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Biomedical subjects

M R Loken

Publications and source records attributed to M R Loken.

At least 73 records · Page 4Linked to original sources
PubMed1984

Control of cloned alloreactive T lymphocyte proliferative responses. A possible role for cell-surface-bound alloantigen.

Previous studies using flow cytofluorometry demonstrated that passively acquired alloantigens encoded by the I-A and H-2K/D regions of the MHC could be identified on the surface of murine cloned T lymphocytes. The present studies were designed to further characterize the nature of that bound alloantigenic material on cloned lines of alloreactive amplifier T cells (Th). Immune precipitation and polyacrylamide gel electrophoresis were used to substantiate that allogeneic I-A and H-2K/D antigens were present on he surface of these cloned Th. Using proteolytic digestion with papain, bound allogeneic I-A and H-2K/D was removed from the surface of a cloned Th designated L2. Allogeneic I-A or surface of a cloned Th designated L2. Allogeneic I-A or H-2K/D antigens were not reexpressed if papain digested cells were incubated overnight in culture medium without addition of fresh alloantigen. Further, cloned cells did not release detectable amounts of interleukin 2 (IL-2) in response to Con A stimulation immediately following enzymatic digestion. After overnight culture, papain-digested cells released amounts of IL-2 similar to untreated controls. The proliferative response of these cells measured by 3H-thymidine incorporation, however, was significantly less than that observed using undigested controls, unless alloantigen was provided in the form of fresh irradiated stimulating cells. The addition of partially purified preparations of monoclonal anti Ia antibodies to cultures of cloned Th resulted in marked reduction in incorporation of 3HTdR. These findings are consistent with the hypothesis that modulation of the Th proliferative response may occur at least partially through binding of allogeneic material to the cell surface.

Animals↗
PubMed1984

Characterization of functionally distinct subpopulations of rabbit T lymphocytes.

Two subpopulations of rabbit spleen and mesenteric lymph node T cells were identified by a monoclonal antibody, 8AC8. These subpopulations were separated on the flow cytometer and were analysed for their response to T cell mitogens and antigens, their responsiveness in mixed lymphocyte cultures, and their ability to function as cytotoxic effector cells. The 8AC8+ T cell subpopulation contained cells highly responsive to T cell mitogens, to antigen, and to allogeneic or autologous stimuli in a mixed lymphocyte reaction. In contrast, the 8AC8- T cell subpopulation was non-responsive to T cell mitogens, and responded, poorly to antigen, and to allogeneic and autologous stimuli in an MLR. Both the 8AC8+ and 8AC8- subpopulations exhibited xenogenic cytotoxic effector function. Thus, the 8AC8 MAb identified a subpopulation of mature differentiated rabbit T cells; these 8AC8+ cells share many of the characteristics of the human OKT4 helper/inducer T cell subpopulation.

Animals↗
PubMed1984

Human lymphocyte subpopulations identified by using three-color immunofluorescence and flow cytometry analysis: correlation of Leu-2, Leu-3, Leu-7, Leu-8, and Leu-11 cell surface antigen expression.

Three-color immunofluorescence has been used to determine the co-expression of cell surface antigens on human peripheral blood lymphocytes. Monoclonal antibodies or avidin were coupled to either FITC (green), phycoerythrin (orange), or Texas Red (red) fluorochromes. These three fluorochromes could be independently measured by using a dual laser FACS IV system equipped with an argon ion laser (488 nm) and a dye laser (600 nm). Human peripheral blood lymphocytes were stained with the following combinations of reagents: (1) FITC anti-Leu-11a + PE anti-Leu-2a + TR avidin/biotin anti-Leu-7; (2) FITC anti-Leu-11a + PE anti-Leu-3a + TR avidin/biotin anti-Leu-7; (3) FITC anti-Leu-8 + PE anti-Leu-2a + TR avidin/biotin anti-Leu-7; and (4) FITC anti-Leu-11a + PE anti-Leu-2 + TR avidin/biotin anti-Leu-8. The light scatter, green fluorescence, orange fluorescence, and red fluorescence signals for each sample were stored by a Consort 40 PDP/11 computer in list mode files. Sequential reanalysis of the data directly demonstrated the existence of several unrecognized subpopulations of lymphocytes. Previously, we reported that the anti-Leu-7 and anti-Leu-11 antibodies can be used to identify discrete subsets of human NK cells with distinct functional capacities. In this report, we show that these subsets can be further subdivided on the basis of Leu-8 and Leu-2 expression. Thus, these studies illustrate how multicolor and multiparameter flow cytometry can further our understanding of cellular heterogeneity within this group of lymphocytes.

Adult↗
PubMed1984

Allotypic specificities of murine IgD and IgM recognized by monoclonal antibodies.

Hybridomas generated from mice immunized with allotype and H-2-incompatible spleen cells were screened by flow cytometry. Monoclonal antibodies (MAb) to four of the five known specificities of IgD were identified. The location of these specificities on the IgD molecule was determined by the ability of a given MAb to bind to cells stripped of the Fab fragment by trypsin proteolysis. Igh-5.1 (present on IgDa and IgDe) and Igh-5.5 (unique to IgDe) were both located on the Fab fragment. Results from cross-blocking experiments suggest that, except for Igh-5.3, MAb which recognize the same specificity bind to the same antigenic determinant. Both the trypsin digest and blocking studies indicate that Igh-5.3 is composed of at least two antigenic determinants. AF6-78.25, a MAb specific for IgM of the b, d, and n haplotypes, was also identified; this MAb defines a new specificity, Igh-6.6. On the basis of the reactivities of these MAb, it appears that although the Igh-Ce and Igh-Cd haplotypes are virtually identical at the Igh-3(gamma 2b), Igh-1(gamma 2a), and Igh-2(alpha) loci, they are highly divergent at the Igh-5(delta) and Igh-6(mu) loci. This indicates that the Igh-Cd haplotype may have resulted from a recombination between Igh-Ce and another haplotype.

Animals↗
PubMed1983

A clone-specific monoclonal antibody that inhibits cytolysis of a cytolytic T cell clone.

Monoclonal antibody 384.5 specifically inhibited cytolysis of P-815 target cells by cloned L3 cytotoxic T lymphocyte (CTL) effector cells. The lytic activity of other cloned CTL that have other distinct specificities was not affected. Antibody 384.5 did not inhibit the cytolytic activity of bulk populations of C57BL/6 mixed lymphocyte culture (MLC) cells. Concanavalin A-facilitated cytolysis by T cell clone L3 but not T cell clone B18 was inhibited by antibody 384.5, whereas phytohemagglutinin-facilitated cytolysis by L3 cells was not strongly inhibited. Antibody 384.5 binds specifically to L3 cells but not to several other T lymphocytes clones, or to a detectable portion of populations of primary MLC cells, normal spleen, thymus, lymph node, or bone marrow cells. In contrast, C57BL/6 anti-B10.A(5R) secondary MLC cells (genetically enriched for reactivity against the H-2Dd region gene products) contained a small population which reacted with the antibody 384.5. The determinant detected by antibody 384.5 was susceptible to trypsin treatment, and was reexpressed after overnight incubation. These results suggest that the monoclonal antibody 384.5 detects an endogenously synthesized clone-specific determinant associated with the cytolytic activity of the L3 CTL clone. These properties make antibody 384.5 an attractive candidate for an antibody that reacts with the antigen-recognition site of a cytolytic T cell antigen receptor.

Animals↗
PubMed1983

Characterization of murine hemopoietic cells using rat anti-mouse monoclonal antibodies.

Three rat anti-mouse monoclonal antibodies reactive with different populations of cells in mouse bone marrow have been extensively characterized with respect to their binding to cells in the lymphohemopoietic system. One antibody identifies lymphocytes committed exclusively to the B lineage. A second antibody identifies cells of the myeloid lineage including granulocytes and macrophages. The third antibody binds to nearly all of the bone marrow cells including erythrocytes, however, quantitative differences in antigenic expression allows the discrimination between early and late erythroid cells and can be used to purify eosinophils. The differential binding of these three monoclonal antibodies to mature cells, immature cells and progenitor cells (CFU-S, CFU-C (GM), BFU-E, and CFU-E) permits the construction of a scheme of lymphohemopoietic differentiation based on the expression of three cell surface antigens.

Animals↗
PubMed1983

Characterization of the murine antigenic determinant, designated L3T4a, recognized by monoclonal antibody GK1.5: expression of L3T4a by functional T cell clones appears to correlate primarily with class II MHC antigen-reactivity.

We describe here the properties of mAb GK1.5, which recognizes a cell surface molecule designated L3T4; the determinant on L3T4 recognized by mAb GK1.5 is designated L3T4a. We present evidence here that: i) the expression of L3T4a by murine T cell clones correlates primarily with class II MHC antigen-reactivity; ii) mAb GK1.5 blocks all class II MHC antigen-specific functions (cytolysis, proliferation, release of lymphokines) by murine class II MHC antigen-reactive T cell clones, although there appears to be clonal heterogeneity in the degree to which these functions are blocked by mAb GK1.5; iii) mAb GK1.5 blocks class II MHC antigen-specific release of IL-2 from cloned T cell hybridomas by blocking class II MHC antigen-specific binding; and iv) L3T4 is very similar to the human Leu3/T4 antigen. The properties of mAb GK1.5 (complement fixation, reactivity with all mouse strains tested, profound blocking of all class II MHC antigen-specific functions by murine T cells, usefulness for FACS analyses, and usefulness for immuno-precipitation/SDS-PAGE analyses) make it suitable for investigating both the role of class II MHC antigen-reactive T cells in various immunological phenomena and the mechanistic basis, at the molecular level, of class II MHC antigen-reactivity by murine T cells.

Animals↗
PubMed1983

Inhibition of proliferation of MIs- and Ia-reactive cloned T cells by a monoclonal antibody against a determinant shared by I-A and I-E.

The interaction between cloned amplifier T cells and stimulating cells has been probed with the use of a monoclonal antibody selected for its ability to inhibit stimulation of Mls- and la-reactive T cells. The antibody recognizes a public determinant on I-A- and I-E-coded molecules of a variety of murine H-2 haplotypes. The antibody inhibits the proliferation of the I-Ab-reactive T cell clone 48.1 and of the Mls-reactive T cell clones L2 and Fa 13. The antibody may be added as late as 3 hr after initiation of culture to cause inhibition of L2 proliferation and release of interleukin 2. A profound inhibition of primary and secondary mixed leukocyte responses also is observed. The results indicate that a large fraction of Mls reactive T cells require interaction with la antigens.

Animals↗
PubMed1983

The skin test antigen stimulated killer (STAK) cell mediating NK like CMC is OKM1 positive and OKT3 negative.

Recently we demonstrated that candida antigen stimulated natural killer cell like cell-mediated cytolysis (NK like CMC) in peripheral blood mononuclear cells (PBMNC) isolated from normal individuals (Tartof et al., 1980). Utilizing monoclonal antibodies directed against human mononuclear cell subpopulations in conjunction with a fluorescence activated cell sorter (FACS) we determined that, similar to the previously described NK cell, the skin test antigen stimulated killer (STAK) cell is a larger OKM1 positive, OKT3 negative cell. We obtained similar results using two different skin test antigens. Thus, stimulation of NK like CMC in PBMNC by skin test antigens probably represents activation of NK or NK like cells.

Antibodies, Monoclonal↗
PubMed1983

T-cell lineage of a Friend virus-induced lymphatic leukemia in athymic rats.

Athymic (rnu/rnu) and euthymic rats inoculated with the Friend virus-associated lymphatic leukemia virus developed lymphocytic leukemia. Neoplastic cells from these animals were evaluated by means of indirect immunofluorescence and flow cytofluorometry with monoclonal antibodies Ox-1, Ox-7, and W3/25, which react with surface antigens present on normal rat lymphoid cell populations. Lymphoid cells from leukemic animals revealed characteristic alterations in cell surface fluorescence profiles when compared to normal, healthy controls. Athymic and euthymic leukemic rats were similar in that many cells from both the spleen and bone marrow had markers on the cell surface normally found on thymocytes but not on mature peripheral lymphocytes. These studies provided evidence supporting the presence of T-lineage lymphocytes in the athymic rat. Further, this population of early or "pre"-T-lymphocytes included the predominant leukemia cell type induced by the Friend virus-associated lymphatic leukemia virus.

Animals↗
PubMed1983

Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule.

Monoclonal antibody GK1.5 recognizes a previously undescribed murine T cell surface molecule, designated L3T4, which migrates on SDS-PAGE under reducing conditions as a single band with an apparent m.w. of 52,000. L3T4 is expressed by approximately 80% of thymocytes and by approximately 20% of spleen cells. There appears to be poor correlation between expression of L3T4 by functional T cell clones and expression of Lyt-2, expression of the cytolytic phenotype, and class I MHC antigen reactivity. On the other hand, both a class II MHC antigen-reactive HTL clone and an Lyt-1- Mls-reactive HTL clone express L3T4. Analysis of the effect of mAb GK1.5 on PFC responses in adoptive transfer suggests that L3T4 is expressed by the helper/inducer subset of murine T cells. Expression of L3T4 by murine T cells, however, may correlate primarily with class II MHC antigen reactivity rather than with functional phenotype; mAb GK1.5 profoundly blocks antigen-specific cytolysis by the cloned class II MHC antigen-reactive CTL line A15-1.17. Antigen-specific cytolysis by A15-1.17 is blocked by mAb GK1.5 at a step before the lethal hit. Collectively, the flow cytometric, functional, and biochemical data indicate that L3T4 is similar to the human Leu-3/T4 molecule.

Animals↗
PubMed1983

Nonrandom escape of tumor cells from immune lysis due to intraclonal fluctuations in antigen expression.

Considerable heterogeneity in the amount of surface antigen can regularly be demonstrated by cytofluorometric analysis among genetically identical cells in a tumor clone. We have used monoclonal idiotype-specific antibodies to investigate the patterns of change in amounts of an idiotypic tumor antigen and how such changes affect the immune escape of malignant B cells expressing this antigen. By the use of fluorescence-activated cell sorting, we separated cells expressing either very large or very small amounts of the idiotypic target antigen and then analyzed these subpopulations of tumor cells at various times after isolation for expression of idiotype. We found that the differences in amount of antigen expression were not heritable, and that over a period of about 7 days of continuous growth in vitro, the fluorescence-activated cell sorted populations gradually came to express normal amounts of idiotype. The mechanisms regulating the quantity of surface idiotype were independent of those affecting the amounts of other membrane-associated molecules such as H-2 antigen. Furthermore, these nonheritable intraclonal differences in amounts of antigen expression were unrelated to stages of the cell cycle but clearly did affect the susceptibility of the cells from immune lysis. Thus, tumor cells expressing the lowest amount of surface idiotype were much more resistant to the lytic effects of antiidiotypic antibody and complement but had the same cell cycle distribution as did unseparated cells. These results demonstrate that nonheritable, non-cell cycle-related heterogeneity in amount of tumor antigen expression can significantly determine which cells in a cloned malignant cell population preferentially escape monoclonal tumor-specific antibody therapy.

Animals↗
PubMed1982

Two color light scattering identifies physical differences between lymphocyte subpopulations.

Forward angle light scattering of two different wavelengths by cells in a flow cytometer was used to investigate physical differences between lymphocytes of different lineage, functional subclass and developmental stage. Correlation of the ultraviolet (UV: 351 nm and 364 nm) and 488 nm light scattering signals produced by lymphoid cells demonstrated that the two signals were not equivalent and that they placed different emphasis on the physical parameters characterizing lymphocytes. Both small T and B lymphocytes from peripheral lymphoid tissues and mitogenically activated large T and B lymphocyte blasts were discriminated by both wavelengths. Differences between the Lyt-2 negative and Lyt-2 positive T lymphocyte subsets were also apparent. Two color light scattering could also discriminate between immature thymocytes and mature peripheral T cells and between small bone marrow cells and mature peripheral B cells. In bone marrow an increase in UV light scattering coincided with the appearance of cell surface immunoglobulin on small cells. These data establish that two color light scattering is a sensitive probe for distinguishing cells of apparently similar morphology and that it can be used to study the physical changes that occur during lymphoid cell differentiation.

Animals↗
PubMed1982

High molecular weight lymphocyte surface proteins are structurally related and are expressed on different cell populations at different times during lymphocyte maturation and differentiation.

HMW lymphocyte surface proteins T20 and B220, which are expressed on T and B cells, respectively, have been found to be extensively related in structure, beyond what would be expected by the sharing of the determinant detected by monoclonal antibody 13/2. In addition, another HMW protein (T200A) has been discovered, which is preferentially expression proliferating T cells, and which exhibits significant structural homology with T200, but does not express the 13/2 determinant. T200A bears a novel antigenic determinant, detected by monoclonal antibody BS-S28E, which is not shared by either T200 or B220. A fourth HMW protein (170/100) has been found, which is expressed on most lymphocytes, but which is neither structurally nor antigenically related to T200A, T200, or B220. Monoclonal antibodies reactive with 170/100 inhibit T cell-mediated cytolysis, although monoclonal antibodies reactive with T200A, T200, or B220 do not.

Animals↗
PubMed1982

I-A antigens on cloned alloreactive murine T lymphocytes are acquired passively.

Cloned lines of alloreactive cytolytic and amplifier T lymphocytes have been examined for expression of I-A antigens. Using flow cytofluorometry and monoclonal antibodies, MHC antigens representing both I-A and H-2K or D determinants were reproducibly detected on the cell surface of these cloned T lymphocytes. All cloned lines examined expressed H-2K or D antigens representing the MHC haplotype appropriate to their strain of origin. However, I-A as well as H-2K or D antigens appeared to be acquired passively from the stimulating cell population. Within the lower limits of detection using the fluorescence-activated cell sorter, evidence suggesting endogenous synthesis of I-A antigens by these cloned T lymphocytes could not be demonstrated. Further, the observed binding appeared to be nonspecific, because material originating from allogeneic, syngeneic, or third-party stimulating cells could be observed under appropriate conditions.

Animals↗