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R Chervenak

Publications and source records attributed to R Chervenak.

49 records · Page 3Linked to original sources
PubMed1991

Characterization of the progeny of pre-T cells maintained in vitro by IL-3: appearance in the periphery and V beta utilization in vivo.

We have recently demonstrated that bone marrow-resident cells, which are able to repopulate the thymus of irradiated recipient mice (pre-T cells), can be maintained in vitro for at least 2 weeks in the presence of exogenous IL-3. Because this marrow culture system can be applied to the study of early T cell differentiation, it is important to ascertain the extent to which in vitro culture of the pre-T cells might alter the T cell progeny which can develop from them. In previous work, we showed that the progeny of cultured pre-T cells appeared to develop in a kinetically normal fashion within the thymus of recipients and that the acquisition of key developmental markers (IL-2R and CD3) was identical in the progeny of fresh and cultured pre-T cells. Here, we report the results of experiments carried out to characterize the progeny of cultured pre-T cells which were found in the peripheral lymphoid tissues several weeks following intrathymic transfer to irradiated recipients. We found no remarkable differences between the progeny of cultured or fresh marrow cells with respect to the timing of their appearance in the periphery nor their expression of CD4 or CD8. By studying the patterns of utilization of five different V beta gene products by the T cells derived from fresh or cultured bone marrow, we were able to test the susceptibility of both sets of progeny to both positive and negative selection pressures during their in vivo maturation. These experiments established that the progeny of cultured marrow cells were equally susceptible to TCR repertoire selection, as were the progeny of fresh bone marrow cells, and that the process of in vitro growth did not alter the potential TCR repertoire of the pre-T cells.

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PubMed1991

Characterization of the progeny of pre-T cells maintained in vitro by IL-3: expression of the IL-2 receptor and CD3 during thymic development.

We have recently described a bone marrow culture system which is able to maintain, for at least 2 weeks, cells which have the capacity to repopulate the thymus of irradiated recipient mice (pre-T cells). Because this culture system depends upon the addition of an exogenous growth factor (IL-3) which may potentially influence the differentiation of the cultured pre-T cells, it is important to determine whether or not the progeny of cultured marrow cells are able to develop within the thymus in a kinetically normal fashion. Here we report the results of an analysis of the progeny of those cultured progenitor cells at 2, 3, and 4 weeks following intrathymic transfer. The passage of cultured donor-derived cells through critical early (expression of the IL-2 receptor) and late (expression of high levels of CD3) intrathymic events was assessed in these studies and compared with the pattern observed in the progeny of fresh bone marrow cells. The results of these studies showed that the progeny of cultured pre-T cells were able to develop expression of the IL-2 receptor and CD3 surface antigen during their residency within the thymus. In addition, both the timing and levels of expression of these surface markers were virtually identical on the progeny of fresh and cultured pre-T cells. These data suggest that cultured pre-T cells are not dramatically altered by their passage in vitro and are able to give rise to normally developing thymocytes upon in vivo transfer.

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PubMed1990

In vitro maintenance of bone marrow-resident T cell precursors in cultures supplemented with exogenous IL-3.

T cell precursors (pre-T cells) from adult murine bone marrow were propagated in vitro using a newly developed cell culture system. This system differs from "classical" bone marrow culture techniques in that it does not require the establishment of a monolayer of bone marrow stromal cells nor the presence of an irradiated feeder layer for the maintenance of the pre-T cell compartment, but rather depends on the continuous presence of exogenous interleukin-3 (IL-3) in the culture. Cells harvested from these cultures retain the capacity to repopulate the thymuses of sublethally irradiated recipient mice, following intrathymic transfer, for at least 2 weeks after the initiation of the cultures. Interestingly, pre-T cells maintained under these culture conditions rapidly lose their capacity for thymic repopulation following intravenous transfer, suggesting a potential defect in recirculation and/or homing properties. This culture system should prove useful in the investigation of bone marrow-resident pre-T cells.

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PubMed1988

Target cell expression of MHC antigens is not (always) a turn-off signal to natural killer cells.

Recent evidence has demonstrated that the lytic function of natural killer cells might be regulated by potential target cells through the target cells' expression of cell surface components that are able to inhibit the lytic process. Specifically, it has been shown in many target cell systems that the expression of class I MHC proteins by target cells is inversely proportional to their susceptibility to lysis by NK cells. It has been suggested, therefore, that MHC proteins may act as important negative regulatory elements in the ongoing control of NK cell function. Herein, we examined two closely related murine lymphoma cells (ASL1 and ASL1w), both in terms of their susceptibility to lysis by NK cells as well as their expression of both H-2K and H-2D class I MHC proteins. The results of these studies showed that whereas ASL1 and ASL1w cells differed greatly in their susceptibility to NK cell lysis (ASL1 was much more NK resistant than ASL1w), both expressed high levels of H-2K and D proteins. In contrast to what might have been predicted base on reports from other target cell systems, the more NK susceptible ASL1w cells expressed somewhat higher levels of H-2K Ag than did ASL1 cells. These results indicate that expression of H-2 class I proteins by target cells, in and of itself, is not sufficient to inhibit the lytic activity of murine NK cells.

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PubMed1987

Influence of MHC on thymus repopulation following intrathymic transfer of mouse T-cell precursors.

T-cell precursors (pre-T cells) traditionally have been detected by their ability to repopulate the thymus of heavily irradiated mice following intravenous injection. Recently, an assay system involving the direct injection of pre-T cells into the thymus of sublethally irradiated animals has been described. Here we report the results of experiments designed to evaluate the ability of bone marrow cells to produce thymic repopulation following intrathymic injection in a wide range of donor-host strain combinations. Irradiated (600 R) mice were injected intrathymically with 2 X 10(6) bone marrow cells which differed from the recipient with respect to their Thy-1 allotype and the percentage of thymus cells expressing either donor- or recipient-type Thy-1 was determined 9 to 23 days after injection. The results of these experiments showed that thymocytes expressing the Thy-1 allotype derived from the donor marrow were only detected when the donor and host were matched at MHC. By contrast, thymic repopulation by MHC-mismatched donor marrow cells could readily be observed when these cells were given intravenously.

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PubMed1986

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells.

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.

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PubMed1985

Prethymic T cell precursors express receptors for antigen.

An anti-idiotype serum raised in BALB/c mice against syngeneic lymph node T cells from 2,4-dinitrofluorobenzene (DNFB)-sensitized mice was used to study the early expression of antigen receptors on developing T cells. Normal BALB/c bone marrow cells were treated with either anti-Thy-1.2 plus complement or anti-Thy-1.2 and anti-idiotype plus complement before use in the reconstitution of lethally irradiated syngeneic mice. Five weeks after reconstitution, recipient mice were assayed for both contact sensitivity (CS) and in vitro proliferative responses to DNFB. Mice reconstituted with bone marrow cells treated with both anti-Thy and anti-idiotype sera showed a significant decrease in reactivity to DNFB in both assay systems when compared with mice reconstituted with marrow treated with anti-Thy only. CS response to the noncross-reacting hapten oxazolone was identical in both recipient groups. Bone marrow mixing experiments showed no evidence of anti-idiotype-induced suppressor cells in these experiments. These data provide strong evidence that at least some T cell precursors express receptors for antigen prethymically.

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PubMed1983

Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis.

Within minutes of exposure of target cells to cytotoxic T lymphocytes, their nuclear DNA begins to be fragmented. This phenomenon precedes 51Cr release by at least an hour. DNA fragmentation occurs only when appropriately sensitized cytotoxic T cells are used and is not merely a result of cell death because killing of target cells by heating, freeze/thawing, or lysing with antibody and complement did not yield DNA fragments. Agarose gel electrophoresis of target cell DNA showed discrete multiples of an approximately 200-base-pair subunit, suggesting that fragmentation was the result of activation of a specific endonuclease. A similar pattern of DNA fragments is observed during glucocorticoid-induced killing of mouse thymocytes. The endonuclease in that case is inhibited by zinc ions, and we find that Zn2+ also inhibits DNA fragmentation and 51Cr release induced by cytotoxic T cells, suggesting a final common biochemical pathway for both types of cell death.

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PubMed1983

Clonal abortion of bone marrow T cell precursors: T cells acquire specific antigen reactivity prethymically.

Lethally irradiated (900 R) mice were reconstituted with bone marrow cells from syngeneic donors that had been tolerized 2 to 3 wk earlier to either DNP or TNP compounds. Five weeks after reconstitution, these animals were tested for their ability to mount a delayed hypersensitivity (DH) response to the tolerizing haptens. Recipient mice were specifically tolerant to the hapten that was used to induce tolerance in the marrow donor. Mixing experiments in which mice were reconstituted with marrow from DNP-tolerant and TNP-tolerant donors showed no indication of active suppression or effective antigen carry-over in this system. This observation held true even in experiments in which mice were reconstituted with a mixture of marrow from tolerant and normal donors at a ratio of 5:1. Thus the reduced responsiveness in recipient mice seemed to be due to the functional elimination of hapten-responsive T cell precursor (pre-T) clones. Recipient unresponsiveness was also shown to be MHC restricted. Maintenance of unresponsiveness appeared to be due to the restricted access of regenerating pre-T cell clones to the maturational influence of the recipient's thymus.

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PubMed1982

Peanut lectin binding as a marker for activated T-lineage lymphocytes.

A series of experiments was undertaken to ascertain the reliability of peanut agglutinin (PNA) binding as a means of distinguishing mouse pre-T cells from other cell types. Normal bone marrow, thymus and spleen cells were stained with graded doses of fluoresceinated lectin (FITC-PNA) and subsequently examined by fluorescence microscopy to determine the number of lectin-binding cells. In addition, the PNA binding properties of mitogenically activated lymphocytes were examined in a similar manner. The results of this study show that PNA binding does not define a fixed population of cells, as the percentage of PNA+ cells detected in normal mouse tissues varied directly with the concentration of PNS used for staining. Nor is PNA able to distinguish between pre-T cells and mature T cells after mitogenic stimulation, both of which are shown to bind PNA. This lectin may , however, have value as a general marker of lymphoid precursors and activated lymphocytes of the T lineage.

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PubMed1978

Colony-forming units in the spleen and bone marrow of young (C57BL/6xDBA/2)F1 hybrid mice.

Spleen or bone marrow cells from (C57BL/6 X DBA/2)F1 hybrid mice ranging in age from 1 to 32 days were transplanted into syngeneic irradiated adult recipients. By enumeration of macroscopic hemopoietic spleen colonies 8 days after transplantation, the colony-forming unit(CFU) content of the spleen and bone marrow of the young donor mice was determined. The CFU concentration in the bone marrow, initially at a level of 15.1--17.0 CFU/105 nucleated cells, increased during the third week of life and reached a value comparable to that of the adult during week 4. In contrast, the CFU concentration of the spleen decreased during the first 2 weeks and reached a plateau at a level of 4.45--6.66 CFU/105 nucleated cells during the third and fourth weeks of age. This was still significantly higher than that of the adult hybrid. The rise in prominence of the bone marrow as a hemopoietic organ during this period appeared to be due to an increase in total cellularity coupled with an increase in CFU concentration in this hybrid. While the cellularity of the spleen also increased during this time, the CFU concentration decreased, resulting in little change in the total CFU content of this organ.

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