The majority of human CD3 epitopes are conferred by the epsilon chain

Transgenic mouse T cells expressing the human CD3ε chain bind the majority (29/36) of monoclonal antibodies (mAbs) specific for human CD3. A proportion of these mAbs are also able to recognize isolated CD3ε in a soluble, recombinant form. Thus, CD3ε can confer most CD3 epitopes on the TCR-CD3 complex, but many determinants may require assembly of the complex for their formation. A number ot mAbs did not recognize ε-transgenic T cells and probably need other CD3 subunits for binding. CD3-specific mAbs from each of the three groups defined here, as well as mAbs directed against the TCRαβ heterodimer, are all able to activate T cells. Therefore mAb attachment at several different sites on the TCR-CD3 complex can give rise to activation signals. This suggests that the cross-linking function of mitogenic antibodies may be their most significant property, rather than the perturbation of a particular ‘functional epitope

Self-limited autoimmune disease related to transient donor B cell activation in mice neonatally injected with semi-allogeneic F1 cells

BALB/c mice injected at birth with 108 semi-allogeneic (C57BL/6 x BALB.IgHb)F1 spleen cells develop a lupus-like syndrome in which autoantibodles bear exclusively the donor allotype. We have analyzed the evolution of donor B cell chimerism and the autoimmune manifestations during the first year of life in these mice. Anti-DNA, -histone, and -cardiolipln IgG antibodies as well as circulating immune complexes appeared in the second week of life, reached the highest values around the sixth week, and then progressively dropped to normal values after the sixth month in most mice. The kinetics of the evolution of the autoimmune manifestations, as well as the kinetics of serum donor Ig allotype, were parallel to the kinetics of donor B cell chimerism, which was particularly prominent in the spleens in early weeks of life, and progressively decreased after remission of the autoimmune syndrome. Membrane-proliferative glomerulonephritls, which was followed as the more representative histological abnormality in this model, was particularly evident after 10 weeks of life, but disappeared by the end of the follow-up. Interestingly, when mice with a self-limited disease were re-injected with 108 F1 spleen cells i.v., a flare in the serologlcal manifestations was observed. In these re-injected mice a predominance of anti-DNA, lgG1 antibodies bearing exclusively the donor allotype was also observed, as in the early weeks of life. These results emphasize the central role of donor B cell chimerism in the development and in the self-limitation of the autoimmune disease in parental mice neonatally injected with F1 cells and Indicate that the capacity to react with F1 cells, to generate a renewed burst of symptoms, persists in these mice after the disappearance of autoimmune finding

Delineation of human thymocytes with or without functional potential by CD1-specific antibodies

Hematopoletic precursors lacking T cell antigen receptors TCR-CD3−) and CD4 and CD8 surface markers (i.e. double-negative thymocytes) give rise to functionally mature T iymphocytes. Yet their major progeny are immunologically unresponsive thymocytes in spite of having acquired TCR-CD3 and CD4-CD8. Because only mature thymocytes migrate to peripheral lymphoid organs and most thymocytes die in situ, the knowledge of the events associated with functional maturation in the double-negative thymocyte progeny is a fundamental question in T cell development. We reasoned that a clue to trace the fate of early human thymocytes may perhaps come from the study of the developmental acquisition of CD1 antigen, currently used to define better the functionally inert CD4+8+ (double-positive) stage and absent in mature, medullary thymocytes and peripheral T cells. By using antibodles specific for CD1 (HTA 1/T6) we show here that a large fraction of double-negative thymocytes also express CD1. CD1+3−, CD1+3+, CD1−3+, and CD1−3− subsets all exist. The CD1+3− subset generates CD1+3−4−8+ precursors of CD1+ double-positive cells. A large portion of the CD1+3+ subset bears TCRγδ-CD3 complexes. The CD1− subsets are responsive in assays of function, in which they can be stimulated to use the interleukin 2 pathway of prollferation and to mediate cytotoxicity. in contrast, all CD1+ thymocytes behave as functionally inert cells. Thus, the CD1 surface marker delineates human thymocyte precursors and their products which lack, or possess, functional potential in vitro, on both αβ and γδ lineage

Function of CD44(Pgp-1) homing receptor in human T cell precursors

T cell precursors migrate from extrathymic hematopoietic tissues and dlfferentlate after encountering the thymic microenvironment. We asked whether human T cell precursors express the CD44(Pgp-l/gp90HR) class of homing receptors that have been implicated in the traffic of hematopoietic cells, such as lymphocyte entry to peripheral lymphold organs. Flow cytometry and immunoprecipitation studies demonstrate that CD7+34+, CD1-2-3-4-8- 14-16-20- cells in bone marrow and thymus, which have been shown to exhibit features of T cell precursors, bear CD44. Immunohlstologoical studies show that clusters of thymocytes in the subcapsular and the inner cortex and most medullary thymocytes are clearly CD44+, whereas the expression of CD44 is selectively downregulated in CD3- and CD3low functionally incompetent cortical thymocytes. The expression of CD44 is not restricted to T cell precursors but also occurs in thymic stroma, which bear a different molecular species of CD44. CD44-specific antibodies exert stimulatory effects on T cell precursors, a process that is dependent on stromal cells. We postulate that CD44 might be an adhesion molecule for precursor homing to thymus and that it participates in cell-to-cell interactions within the thymic environmen

Expansion of CD34+ human hematopoietic cells from umbilical cord blood using roller bottles

"El transplante de células madre hematopoyéticas está limitado por la cantidad de células CD34+ presentes en las unidades de sangre de cordón umbilical; el objetivo de este trabajo fue obtener la expansión in vitro de células madre hematopoyéticas. Se establecieron cultivos de células CD34+ de sangre de cordón umbilical en medio IMDM suplementado con citocinas para promover la expansión de progenitores hematopoyéticos. En 5 días de cultivo en frascos giratorios se obtuvo la máxima expansión de unidades formadoras de colonias (UFC) totales de 16.94±2.82 veces, mientras que en cultivo estático fue de 17.28±4.47. La máxima expansión de células totales fue de 20.31±6.18 veces en frasco giratorio y de 26.45±9.89 veces en cultivo estático a los 10 días de cultivo. Se demostró la eficacia del sistema de frascos giratorios con medio IMDM para el cultivo a corto plazo de células enriquecidas CD34+ provenientes de sangre de cordón umbilical y para la expansión de progenitores hematopoyéticos, potencializando el uso de este sistema para otros experimentos y aplicaciones clínicas a futuro."Hematopoietic stem cell (HSC) transplantation is limited by the initial CD34+ cell content in cord blood units; the aim of this work was the in vitro expansion of HSC to overcome this issue. Supplemented IMDM roller bottle cultures of CD34+ cells from human umbilical cord blood were established for hematopoietic progenitor expansion. The maximum total colony forming cells (CFC) expansion was achieved after 5 days of culture, being 16.94±2.82 folds in roller bottles and 17.28±4.47 in static cultures. However, the maximum total cell fold expansion was attained after 10 days of culture. It was of 20.31±6.18 for the roller bottles and 26.45±9.89 for the static cultures. The efficacy of the roller bottles system for short-term cultures of CD34+ cells and expanding hematopoietic progenitors in IMDM was demonstrated; encouraging the use of this culture system for other experiments and may be used for future clinical applications.

Thymic stroma is required for the development of human T cell lineages in vitro

Development of the T cell lineage is characterized by the homing of hematopoietic precursors to thymus, followed by their acquisition of receptors for antigen. T cell receptors are αβ or γδ heterodimers associated with CD3 (TCR-CD3). Very early T cell precursors in humans have been characterized as CD7+45+ cells which lack the T cell differentiation antigens CD1, CD2, CD3, CD4, and CD8. A phenotypically equivalent early thymocyte population also occurs in postnatal life, and we have previously shown that interleukin 2 (IL2) promotes the development in vitro of both the αβ and the γδ T cells from these early thymocytes. Here we have analyzed the requirements of the induction of the IL2 pathway in early thymocytes, and their developmental potential. We show that: (I) thymic stromal cells, which are present in thymocyte suspensions, are necessary to induce the IL2 pathway and the development of αβ or γδ T cell lineages from early thymocytes in vitro; and (II) when removed from the in vivo environment, early thymocytes can develop in vitro into TCR-CD3− cells of the natural killer (NK) lineage. We conclude that CD7+45+, CD1-2-3-4-8- early thymocytes are multipotential progenitors that, at least, have the capacity to develop into αβ or γδ T cell and NK lineages. The analysis of the mechanisms of generation and selection of human T and NK cell diversity, not feasible in bone marrow cultures, is now possibl

Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci

CD19+CD10+ human B lineage bone marrow cells were separated into cycling or resting cells, which differ in their expression of CD34, V(preB), recombination activating gene (RAG-1), and terminal deoxynucleotidyl transferase (TdT). Polymerase chain reaction analyses developed for D(H)J(H) and V(K)J(K) V(K)J(K)K((de)) and V(K)K((de)) rearrangements with DNA of single cells and a comparison with B lineage cell development in mouse bone marrow, allow to delineate the human B lymphocyte pathway of development as follows: CD34+V(preB)+RAG-1+TdT+, D(H)J(H)-rearranged, KL germline cycling pre-B I cells → CD34- V(preB)+μH chain+ (pre-B receptor+) RAG- 1+ TdT, V(H)D(H)J(H)-rearranged, KL germline, cycling pre-B II cells → CD34- V(preB)-, intracytoplasmic μH chain+ (pre-B receptor) RAG-1+ TdT, V(H)D(H)J(H)-rearranged, mainly KL germline cycling pre-B II cells → CD34+ V(preB)- intracytoplasmic μH chain+, RAG-1+ TdT, V(H)D(H)J(H)-rearranged, V(K)J(K)-rearranged, IgM-, resting pre-B II cells → CD34+ V(preB)-, sIgM+, RAG-1+ TdT-, V(H)D(H)J(H)- and V(K)J(K)-rearranged IgM+ immature B cells → CD34+, CD10- sIgM+/sIgD+ mature B cells. This order, for the first time established for human B lineage cells, shows striking similarities with that established for mouse B lineage cells in bone marrow.We thank Drs. Rod Ceredig and Thomas Winkler for critical reading of this manuscript. We are grateful to Marcus Dessing for his outstanding skill at the FACSÒ sorter and his extraordinary help during long, unusual hours. We thank Prof. A. Gratwohl, Dr. E. Signer, and Dr. U. Ramenghi for providing the bone marrow samples and Prof. F. Caligaris Cappio for continuous encouragement and discussions. We gratefully acknowledge Ms. Nadia Straube’s technical experience in DNA sequencing. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland. E. Sanz was supported by contracts from the CSIC and grant CAM92/126, and A. de la Hera was supported by grants SAF-93-0925 and SAF-96-0201 from the CICY.Peer reviewedPeer Reviewe

Expansion of human hematopoietic cells from umbilical cord blood using roller bottles in CO2 and CO2-free atmosphere

"In this work, we evaluated the expansion of human hematopoietic stem cells from umbilical cord blood in roller bottles. The Iscove's modified Dulbecco's medium, the Stem Pro 34-SFM medium, and the L-15 Leibovitz's medium for cultures in CO2-free atmosphere were assessed. At day 5 of culture, total colony forming unit expansions of 14.44 ± 3.74, 11.20 ± 6.37, and 17.25 ± 3.65-folds were attained, respectively. The expansion reached using L-15 medium in roller bottles was around 10 times higher than that achieved in the static control cultures. To our knowledge, this is the first report of cultures in CO2-free atmosphere to expand cord blood human hematopoietic stem cells and it opens a new branch of possibilities for culturing and clinical applications.

Solid – Liquid separation of dairy manure: distribution of components and methane production

Chemical treatment and screening can be an effective technique for separation of dairy cattle manure into a liquid fraction (LF) and a nutrient-rich solid fraction (SF). The optimum loading of a strong cationic polyacrylamide was found to be 43.9 g kg−1 of dry excreta. The separated SF contained 29.1% of the initial mass present in the manure and the chemicals added. The Volatile Solids (VS)/Total Solids (TS) ratio, which was 0.78 for the manure, rose to 0.82 for the SF and decreased to 0.63 in the LF. Furthermore, the SF retained 76.1, 79.9, 59.4 and 87.4% of TS, VS, Total Kjeldahl Nitrogen and Total Phosphorus, respectively. In the LF, the ratio of filtrate chemical oxygen demand (CODfiltrate) and COD due to volatile fatty acids (CODVFA) in relation to total COD (CODT) were 0.86 and 0.76, respectively. The percentage of anaerobically biodegradable chemical oxygen demand (CODBD) for the LF was 83.0%. Treatment of the LF in high loading anaerobic reactors would be possible due to these COD characteristics. Specific methane production in terms of VS for the separated LF was 0.580 m3 kg−1. For dairy manure and SF, it was 0.320 and 0.258 m3 kg−1, respectivel