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Biomedical subjects

P L Witte

Publications and source records attributed to P L Witte.

32 records · Page 2Linked to original sources
PubMed1987

Characterization of B lymphocyte lineage progenitor cells from mice with severe combined immune deficiency disease (SCID) made possible by long term culture.

A single gene mutation results in near absence of B and T lymphocytes and their immediate progenitors in mice with severe combined immunodeficiency disease (SCID). However, long term culture conditions allowed rapid outgrowth of lymphocytes from SCID bone marrow suspensions, and this permitted their detailed analysis. The cells were judged to be committed to the B lymphocyte lineage on the basis of expression of the BP-1 antigen, as well as by the density and pattern of expression of other markers. Cultured SCID lymphocytes were indistinguishable from control BALB/c cells in terms of morphology, typing for 13 cell surface markers, and changes in cell surface antigen expression with time in culture. In contrast to cultures of normal cells, which always included IgM synthesizing cells, SCID lymphocytes rarely expressed mu heavy chains. Southern blot analysis demonstrated that at least the first Ig gene rearrangement step had occurred in most of the cultured cells. The patterns of JH gene rearrangements suggested that relatively limited population diversity existed in individual cultures of SCID and normal BALB/c marrow. In addition, there was evidence that abnormal Ig heavy chain gene rearrangements had taken place in lymphocytes from approximately 25% of the SCID cultures. These cells were distinguished by the absence of detectable JH gene segments. kappa light chain genes appeared to be unrearranged in SCID cultured lymphocytes. We conclude that the lymphopoietic microenvironments of SCID mice are probably normal, and the animals have infrequent progenitors of B cells. Aberrant or nonproductive IgH gene rearrangements may account for the absence of pre-B and B cells in SCID mice. This study demonstrates the usefulness of long term culture methodology for isolating rare subsets of non-transformed lymphoid cells from normal and genetically defective hemopoietic tissues.

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PubMed1987

Relationships between B-lineage lymphocytes and stromal cells in long-term bone marrow cultures.

In long-term culture of mouse bone marrow, the growth and differentiation of B-lineage lymphocytes depends on interaction with adherent cells or their products. The objectives of these studies were to characterize the types of cells present in the supporting adherent layer as well as the physical relationships of these cells with lymphocytes. With an extensive panel of antibodies against hemopoietic and lymphocyte antigens, two discrete nonlymphoid populations were identified: macrophages and undefined, large cells which we termed "stromal cells". Lymphocyte clusters grew in actual contact with the latter cells only. Stromal cells lacked expression of most hemopoietic antigens, including the common leukocyte antigen, J11d, heat stable antigen (M1/69), Thy-1 and BP 1. Antigens expressed by stromal cells were detected by AA4.1, our 94.2 antibody, and antibody to the Forsmann antigen, but the most distinguishing characteristics of the lymphocyte-binding stromal cells were production of basement membrane components, laminin and collagen IV, and the extremely low uptake of acetylated low density lipoprotein (LDL). Using acetylated LDL uptake as a sorting criterion, the lymphocyte-binding stromal cells were separated from the macrophages, recultured and shown to support lymphocyte proliferation. We found the binding between stromal cells and lymphocytes to be highly selective and dependent on divalent cations; hence, specialized adhesion mechanisms may have a role in B cell development. Moreover, our studies suggest that phosphatidylinositol-anchored cell surface molecules may be involved in this adhesion. Our findings demonstrate the possibility that a single cell type provides physical support and proliferation stimuli for early B-lineage cells. This accessory cell is not a macrophage; rather, it has features of an endothelial or epithelial cell.

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PubMed1986

Effects of 5-fluorouracil on B lymphocyte lineage cells.

The aim of these studies was to determine the sensitivity of B lymphocyte lineage precursor cells in mice to 5-fluorouracil (5-FU). Selective effects could be very helpful in dissecting precursor-product relationships between these and the rare multipotential stem cells from which they derive. Numbers and functions of particular types of cells were determined at intervals after a single treatment (3 mg) and, as expected, myeloid-committed stem cells were very severely affected. Day 8 spleen colony-forming cells (CFU-S) and colony-stimulating factor-responsive macrophage progenitors were reduced by 98% within 24 hr, whereas presumptive early stem cells (day 14 CFU-S) were much more resistant. B cells, which were probably recently formed in bone marrow and which are not thought to be actively dividing, were also 5-FU-sensitive, but perhaps less so than pre-B cells and immunoglobulin-negative lymphocytes bearing a B lineage marker. Approximately 5 wk were required for the normal cellular composition of marrow to return to normal. Transplantation of marrow from 5-FU-treated mice suggested that the slow regeneration of B lymphocytes might partially result from residual drug effects or damage to microenvironmental elements which are required for B lineage differentiation. Acute reductions of lymphocytes in the thymus were also documented, and the larger cells declined most rapidly and regenerated most slowly in that tissue. Of particular interest was the differential susceptibility of B cells in various lymphoid tissues to 5-FU. Whereas lymph node B cells were minimally affected, one-half of the splenic B cells disappeared within 48 hr of drug injection. Intrinsic differences in 5-FU sensitivity were confirmed by treatment of lymphocytes in vitro, and this suggests that particular B cell sets may be metabolically distinct. This drug should find additional experimental application in studies of B lymphocyte formation and functional heterogeneity.

Animals↗
PubMed1986

Development and ontogeny of hamster T cell subpopulations.

The Syrian hamster is unique among laboratory animals because products of class I MHC genes are monomorphic. Thus, this species may be a model in which to test the relationship between MHC polymorphism and the T cell antigen receptor repertoire. Recently, cytotoxic and helper T cell subpopulations have been distinguished on the basis of cell surface phenotype detected with monoclonal antibodies (mAb). We used these reagents (mAb 110 detects all peripheral T cells and mAb 38 detects cytotoxic T cells) to dissect and categorize thymic populations according to relative maturational status. The two mAb divide thymocytes into four subpopulations in the young adult. Two (110+ 38+, 110+ 38-) were peripheral-like and were housed in the medulla, exclusively; another subset (110- 38+) consisted almost entirely of TdT+ cortical thymocytes. The fourth subset (110- 38-), bearing neither marker, was heterogeneous and consisted mostly of medium-large-size thymocytes, including cells with an early phenotype (nuclear TdT+). Cells with the cortical phenotype proved to be the most sensitive to cortisone treatment, whereas those which expressed the medullary marker, 110, were most resistant. To ascertain the relationship between 110- and 110+ T lineage cells, we followed the appearance of the four thymic subpopulations during ontogeny of the hamster thymus. Adult-like thymic architecture (delineation of cortex and medulla) as well as the two 110- subsets were established before expression of 110 antigen was apparent in the thymus. However, lymphocytes bearing the 110 antigen were found in lymph nodes prior to thymus during ontogeny, concomitant with developing T cell function in peripheral tissue. This finding implies that cells lacking 110 antigen were exported from the thymus and subsequently acquired expression of the molecule in the periphery, and we suggest that acquisition of 110 antigen may be a stage of postthymic maturation. Although 110+ cells appeared to be the most mature subset by several criteria, all functional thymocytes of adults or neonates were not 110+. Thus, we conclude that the 110 marker is acquired after T cells reach functional maturity. Moreover, the response profile of isolated 38+ thymocytes was analogous to peripheral 38+ T cells, suggesting that the dichotomy of function detected with our mAb also occurs before acquisition of 110 antigen. We have modeled what is known about hamster T cell development into a hypothetical scheme.

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PubMed1986

Interculture variation and evolution of B lineage lymphocytes in long-term bone marrow culture.

A recently described long-term culture system offers a unique experimental approach for dissecting regulatory mechanisms that control the developmental progression of B-lineage lymphocytes. Lymphoid cells, including B cells and their precursors, can be maintained for prolonged periods in strict dependence on a layer of adherent cells. However, before this system can yield to interpretable manipulation, much information is needed as to the identity and temporal phenotypic stability of both lymphoid and nonlymphoid cells. The findings reported here provide answers to some of those important questions. Successful establishment of lymphoid cells in culture was extraordinarily dependent on the batch of fetal calf serum used in the medium, and some undesirable serum lots supported cultures that were virtually all myeloid. With standardized culture conditions, various populations of lymphoid cells were identified on the basis of B-lineage differentiation markers and culture to culture variation was assessed. Lymphocytes that were firmly attached to the adherent cells were carefully compared to nonadherent lymphocytes in terms of cycle status, phenotype, size, and transferrin receptor expression. They were essentially identical in all of these respects and a partitioning of proliferating cells and their progeny in the cultures was therefore not apparent. It is also noteworthy that although a high mitotic rate was maintained, a majority of the cells were small lymphocytes. The outgrowth of identifiable B-lineage cells (detected with monoclonal 14.8 antibodies) in replicate cultures was initially similar, but the extent of interculture variation increased dramatically during the period 4-6 weeks after initiation of culture. Replicate cultures established from the same marrow cell pool often differed as much as 20-fold in numbers of 14.8-positive cells. After this time, the composition of individual cultures evolved much more gradually, and numbers of B cells and pre-B cells remained relatively constant. This indicates that subsets of lymphocytes become established in each culture dish during a discrete phase. At least two types of supporting adherent cells predominated in these cultures: typical macrophages and very large, nonphagocytic cells resembling adventitial reticular cells. The latter included subpopulations resolved on the basis of alkaline phosphatase content. In contrast to the lymphoid populations, proportions of these adherent cell types were relatively invariant among replicate cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1985

Local cellular defenses in influenza-infected lungs.

The functional capacities and surface phenotype of the cells that accumulate in the lungs of hamsters during influenza A virus (PR/8/34) infection were studied to determine the cellular mechanisms that may limit the viral infection in the lung. Nonspecific natural killer (NK) cytotoxicity was augmented early (3 days) after infection in the lung but was undetectable at 6 days postinoculation. Virus-specific cytotoxic cells were detected within populations of mononuclear cells harvested from the lung but not from the hilar lymph node or spleen of influenza-infected hamsters following intratracheal inoculation. In contrast to virus-specific cytotoxic activity which remained locally, delayed-type hypersensitivity (DTH) activity was detected in assays in which cells were used from lung, hilar lymph nodes, or spleen. Depletion studies using rabbit anti-asialo GM1 and newly developed mouse monoclonals WI20 and WI38, which detect surface antigens on hamster T-lymphocyte populations, demonstrated that in the hamster NK cells are asialo GM+, WI20-, WI38-; DTH lymphocytes are asialo GM-, WI20+, WI38-; and cytotoxic T lymphocytes are asialo GM-, WI20+, WI38+. Together these data suggest that antigen-specific cytotoxic T cells can be induced locally within the hamster lung during influenza infection, but that they appear to be unable to circulate systemically, unlike the T cells that mediate DTH. Thus while the lung appears to share some immune responses to local infections with peripheral lymphoid organs, effector cells can be induced to develop locally and may be regulated locally without a mandatory involvement of the systemic immune system.

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PubMed1985

Description of phenotypically distinct T-lymphocyte subsets which mediate helper/DTH and cytotoxic functions in the Syrian hamster.

Recent studies describe aberrations in the functions of T lymphocytes from Syrian hamsters. A current proposal links the apparent functional deficiencies of cytotoxic and suppressor T cells with anomalies found in class I molecules of this species (no polymorphism is detected) and speculates that hamsters possess limited heterogeneity of T cell subpopulations, particularly a class I-restricted subset. The present work tests this hypothesis by examining the extent of T cell heterogeneity defined by differential cell surface antigen expression. A panel of mouse monoclonal antibodies against hamster lymphocyte antigens was generated. MAb #20 and #110 bound to most, if not all, peripheral T cells; a third antibody, #38, divided T cells into two subpopulations which were functionally distinct. Cells within the #38-negative subset produced easily detectable IL 2 and mediated delayed-type hypersensitivity to influenza virus. In contrast, isolated 38+ cells produced little IL 2 and required the addition of exogenous T cell growth factor for proliferation to Con A. Treatment of immune cells with mAb #38 and complement abrogated cytolysis to TNP-haptenated or influenza-infected targets. Thus, Syrian hamsters possess at least two T cell subpopulations of discrete functional ability and unique cell surface antigen expression. Although the data suggest that T cells analogous to those of the class I-restricted, Lyt 2+ subset are present in the hamster, it is predicted that the scope of their composite antigen receptor repertoire may be limited by the monomorphism of class I molecules in this species.

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PubMed1983

Thy-1 antigen is present on B and T lymphocytes of the Syrian hamster.

A widely used murine monoclonal antibody (mAb) to the Thy-1.2 allele and a highly absorbed xenoantiserum to hamster thymocytes bind to all thymocytes and a subpopulation of peripheral lymphoid cells of the Syrian hamster (Mesocricetus auratus). The mAb appears to detect the hamster Thy-1 homologue because hamster and mouse brain tissue each completely absorb the reactivity to hamster lymphocytes. Previous studies suggested that anti-Thy-1.2 identifies hamster T lymphocytes exclusively. Fluorescence analysis, however, demonstrates a large overlap of Thy-1.2+ and slg+ lymph node and spleen cells. Moreover, highly enriched (greater than 97%) Thy-1+ lymph node cells are greater than 50% slg+, give enhanced proliferative responses to LPS and anti-Ig in vitro, and reexpress normal amounts of slg 18 to 24 hr after Ig is removed from the cell surface. Thus, the hamster Thy-1 homologue is distributed not only on T cells but also on the majority of resting, peripheral B cells, and therefore cannot be utilized adequately as a T cell-specific marker in this species.

Animals↗
PubMed1983

Monoclonal antibodies to hamster class II MHC molecules distinguish T and B cells.

Monoclonal antibodies were prepared against cell surface antigens present on Syrian hamster lymphocytes and a hamster B cell lymphoma line, GD-36. One of these antibodies, S11, precipitated glycoproteins of 29,000 and 39,000 m.w. These glycoproteins were shown to be identical to or a subset of la-like glycoproteins precipitated by hamster alloantisera; however, molecules identified by S11 differed from the predominant hamster la homologues detected with a cross-reactive monoclonal antibody to murine la.7. The immunofluorescence pattern of both anti-la reagents, S11 and anti-la.7, on hamster lymphoid cells is similar by fluorescence-activated cell sorter analysis. A subpopulation of spleen and lymph node cells stains brightly with these antibodies. By two-color fluorescence, this peripheral lymphocyte subpopulation, identified with monoclonal anti-hamster la, also bears surface immunoglobulin (IgM). These data strongly suggest that hamster resting peripheral B cells, and not T cells, express la antigens and can be identified and isolated differentially by using this marker.

Animals↗
PubMed1983

Improved efficiency of hybridoma ascites production by intrasplenic inoculation in mice.

Intrasplenic inoculation of small numbers (10(4)) of antibody-producing hybridoma cells into unprimed BALB/c mice resulted in high-titered ascites. By intrasplenic injection, the low number of cells needed to produce ascites was 2-3 logs fewer than that required by ip inoculation. Hybridoma cells suspended in the ascitic fluid could be reestablished in vitro. Furthermore, cells obtained directly from individual, cloned colonies or from previously frozen samples also resulted in high-titered ascites, if the cells were inoculated intrasplenically. This method reduces time and expense required to grow large numbers of hybridoma cells for ip-derived ascites production and allows ready recovery of frequently unstable frozen-thawed cells.

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