known to decrease natural killer cell activity, resulted in human thymopoiesis in up to 60% of the mice. T-cell development was phenotypically normal and resulted in polyclonal, mature, and functional CD1−TCRαβ+ CD4+ or CD8+single-positive T cells. In mice with ongoing thymopoiesis, peripheral T cells were observed. TREC analysis showed that T cells with a naive phenotype (CD45RA+) emerged from the thymus. In approximately half of these mice, the peripheral T cells included a pauciclonal outgrowth of CD45RO+ cells. These data suggest that all elements of a functional immune system were present in these animals.
Objectives: To summarize important findings from research on chimeric antigen receptor (CAR) T-cell immunotherapy in cancer. We discuss CAR design, cell products, toxicity management, heterogenous ...solid tumors and allogeneic transfer.
Methods: A review of literature was conducted. The available literature was selected on original research, state-of-the art design, relevance to the objective and journal impact factor.
Results: First-generation CARs provide patient T cells with tumor-specific antigen recognition. Second- and third-generation CARs incorporate costimulatory domains for enhanced T-cell persistence and antitumor activity. Fourth-generation CAR T cells (TRUCKs) include a cytokine production cassette, and hold promise in the treatment of heterogenous solid tumors. Transduced cell phenotype and subset composition are important factors. Suicide genes and safety switches are designed to decrease potential toxicity. Multi-specific CAR T cells can address heterogenous tumors. Allogeneic, off-the-shelf CAR T cells might reduce the production delay.
Conclusion: CAR T cells have revolutionized the immunotherapeutic treatment of cancer: exciting results in refractory and relapsed B-cell malignancies have been published. Neurologic complications, solid tumor management and allogeneic constructs require further research. In conclusion, further design adjustments will enable CAR T cells to decisively reshape the field of cancer immunotherapy.
The Ly49E NK receptor is a unique inhibitory receptor, presenting with a high degree of conservation among mouse strains and expression on both NK cells and intraepithelial-localised T cells. Amongst ...intraepithelial-localised T cells, the Ly49E receptor is abundantly expressed on CD8αα-expressing innate-like intestinal intraepithelial lymphocytes (iIELs), which contribute to front-line defense at the mucosal barrier. Inflammatory bowel diseases (IBDs), encompassing Crohn's disease and ulcerative colitis, have previously been suggested to have an autoreactive origin and to evolve from a dysbalance between regulatory and effector functions in the intestinal immune system. Here, we made use of Ly49E-deficient mice to characterize the role of Ly49E receptor expression on CD8αα-expressing iIELs in the development and progression of IBD. For this purpose we used the dextran sodium sulphate (DSS)- and trinitrobenzenesulfonic-acid (TNBS)-induced colitis models, and the TNFΔARE ileitis model. We show that Ly49E is expressed on a high proportion of CD8αα-positive iIELs, with higher expression in the colon as compared to the small intestine. However, Ly49E expression on small intestinal and colonic iIELs does not influence the development or progression of inflammatory bowel diseases.
This workshop aims to present the outlines of a research project situated in the interdisciplinary research domain of bibliotherapy: the use of literature in healthcare contexts. With this project we ...want to demonstrate that different syndromes and pain experiences require a diversification of the (prescribed or non-prescribed) texts and the associated reading practice. On the one hand, the project aims to refine the international discourse of bibliotherapy on this central point. On the other hand, through concrete reading and co-reading sessions with participants from different medical backgrounds (cancer patients, the elderly, patients in rehabilitation, renal dialysis patients), we aim to gain a sharper insight into various bibliotherapeutic needs associated with specific syndromes. The project brings together a team of literary scholars and medical experts who, with their joint efforts, want to make a significant contribution to the interdisciplinary domain of health humanities and narrative medicine.
In the project we want to find out how literary texts have a therapeutic effect in specific care contexts and we want to increase that effect through a better understanding of the functioning of that text. We assume that the therapeutic effect of literary texts is determined by an interplay between the text itself (with its specific characteristics), the way and circumstances of reading and the mental and physical state of the patient-reader.
In the workshop we want to present the outline of the project and together with participants simulate a shared reading session.
Human hemopoietic stem cells (HSC) have been shown to engraft, differentiate, and proliferate in the hemopoietic tissues of sublethally irradiated NOD/LtSZ scid/scid (NOD/SCID) mice. We used this ...model to study homing, survival, and expansion of human HSC populations from different sources or phenotype. We observed that CD34+ cells homed specifically to bone marrow (BM) and spleen, but by 3 days after injection, survived only in the BM. These BM-homed CD34+ cells proliferated intensively and gave rise to a 12-fold, 5.5-fold, and 4-fold expansion in 3 days for umbilical cord blood, adult mobilized peripheral blood, and adult BM-derived cells, respectively. By injection of purified subpopulations, it was demonstrated that both CD34+38+ and CD34+38- umbilical cord blood HSC homed to the BM and expanded. Importantly, kinetics of expansion were different: CD34+38+ cells started to increase in cell number from day 3 onwards, and by 4 wk after injection, virtually all CD34+ cells had disappeared. In contrast, CD34+38- cells remained quiescent during the first week and started to expand intensively from the third week on. In this paper, we have shown that homing, survival, and expansion of stem cells are three independent phenomena important in the early phase of BM engraftment and that kinetics of engraftment differ between CD34+38+ and CD34+38- cells.
Human bone marrow and umbilical cord blood are sources of allogeneic hematopoietic stem cells for transplantation, which is a life-saving treatment in a variety of diseases but is burdened by delayed ...T-cell reconstitution. Observational studies evaluating T-cell reconstitution in post-transplant recipients suggest that cord blood hematopoietic stem cells have a more effective capacity for T-cell reconstitution. This study focuses on the comparison of the capacity of cord blood and bone marrow hematopoietic stem cells to generate T cells in vitro.
Hematopoietic stem cells were cultured in OP9-delta-like-1 and OP9-green fluorescent protein co-cultures to estimate T and myeloid generation capacity, respectively. Phenotypic markers of T-lineage or myeloid differentiation were measured by flow cytometry and used to analyze their kinetics as a function of culture time. Hematopoietic stem cells were labeled with carboxyfluorescein diacetate succinamidyl ester and analyzed after culture to track their phenotypic progression in consecutive generations. Mixed OP9-delta-like-1 co-cultures were done with either carboxyfluorescein diacetate succinamidyl ester-labeled bone marrow and unlabeled cord blood hematopoietic stem cells, or vice versa, to evaluate their mutual influence on T-lineage differentiation. The T-cell potential of hematopoietic stem cells was addressed quantitatively by limiting dilution analysis.
Bulk cultures showed faster and more extensive T-cell differentiation by cord blood hematopoietic stem cells. Furthermore, the T-lymphoid differentiation capacity of cord blood and bone marrow hematopoietic stem cells can be discriminated very early based on the coordinated expression of CD34 and CD7. Mixing experiments with cord blood hematopoietic stem cells and bone marrow hematopoietic stem cells showed that these differences are cell intrinsic. Quantitative clonal analyses demonstrated that CD34(+)CD38(-/lo) hematopoietic stem cells from cord blood contained a two-fold higher T-lineage generation capacity than CD34(+)CD38(-/lo) bone marrow hematopoietic stem cells, whereas the myeloid differentiation was similar.
Our data shows that cord blood hematopoietic stem cells have higher T-lymphoid differentiation potential than bone marrow hematopoietic stem cells and that this property is cell autonomous.
Abstract 3670
Poster Board III-606
Human 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.
No relevant conflicts of interest to declare.
In vitro generation of mature T cells from human hematopoietic stem and progenitor cells (HSPC) could fulfill two existing needs. First, it could enhance and quicken T cell immune reconstitution ...after stem cell transplantation, which is very slow and generates a skewed TCR repertoire. Second, by generation of tumour antigen specific T cells it could provide an efficient therapy for numerous malignancies and could enhance GVT effect in the context of allogeneic SCT, without aggravating GVHD. T cells can be generated from human HSPC by culturing them on the murine stromal cell line OP9-transduced with the Notch ligand Delta-like-1 (OP9-DL1). Notch receptor activation is essential for T cell development. However, it is unclear whether Notch activation is sufficient for end maturation into functionally and phenotypically mature TCR positive cells. It was shown that human CD34+ cells cultured on OP9-DL1 differentiate to T cells which can proliferate and produce interferon-g upon polyclonal stimulation. The nature of the mature cells generated in these cultures, however, has not been well studied. CD34+ HSPC from postnatal thymus (PNT) or cord blood were cocultured with OP9-DL1, in the presence of the cytokines Flt-3L (5 ng/ml), SCF (2.5 ng/ml) and IL-7 (5 ng/ml). Every 3–5 days cells were harvested and transferred to fresh OP9-DL1 cells. At repetitive timepoints, an aliquot of the cells was analysed phenotypically. In some experiments, IL-15 was added to the culture. For some experiments, cells harvested from OP9-DL1 at the timepoint mature T cells were observed (usually about d 40 of culture), were transferred to feeder cells, consisting of JY cell line (5.104 cells/ml irradiated with 50 Gy and PBMC (5.105/ml irradiated with 40 Gy), in the presence of PHA (1 mg/ml). After 7 days, IL-2 (50 IU/ml) was added to the culture. Every 14 days, cells were restimulated with new feeders (irradiated JY and PBMC) and new addition of PHA. After 3 weeks of stimulation cells were stimulated overnight with 15 ng/ml PMA and 1500 ng/ml ionomycin, and 18 hours later cells were checked for intracellular presence of cytokines. We investigated whether the T cell population generated in these cultures contains mature cells with the characteristics of TCRγδ cells and of positively selected CD8 or CD4 single positive (SP) TCRαβ cells as observed in the human thymus. We found that under the described conditions, HSPC mature into CD1-CD27+ phenotypically mature T cells, with the TCRγδ fraction maturing faster and more efficiently compared to the TCRαβ fraction. Consistent with a mature phenotype, TCRγδ cells were mostly CD8αα or double negative (DN). No mature CD4 SP TCRαβ cells were observed and the mature CD8 SP cells co-expressed variable ratios of CD8αβ and CD8αα dimers, suggesting that these cells are not conventional positively selected TCRαβ cells. In support of this hypothesis, both mature CD1- TCRαβ and TCRγδ cells expressed the IL2Rβ receptor consitutively and both populations proliferated on IL-15 without prior antigen stimulation, CD8αα (TCRαβ and TCRγδ) cells being the most IL-15 responsive. Mature activated T cells secreted IFN-γ and TNFα, little or no IL-2 and IL-4, with no difference observed between TCRαβ and TCRγδ cells. These data suggest that CD8 TCRαβ cells generated in these cultures are unconventional CD8 cells possibly maturated through agonist selection. However, when cells harvested after 40 days of culture on OP9-DL1 were stimulated with PHA and IL-2 for 3 weeks, conventional appearing CD8αβ cells emerged, with a cytokine production profile similar to that of thymic CD8αβ TCRαβ T cells, with the majority of cells secreting IFN-γ and IL-2. We can conclude from these data that OP9-DL1 supports the development of both unconventional and conventional CD8+ TCRαβ cells, of which the generation and selection process are currently being investigated. Also the in vitro anti-tumor capacities of both populations need to be addressed.
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