In vitro-differentiated T/natural killer-cell progenitors derived from human CD34+ cells mature in the thymus

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a treatment option for patients with hematopoietic malignancies that is hampered by treatment-related morbidity and mortality, in part the result of opportunistic infections, a direct consequence of delayed T-cell recovery. Thymic output can be improved by facilitation of thymic immigration, known to require precommitment of CD34(+) cells. We demonstrate that Delta-like ligand-mediated predifferentiation of mobilized CD34(+) cells in vitro results in a population of thymocyte-like cells arrested at a T/natural killer (NK)-cell progenitor stage. On intrahepatic transfer to Rag2(-/-)gamma(c)(-/-) mice, these cells selectively home to the thymus and differentiate toward surface T-cell receptor-alphabeta(+) mature T cells considerably faster than animals transplanted with noncultured CD34(+) cells. This finding creates the opportunity to develop an early T-cell reconstitution therapy to combine with HSCT

The prevalence of antibodies against the HLA-DRB3 protein in kidney transplantation and the correlation with HLA expression

Human leukocyte antigen (HLA)-DRB3 is a functional HLA class II gene, which has a limited allele diversity in the human population. Furthermore, the HLA-DRB3 gene is only present in a subset of individuals. Therefore, in organ transplantation, this HLA molecule is frequently mismatched between patient and graft donor and thus antibodies against this mismatched HLA molecule can develop. In this study, we aimed to evaluate the prevalence and reactivity of these antibodies and aimed to identify factors that underlie antibody formation against HLA-DRB3. We showed in our patient cohort that HLA-DRB3 antibodies are identified in about 7% of all patients that were screened with solid phase assays. In these assays, we observed multiple antibody reactivity patterns indicating that HLA-DRB3 harbours multiple epitopes. In those cases, where we succeeded at tracing back the induction of these antibodies to the molecular HLA typing of the immunogenic event, we noticed a different frequency of HLA-DRB1 allele groups in the donors as compared to a control group. To a certain extent this distribution (e.g. HLA-DRB1* 11 individuals) could be linked to an altered expression level. However, it also appears that different HLA-DRB3 alleles (e.g. HLA-DRB3* 01 group) vary in their immunogenicity without having an expression difference. In conclusion, our study provides information on the immunogenicity and reactivity patterns of antibodies against HLA-DRB3 in kidney transplantation, and it points towards the possibility of HLA expression as a factor underlying antibody formation

Differential Th2 polarizing capacities of differently matured moDC.

<p>Naive CD4⁺ T cells were co-cultured for 7 days with PGE₂/TNF-α (dark gray triangle), LPS/IFN-γ (light gray circle) or FMKp/IFN-γ (black square) matured DC. Expression of GATA3, IL-4, IL-5 and IL-13 and production of IL-5 and IL-13 were monitored. IL-5 and IL-13 protein production by naive T cells co-cultured with differently matured DC was determined in the supernatant of the co-culture. Graphs are representative of 5 independent experiments.</p

Differential Th1 polarizing capacity of differently matured DC.

<p>Naive CD4⁺ T cells were co-cultured for 7 days with PGE₂/TNF-α (dark gray triangle), LPS/IFN-γ (light gray circle) or FMKp/IFN-γ-(black square) matured DC. (A) Expression of T-bet and IFN-γ and production of IFN-γ were monitored. Graphs are representative of 11 independent experiments. (B) Comparison of the expression of T-bet and IFN-γ on day 5 and IFN-γ production on day 7 of differently matured DC cultured with naive CD4⁺ T cells. 11 independent experiments and their median levels are shown. Wilcoxon signed-rank test significance **<i>P</i>≤0.01, ***<i>P</i><0.001 (C) Correlation between T-bet and IFN-γ expression on day 5 and IFN-γ secretion on day 7 of FMKp/IFN-γ-matured DC in co-culture with naive CD4⁺ T cells. Nonparametric Spearman correlation test significance indicated in the graphs. (D) Expression of CD45RO, CD25, and intracellular IFN-γ of naive CD4⁺ T cells cultured for 7 days with differently matured DC as indicated above the graphs. On day 6 GolgiPlug and GolgiStop were added to the co-culture and the staining was performed on day 7. Cells shown in the plots represent living singlet cells gated on CD3⁺CD4⁺ (dark gray population). T cells positive for IFN-γ are shown in black. Dot plots are representative graphs of 3 independent experiments.</p

FMKp/IFN-γ-matured DC polarize a subpopulation of naive T cells into the Th1 lineage.

<p>iDC or FMKp/IFN-γ-matured DC were pulsed with PADRE for 1 h before the addition of autologous CD4⁺CD45RA⁺ T cells to the 7-day co-culture. (A) Expression of T-bet and IFN-γ and IFN-γ production of naive CD4⁺ T cells co-cultured with iDC (□) or FMKp/IFN-γ-matured DC (▪) are compared. CD3ε was used as housekeeping gene and the expression data were normalized to the relative mRNA content of naive T cells on day 0. IFN-γ production by naive T cells co-cultured with differently matured DC was determined in the supernatant of the co-culture on day 1, 3, 5 and 7 by CBA. Graphs are representative of 11 independent experiments. (B) On day 7, T cells were stained for expression of CD45RO and CD25 and analyzed by flow cytometry. Cells are gated in FSC/SSC on lymphocyte gate, excluding dead cells and doublets (light gray dots), and selected for CD4⁺ cells (dark gray and black dots). Percentages of CD45RO and CD25 positive populations are indicated in the plots. Dot plots are representative of 5 independent experiments. (C) On day 6 of the co-culture, GolgiPlug and GolgiStop were added to T cells with or without PMA/ionomycin restimulation. The next day, intracellular IFN-γ staining was performed and cells were analysed by flow cytometry. Representative dot plots of CD45RO and IFN-γ expression of CD4⁺ T cells co-cultured with FMKp/IFN-γ-matured DC are shown. Data are representative of 5 independent experiments.</p

Differential Th1 polarizing capacity of differently matured plasmacytoid DC.

<p>pDC, isolated from fresh blood, were stimulated overnight with different cocktails and 24 h later autologous naive CD4⁺ T cells were added and the expression of T-bet and IFN-γ and secretion of IFN-γ were monitored during 7 days. (A) pDC were stimulated overnight with IL-3 (full gray line), ODN2216 (dashed black line) or with a combination of both (full black line). (B) Comparison of the capacity of differently matured pDC to induce Th1 polarization. pDC were incubated with PGE₂/TNF-α (dark gray triangle), LPS/IFN-γ (light gray circle) or FMKp/IFN-γ (black square) cocktail in the presence of IL-3. Representative data from 2 independent experiments are shown.</p

Presence of CD4⁺CD45RO⁺ T cells in the DC-CD4⁺CD45RA⁺ T cell co-culture influences Th1 read-out parameters.

<p>(A) CD4⁺, CD4⁺CD45RO⁺, CD4⁺CD45RA⁺ populations have been isolated by negative immunomagnetic separation from freshly isolated PBMC. IFN-γ production of total CD4⁺ (black square), CD4⁺CD45RA⁺ (light gray circle), and CD4⁺CD45RO⁺ (dark gray triangle) T cells co-cultured with FMKp/IFN-γ-matured DC for 7 days as measured by CBA. (B) Contribution of contaminating CD4⁺CD45RO⁺ T cells (2.5, 5 or 10%) to CD4⁺CD45RA⁺-derived IFN-γ-production compared with pure (>99.9%) CD4⁺CD45RA⁺ T cell populations. Data shown are representative of 2 independent experiments.</p

Dependency of DC-induced Th1 polarization on DC-derived soluble factors.

<p>(A) FMKp/IFN-γ-matured DC were either extensively washed (○) or not (•) 6 h after induction of maturation and co-cultured for 7 days with autologous CD4⁺CD45RA⁺ T cells. Expression of T-bet and IFN-γ and total IFN-γ production are shown. Graphs are representative of 5 independent experiments. (B) APC-independent assay using immobilized anti-CD3 allows studying the influence of DC-derived soluble factors on T cell polarization. 5×10⁴ cells CD4⁺CD45RA⁺ T cells were cultured for 5 days without (Δ) or with (▴) washed FMKp/IFN-γ-matured DC-derived supernatant in presence of plate-bound α-CD3 (0.25 µg/ml) in a round bottom 96-well plate. Transcriptional induction of T-bet and IFN-γ and secretion of IFN-γ were determined. Data shown are representative data of 3 independent experiments. (C) FMKp/IFN-γ- and HKLM/IFN-γ-matured DC induce Th1 polarization. iDC were matured with HKLM/IFN-γ (□) or FMKp/IFN-γ (▪) and co-cultured with CD4⁺CD45RA⁺ T cells. Expression of T-bet and IFN-γ and total IFN-γ production are shown. Data are representative of 4 independent experiments.</p

In vitro-differentiated T/natural killer-cell progenitors derived from human CD34+cells mature in the thymus

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a treatment option for patients with hematopoietic malignancies that is hampered by treatment-related morbidity and mortality, in part the result of opportunistic infections, a direct consequence of delayed T-cell recovery. Thymic output can be improved by facilitation of thymic immigration, known to require precommitment of CD34(+) cells. We demonstrate that Delta-like ligand-mediated predifferentiation of mobilized CD34(+) cells in vitro results in a population of thymocyte-like cells arrested at a T/natural killer (NK)-cell progenitor stage. On intrahepatic transfer to Rag2(-/-)gamma(-/-)(c) mice, these cells selectively home to the thymus and differentiate toward surface T-cell receptor-alpha beta(+) mature T cells considerably faster than animals transplanted with noncultured CD34(+) cells. This finding creates the opportunity to develop an early T-cell reconstitution therapy to combine with HSCT. ( Blood. 2010; 115:261-264