In vitro generation of mature, naive antigen-specific CD8(+) T cells with a single T-cell receptor by agonist selection.

Peripheral blood T cells transduced with a tumor-specific T-cell receptor (TCR) face problems of auto-reactivity and lack of efficacy caused by cross-pairing of exogenous and endogenous TCR chains, as well as short term in vivo survival due to activation and growth factor-induced differentiation. We here studied an alternative strategy for the efficient generation of naive CD8(+) T cells with a single TCR. TCR-transduced human postnatal thymus-derived and adult mobilized blood-derived hematopoietic progenitor cells (HPCs) were differentiated to CD4(+)CD8(+) double-positive T cells using OP9-Delta-like 1 (OP9-DL1) cultures. Addition of the agonist peptide induced double positive cells to cross-present the peptide, leading, in the absence of co-stimulation, to cell cycle arrest and differentiation into mature CD8(+) T cells. Comprehensive phenotypic, molecular and functional analysis revealed the generation of naive and resting CD8(+) T cells through a process similar to thymic positive selection. These mature T cells show a near complete inhibition of endogenous TCRA and TCRB rearrangements and express high levels of the introduced multimer-reactive TCR. Upon activation, specific cytokine production and efficient killing of tumor cells were induced. Using this strategy, large numbers of high-avidity tumor-specific naive T cells can be generated from readily available HPCs without TCR chain cross-pairing.

The simultaneous appearance of a nasal natural killer-cell lymphoma and acute myelogenous leukemia.

INTRODUCTION AND AIM: Sinonasal malignant neoplasms are uncommon, with an annual incidence of less than 1/100,000. About 80% of these are squamous cell carcinoma. Adenocarcinoma and adenoid cystic carcinoma are next in frequency. Lymphoma of the nasal cavity, paranasal sinuses and nasopharynx are rare, constituting less than 5% of all extranodal lymphomas. CASE REPORT: A 47-year-old man was referred to our hospital because of severe headache and progressive facial pain. He also complained of right-sided visual acuity. He had a manifest exopthalmia with disturbed eye movements. Nasoscopy showed a large mass with atypical appearance. CT and MRI showed a bilateral ethmoid mass invading the frontal sinuses, the right orbit, the lamina cribrosa and the right frontal cerebral region, and growing posteriorly through the choana. The first biopsies were inconclusive, showing only necrotic cells and purulent inflammation with epithelial elements. A larger biopsy demonstrated a high-grade malignant tumour with necrosis. The differential diagnosis of undifferentiated sinonasal carcinoma, undifferentiated neuro-endocrine tumour or T-cell lymphoma was suggested. In the meantime our patient developed high fever and sudden-onset pancytopenia. Bone marrow punction showed 65% blasts, leading to the diagnosis of AML type M2. He was immediately referred for chemotherapy, but died in intensive care before his first session. The biopsy of the sinonasal mass was diagnosed surprisingly as a natural killer cell lymphoma stage IVB. CONCLUSIONS: Natural killer cell lymphoma is rare in Europe. The simultaneous appearance of a NK-cell lymphoma and acute myelogenous leukemia has, as far as we know, never been described in the English literature before.

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 TCRαß(+) 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 CD8αß SP TCRγδ(+) cells and to TCRαß(+) 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.

Outcome in critically ill patients with allogeneic BM or peripheral haematopoietic SCT: a single-centre experience.

Outcome in haematological patients who develop critical illness has significantly improved over the last two decades, but less so in allogeneic BMT recipients. We prospectively investigated the outcome of 44 haematological patients with allogeneic BM or haematopoietic SCT (ABMT/AHSCT) requiring admission to the intensive care unit (ICU) of Ghent University Hospital between January 2000 and December 2007. We related outcome to the cause of critical illness, which was categorized as documented or clinically suspected bacterial infection, non-bacterial infection and non-infectious disease. Mechanical ventilation was required in 32 patients, and 12 patients received renal replacement therapy. Overall ICU-mortality, in-hospital mortality and 6-month mortality rates were 61, 75 and 80%, respectively. Hospital mortality rates in patients with bacterial infection (n=14), non-bacterial infection (n=13) and non-infectious disease (n=17) were 43, 85 and 94% (P=0.003). After adjustment for severity of illness sequential organ failure assessment (SOFA) score, bacterial infection (odds ratio 0.06, 0.01-0.36, P=0.002) was associated with significantly lower odds for hospital mortality. On the basis of our experience, ICU referral of ABMT/AHSCT patients is justifiable, as an acceptable fraction of these patients have longer-term survival. Documented or clinically suspected bacterial infection as the cause of critical illness is associated with better prognosis in comparison with other causes.

Human intrathymic development: a selective approach.

Human T lymphocytes can be generated from CD34 progenitor cells from different sources. This can be obtained in an in vivo model wherein human thymic tissue and fetal liver is transplanted in an immunodeficient mouse. However, human T cells are also generated in immunodeficient mice without co-transplantation of human thymus or in in vitro hybrid human-mouse fetal thymus organ culture. This shows that xenogeneic mouse thymus tissue supports human T cell differentiation. Finally, human T cells are generated on co-culture with murine stromal cells that express the Delta-like1 ligand for the Notch receptor. How these different environments influence the human T cell repertoire is reviewed and discussed.

Both CD34+38+ and CD34+38- cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion.

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.

Signals from the IL-9 receptor are critical for the early stages of human intrathymic T cell development.

Highly purified human CD34+ hemopoietic precursor cells differentiate into mature T cells when seeded in vitro in isolated fetal thymic lobes of SCID mice followed by fetal thymus organ culture (FTOC). Here, this chimeric human-mouse FTOC was used to address the role of IL-9 and of the alpha-chain of the IL-9 receptor (IL-9Ralpha) in early human T cell development. We report that addition of the mAb AH9R7, which recognizes and blocks selectively the human high affinity alpha-chain of the IL-9R, results in a profound reduction of the number of human thymocytes. Analysis of lymphoid subpopulations indicates that a highly reduced number of cells undergo maturation from CD34+ precursor cells toward CD4+CD3-CD8-CD1+ progenitor cells and subsequently toward CD4+CD8+ double positive (DP) thymocytes. Addition of IL-9 to the FTOC resulted in an increase in cell number, without disturbing the frequencies of the different subsets. These data suggest that IL-9Ralpha signaling is critical in early T lymphoid development.

Human T lymphopoiesis. In vitro and in vivo study models.

Successive steps in T lymphocyte differentiation and T potential of human stem cells (HSC) can be tested in the following models: (a) the infusion of cells in NOD-SCID mice, (b) the injection of cells in renconstituted SCID/hu mice, (c) the differentiation of cells in fetal thymus organ culture (FTOC), and (d) on thymic stromal layers. Using mixed human-murine FTOC, we showed (a) TCR alpha beta, TCR gamma delta lymphocytes, NK cells, and dendritic cells complete their differentiation, (b) IL-7R alpha signaling and IL-7 are essential, (c) a detailed phenotypic and functional analysis of discrete successive steps of positively selected thymocytes, (d) an efficient transduction of genes in HSC with persistent gene expression throughout the T-lymphocyte differentiation, and (e) adaptation to submerging high oxygen culture increases the test sensitivity to a clonal assay. Other approaches are the in vivo SCID/hu reconstitution model. With this method small fragments of human fetal liver and thymus are implanted under the kidney capsule of an adult SCID mouse with result in an impressive human thymus organ, six months after transplantation. We use this model to study thymus T-cell developmental kinetics, development of gene-marked precursor cells and thymic homing of precursor cells.

Thymic repopulation by CD34(+) human cord blood cells after expansion in stroma-free culture.

Thymic repopulation by transplanted hematopoietic progenitor cells (HPC) is likely to be important for long-term immune reconstitution and for successful gene therapy of diseases affecting the T-cell lineage. However, the T-cell progenitor potential of HPC, cultured in vitro for cell number expansion and gene transfer remains largely unknown. Here, we cultured highly purified human umbilical cord blood (CB) CD34(+)CD38(-) or CD34(+)CD38(+) cells for up to 5 weeks in stroma-free cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3), and IL-6 and investigated thymus-repopulating ability of expanded cells in vitro and in vivo. After up to 5 weeks of culture in IL-3 + SCF + IL-6 or TPO + FL + SCF supplemented medium, the progeny of CD34(+)CD38(-) CB cells generated T cells and natural killer cells in the thymus. Limiting dilution experiments demonstrated increase in the number of T-cell progenitors during culture. After 3 weeks of culture, gene marked CD34(+)CD38(-) CB cells injected in the human thymus fragment transplanted in severe combined immunodeficient (SCID) mice (SCID-hu) generated thymocytes expressing the retroviral encoded marker gene GFP in vivo. Thus, our results show that the progeny of CD34(+)CD38(-) CB cells cultured for extensive periods, harbor thymus-repopulating cells that retain T-cell progenitor potential after expansion and gene transfer.

Human immunodeficiency virus nef gene expression affects generation and function of human T cells, but not dendritic cells.

Human immunodeficiency virus (HIV)-infected individuals develop an acquired immune deficiency syndrome (AIDS) due to loss in their lymphocyte numbers and cellular defects in T cells and antigen-presenting cells (APC). HIV infection of the thymus results in deficient replenishment of the peripheral naive T-cell pool. The HIV nef gene was shown to be important for progression towards AIDS and cellular depletion of the infected thymus. Here, we demonstrate by retroviral gene transfer that nef expression, in the absence of other HIV genes, impaired human thymic T-cell development. Thymocytes were generated in reduced numbers and downmodulated CD4 and CD8beta cell surface expression. T cells grown from nef-expressing thymocytes were hyperproliferative in vitro upon T-cell receptor triggering. Mature dendritic cells (DC) were functional and had normal surface CD4 levels despite nef expression. Thus, nef expression alone may contribute to AIDS development by reduced T-cell generation and T-cell hyperresponsiveness.

Bone marrow CD34 cells generate fewer T cells in vitro with increasing age and following chemotherapy.

To assess the influence of high-dose chemotherapy and age on the intrinsic capacity of stem cells to generate T cells, CD34+ cells derived from bone marrow used in clinical transplantation were evaluated in an in vitro T-cell assay using a mouse thymic microenvironment. CD34+ cells were sorted from healthy donor and autologous back-up bone marrow after density gradient centrifugation and depletion for CD1, 3, 4, 7, 8, 19 and glycophorin A using magnetic beads. CD34+ cells were then introduced in day 14-15 fetal SCII) mouse thymus lobes by incubation in hanging drops for 48 h. After transfer to gelfoam rafts they were cultured for variable time periods. The lobes were then homogenized in a tissue grinder for flow cytometric analysis gating on human cells. These were evaluated for CD4, CD8, CD3 and HLA-DR surface expression. 51 samples were analysed and three patterns of T-cell precursor development could be observed. In pattern A no human cells could be recovered, in pattern B maturation stopped at the CD4+ CD8- CD3- pre-T-cell stage, and in pattern C maturation to double-positive CD4+ CD8+ thymocytes was reached. In 25 healthy donors under age 40 three showed pattern A, 12 pattern B and 10 pattern C, whereas in 16 healthy donors over the age 40 there were respectively four with A, seven with B and only five with C (P=0.01). In 10 patients who had previously received chemotherapy, none developed pattern C, five pattern B and five pattern A, in contrast to 15/41 pattern C, 19/41 pattern B and 7/41 pattern A in healthy donors. These data suggest an intrinsic loss of T-cell generation capacity from adult bone marrow stem cells in comparison to reports on stem cells of fetal origin. This loss correlated weakly with age, irrespective of thymic involution, and may be further reduced by prior chemotherapy.

Gallstone recurrence after successful shock wave therapy: the magnitude of the problem and the predictive factors.

OBJECTIVE: Reports concerning long term recurrence of gallstones after successful extracorporeal shock wave lithotripsy (ESWL) show a high probability of stone recurrence. There is still discussion on the factors influencing stone recurrence. In this study we wanted to evaluate the long term recurrence of gallstones after stone clearance with ESWL and oral bile acids, and to assess possible risk and preventive factors of stone recurrence. METHODS: A total of 322 consecutive patients with stone clearance between December 1988 and December 1995 were included. All patients were contacted for ultrasonography and were interviewed for additional information on daily intake of aspirin, NSAIDs, cholesterol lowering medication, estrogen therapy, and biliary pain during follow-up. RESULTS: A total of 187 patients were still stone-free after a mean follow-up of 35 months (range: 3-89 months); 135 patients had recurrence. There was a significant association between stone recurrence and estrogen intake (p = 0.04), number of lithotripsy sessions (p = 0.0007), time until stone disappearance (p = 0.0003), and biliary pain (p < 0.0001). There was no difference in recurrence rate between solitary and multiple stones. CONCLUSIONS: Long-term recurrence of gallstones after lithotripsy is high: < or = 69% after 6 yr. We found a significant association of stone recurrence with estrogen intake, number of lithotripsy sessions, and time until stone disappearance. Intake of aspirin or NSAIDs was not associated with decreased stone recurrence. Of the patients with recurrent stones, 57% had biliary pain.

In vitro intrathymic differentiation kinetics of human fetal liver CD34+CD38- progenitors reveals a phenotypically defined dendritic/T-NK precursor split.

Human CD34+CD38- hematopoietic precursor cells from fetal liver are able to develop into T, NK, and dendritic cells in a hybrid human/mouse fetal thymic organ culture (FTOC). In this report, we pay particular attention to the early events in differentiation of these precursor cells. We show that the CD34+CD38- precursor cells, which are CD4-CD7-cyCD3-HLA-DR-/++ (cy, cytoplasmatic), differentiate into a CD4+ population that remained CD7-cyCD3-HLA-DR++ and a CD4- population that expressed CD7 and cyCD3. The CD4+CD7-cyCD3- cells differentiate into phenotypically and functionally mature dendritic cells, but do not differentiate into T or NK cells. The CD4-CD7+cyCD3+ population later differentiates into a CD4+CD7+cyCD3+HLA-DR- population, which has no potential to differentiate into dendritic cells but is able to differentiate into NK cells and gammadelta and alphabeta T lymphocytes. These findings support the notion that the T/NK split occurs downstream of the NK/dendritic split.