HL-A LD (Lymphocyte Defined) Typing: A Rapid Assay with Primed Lymphocytes

Sheehy, Michael J.; Sondel, Paul M.; Bach, Marilyn L.; Wank, Rudolf; Bach, Fritz H.

doi:10.1126/science.124948

Cited by 206 publications

(68 citation statements)

References 17 publications

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“…Sensitization in a mixed lymphocyte culture positively selects for memory cells which give a rapid secondary response upon restimulation with cells from the initial stimulator (3,4). Studies in human families (2,(5)(6)(7) showed that restimulation is caused by the same LD determinants of the major histocompatibility complex (MHC) that induce primary MLC responses.…”

mentioning

confidence: 99%

Genetic and cellular aspects of xenogeneic mixed leukocyte culture reaction.

Lindahl¹,

Bach²

1976

The Journal of Experimental Medicine

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Xenogeneic lymphocytes can induce proliferative responses in the mixed lymphocyte culture (MLC) ~ which are as strong as those induced by allogeneic lymphocytes (1). It is impossible to determine directly which lymphocyte antigens stimulate the xenogeneic MLC response. The recently described "primed lymphocyte typing" (PLT) assay (2) has made it possible to determine indirectly the genetic control of xenogeneic MLC. Sensitization in a mixed lymphocyte culture positively selects for memory cells which give a rapid secondary response upon restimulation with cells from the initial stimulator (3, 4). Studies in human families (2, 5-7) showed that restimulation is caused by the same LD determinants of the major histocompatibility complex (MHC) that induce primary MLC responses.In this paper we present studies of human lymphocytes which were sensitized to cells of a single mouse strain and restimulated with cells from a variety of strains. By choosing combinations of primary and secondary stimulator which shared only certain parts of their genetic material, we could assay the relative importance of non-H-2 and various H-2 antigens for activation of human lymphocytes. The results suggest that the same antigens are important in allogeneic and xenogeneic MLC reactions. The ability of human leukocyte subpopulations to respond in xenogeneic MLC further suggest that the cellular requirements of allogeneic and xenogeneic MLC responses are the same. Materials and MethodsMice. Mice used in this study were bred in our own colony or purchased from The Jackson Laboratory, Bar Harbor, Maine (Table I).Spleen Cells. Spleens were removed aseptically into phosphate-buffered saline (PBS) (Dulbecco's, Ca 2+ and Mg 2÷ free, Grand Island Biological Company, Grand Island, New York), and the cells were pressed through a stainless steel wire screen. The suspension was allowed to settle and big sediments removed. Erythrocytes and dead cells were removed by hypotonic shock (8); the cells were resuspended in PBS and viable cells counted in eosin.

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mentioning

confidence: 99%

Genetic and cellular aspects of xenogeneic mixed leukocyte culture reaction.

Lindahl¹,

Bach²

1976

The Journal of Experimental Medicine

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“…Human T lymphocytes seem to have clonally distributed receptors for HLA-D/DR antigens as demonstrated by the specificity of the recognition and memory phase of the in vitro (MLC) response to allogeneic stimulators (11,12). The present study suggests that T lymphoblasts alloactivated against a given HLA-DR antigen express idiotype-like receptors, as they are capable of inducing an AMLR response specific for the receptor expressed by the AAL.…”

Section: Discussionmentioning

confidence: 79%

Idiotype-like determinants on human T lymphocytes alloactivated in mixed lymphocyte culture.

Suciu‐Foca

Rohowsky²,

Kung

et al. 1982

The Journal of Experimental Medicine

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We have previously (1-3) shown that human T lymphocytes that have been primed to allogeneic stimulators in mixed lymphocyte culture (MLC), acquire the capacity not only of expressing HLA-D and DR antigens but also of stimulating autologous MLC responses (AMLR). This observation has lead us to propose that the Ia-like antigens expressed by primed T cells may be associated with idiotypes that trigger the anti-idiotype lymphocyte response, and that by analogy with the antibody system, such anti-idiotypes may regulate the T cell immune response (3).Because previous studies, however, have shown that resting T lymphocytes respond in AMLR to cells expressing HLA-D/DR antigens, such as B cells and monocytes (4-6), proof to the effect that the autologous MLC reactivity to aUoactivated T cells is truly idiotype-(i.e., receptor-) specific, rather than specific for self-Ia, is required. The present experiments demonstrate that T ceils primed against autologous allostimulated T lymphoblasts (AAL) display specific "memory" responses against the original autostimulator in the primed lymphocyte test (PLT) and fail to exhibit secondary reactivity to autologous T lymphoblasts (AL) primed against a different HLA-D/DR specificity. Materials and MethodsLymphocyte Preparations. Blood was obtained from healthy volunteers with a mean age of 32 yr. Peripheral blood lymphocytes (PBL) containing <5% granulocytes were isolated by FicollHypaque gradient centrifugation. The suspensions were depleted of monocytes by removal of cells adhering to petri dishes. T cells were separated by the thrombin-nylon wool method (7). Further purification ofT cells was accomplished by incubating 1 × 107 lymphocytes with 1/~g of mouse monoclonal OKT-3 antibody (Ortho Pharmaceutical, Raritan, N J), and subsequently rosetting the T lymphocytes with sheep erythrocytes (SRBC) coated with purified anti-mouse IgG (8). After lysing the SRBC, T cells were washed and resuspended in RPMI 1640 culture medium supplemented with glutamine, antibiotics, and pooled normal male serum. Immunofluorescence determination using a Spectrum III Cytofluorometer (Ortho Diagnostics) showed that >95% of the cells contained by these suspensions were OKT-3 positive.Priming of OKT-3-positive T Cells against AAL. OKT-3 + T cells obtained from fresh PBL were used as responders (1 × 107 cells in 10 ml of medium) and were primed in 9-d MLC with an equal number of irradiated AAL, representing the stimulating cells. AAL were obtained from a 5-d MLC between the same responder and irradiated monocyte-depleted lymphocytes * Supported by grants 1 POL-AGO2307 and NOl~A1-82552 from the National Institutes of Health.J. Exp. MEn.

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“…On the fourth day, MLC in the first well was collected, washed with RPMI-1640 medium and then counted. The cell concentration was diluted to 1x10 6 cells/ml with RPMI-1640 containing 20% fetal bovine serum, and the suspension was noted as PMLC (primed mixed lymphocyte culture) (14). An aliquot of frozen D2 (Bm) was washed with RPMI-1640 medium and then counted.…”

Section: Mixed Lymphocyte Culture (Mlc)mentioning

confidence: 99%

Immunosuppression induced by apoptosis of mixed lymphocyte culture is associated with p53

Wei

Cao

Zhang

et al. 2013

Molecular Medicine Reports

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Abstract. A lymphocyte inhibition model was created using a co-culture of donor and host lymphocytes, resulting in apoptosis of the latter, and subsequently, inducing immune tolerance. This method may be used to resolve the immune rejection problem prior to organ transplantation. Using mixed lymphocyte culture (MLC) and/or addition of anti-anti-IL-2 neutralizing monoclonal antibody, we successfully developed a lymphocyte inhibition model in vitro. In this model, the apoptosis of recipient lymphocytes co-cultured with donor lymphocytes was observed by Wright-Giemsa stain, electron microscopy imaging and flow cytometry. The growth and proliferation of mixed lymphocytes were detected by XTT and BrdU assays. Cell viability was determined using CCK-8 cell viability assay. The activity of the recipient lymphocytes was very high when stimulated by antigens alone [PMLC + D2 (Bm) group] but markedly lowered by anti-anti-IL-2 neutralizing monoclonal antibody [PMLC + D2 (Bm) + anti-IL-2 group]. The suppression of recipient lymphocyte activity was due to apoptosis mediated by p53 and caspase-3, and the optimal ratio of donor and recipient lymphocytes for apoptosis was explored. With the exception of the control group, the ratio of apoptotic cells was highest in the PMLC + D2 (Bm) + anti-IL-2 group and lowest in the PMLC + D2 (Bm) group. Blockade of IL-2 with anti-IL-2 neutralizing antibody resulted in an increased number of apoptotic lymphocytes in our experiment, which suggested that IL-2 inhibits the apoptosis of lymphocytes. These data suggest that IL-2 is involved in MLC-induced apoptosis of recipient lymphocytes, and that apoptosis may be associated with p53 and caspase-3 pathways.

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HL-A LD (Lymphocyte Defined) Typing: A Rapid Assay with Primed Lymphocytes

Cited by 206 publications

References 17 publications

Genetic and cellular aspects of xenogeneic mixed leukocyte culture reaction.

Genetic and cellular aspects of xenogeneic mixed leukocyte culture reaction.

Idiotype-like determinants on human T lymphocytes alloactivated in mixed lymphocyte culture.

Immunosuppression induced by apoptosis of mixed lymphocyte culture is associated with p53

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