Human Mixed Lymphocyte Cultures Research Articles

Inhibition of human mixed lymphocyte culture stimulation by anti-HLA-D-associated antisera: studies with primed responding cells.

The inhibitory effects of HLA-D-associated antisera on stimulating lymphocytes when cultured together with allogeneic primed responding lymphocytes were investigated. The responding lymphocytes were primed for either the HLA-Dw2 or Dw3 determinants. The presence of HLA-D-associated (Ia-like) antibodies during the culture period specifically inhibited the stimulatory capability of lymphocytes possessing the HLA-D determinants with which the antisera were apparently reacting, as long as the stimulating cells carried the determinant for which the responding cells were primed. HLA-D-associated antisera of other specificities caused no decrease in the stimulatory capability. These antisera, therefore, apparently contain antibodies which are reactive with determinants closely associated or identical to the HLA-D determinants and may be human analogues to the mouse Ia antigens.

The Xenogeneic Effect

Abstract Studies were conducted on the mechanism of action of a soluble mediator which was generated in human mixed lymphocyte cultures and assayed for helper activity in T cell-deficient murine spleen cell cultures. The mediator, termed xenogeneic reconstitution factor or XRF, restored the anti-sheep erythrocyte plaque-forming cell response of spleen cells from thymectomized, lethally irradiated, and syngeneic bone marrow transplanted (TxB) mice. It was found that in order to obtain a maximal antibody response, the XRF had to be present sometime during the first 24 to 40 hr of the induction process. XRF was also required during the last 24 hr of the incubation period. A striking synergistic effect was obtained by combined exposure of the T cell-deficient spleen cell cultures to XRF during the first and last days of culture. These data suggested a bi-modal mechanism of action of XRF, and raised the possibility that in addition to providing a signal to the B cells, XRF-mediated activation of the residual TxB splenic T cells was crucial to the successful restoration of the antibody response. Treatment of the nonadherent splenocytes from the TxB mice with anti-T cell serum and guinea pig complement completely abrogated the antibody response of these cells in the presence of adherent spleen cells, sheep erythrocytes, and the helper factor XRF. The antibody response of this combination of cells, antigen, and XRF was reconstituted by a population of unimmunized murine T cells which, in the absence of XRF, were totally unable to restore responsiveness. These results suggest that B cell activation to the formation of antibody involves the mandatory co-participation of two functionally distinct helper activities.

Human Mixed-Lymphocyte Culture Reaction: Genetics, Specificity, and Biological Implications

Publisher Summary This chapter deals with the new and exciting developments in MLC (multi-level cell) typing in human histocompatibility studies. The genetics, specificity, and biological implications of human mixed-lymphocyte culture reaction are also discussed. The use of homozygous cells from specific delineates at least six different distinct MLC antigens that can be recognized by B-cell-specific alloantisera and clearly relate to the Ia antigens of the murine system. Certain disease associations and the genes involved in the complement components, appear more closely linked to the MLC genes than to the other components of the HLA (human leukocyte antigen) system. The MHC (major histocompatibility complex) in man HLA is defined by four distinct loci: (1) HLA-A, (2) B, (3) C, and (4) D. Three of these loci code for alloantigen are readily detectable by serological methods (HLA-A, B, and C). The fourth locus (HLA-D) controls lymphocyte responses in the in vitro mixed-lymphocyte culture reaction (MLR). Three important characteristics shared by the genes or gene products of each locus are also discussed. The study of genetic control of immune response and that of clinical transplantations emphasizes the significance of the HLA-D segment of the HLA complex. The chapter summarizes the major achievements in the clarification of the genetic control and polymorphism of the serologically detectable HLA-A, B, and C determinants.

The role of serum in human mixed lymphocyte cultures

Mixed lymphocyte cultures employing human lymphocytes were established in serum-supplemented medium. After an initial incubation of 72 hr or longer, proliferation of the cultures was observed in serum-free medium for an additional 24–48 hr. Thereafter, the proliferation decreased to below values seen in autologous controls. No DNA synthesis was observed in serum-free cultures or in cultures initially incubated with serum for less than 72 hr. Human albumin added to the cultures could not replace serum, although a weak response was obtained. Serum therefore must be present throughout the incubation period in human mixed lymphocyte cultures, if a maximum response is to be elicited.

The human mixed-lymphocyte culture (MLC) interaction after in vivo allo-immunization : II. HL-A specificity of early proliferative response

Human volunteers were immunized with a single intradermal dose of allogeneic buffycoat cells. When donor and recipient differed for four HL-A antigens belonging to the first and second series, an increased MLC proliferation early in the cultures was observed when responding lymphocytes from the immunized individual were confronted with stimulating cells from his donor. These findings were not observed when the incompatibility between donor and recipient involved only one second series HL-A antigen. The specificity of the altered response was studied by confronting responding lymphocytes from the immunized individual with different third-party stimulating cells. Most of these combinations revealed an early increased proliferation compared to control combinations with responding lymphocytes from nonsensitized individuals. However, the early increased responsiveness was significantly more pronounced in combinations where the stimulating cells shared two or more “private” HL-A determinants with the immunizing donor. It is concluded that a major part of the early increased responsiveness is due to proliferation of lymphocytes with receptors for HL-A (SD) determinants.

The human mixed lymphocyte culture (MLC) interaction after in vivo alloimmunization : I. Kinetics

Human volunteers have been immunized with a single dose of allogeneic buffy coat cells given intradermally. The immunization provoked typical alterations in the MLC response, consisting of a markedly increased, early proliferation, when the immune cell population was confronted with the immunizing cells. This change of MLC pattern was detectable 1 wk after immunization, and was subsiding after 3 wk. Specificity for the increased response could be shown. These findings were done in combinations differing for all four HL-A antigens and could not be reproduced when donor and recipient of the immunizing cells differed for only one HL-A antigen. Booster immunization, however, could elicit the changes described in one such combination, while boosting did not affect the MLC response pattern in one four HL-A antigen-different combination.

A quantitative human mixed lymphocyte culture using suspension cultures.

A new quantitative method of mixed lymphocyte culture using suspension cultures of human lymphocytes is described. Compared to methods using settled cells, the new method increased the MLC response on the average 6 times permitting determination of a wider spectrum of MLC differences and a clearer discrimination between MLC responses of different magnitude.

Detection of Migration Inhibitory Activity in Human Mixed Lymphocyte Cultures

Abstract The macrophage migration test represents an in vitro correlate of delayed type hypersensitivity (1). The technique has subsequently been adapted to human peripheral blood leukocyte migration with regard to particulate (2) and water soluble (3, 4) antigens. Inhibition of migration of human peripheral leukocytes has been used as an indication of immunologic reactivity after liver transplantation (5) and after skin grafting (6, 7). Migration is inhibited in mixtures of two cell populations when one of these is sensitized to the transplantation antigens of the other, but direct migration does not demonstrate histoincompatibility since allogeneic mixtures of nonsensitized cells migrate normally (7, 8). It has been proposed, however, that degrees of histoincompatibility between nonsensitized cell types may be measurable by the production of migration inhibition factors (MIF)1 when such cells are cultured together (9).

Inhibition of the mixed lymphocyte reaction with fab fragments from anti-human ALG.

SummaryThe human mixed lymphocyte culture reaction (MLCR) as measured by incorporation of 14C-thymidine is inhibited in the presence of an appropriate concentration of equine anti-human lymphocyte globulin (ALG). Fab fragments prepared from ALG also inhibit the MLC reaction. However, unlike the intact ALG molecules, the Fab fragments are noncytotoxic for human lymphocytes. ALG derived Fab fragments could be potentially useful in suppressing allograft reactivity in vivo.We thank Dr. Gordon R. Dreesman for performing the analytical ultracentrifuge analyses and Mrs. Jane Ann Martin for her technical assistance.

The genetic basis for reactivity in human mixed lymphocyte culture. 1. Studies of HL-A typed siblings.

A high correlation between the results of serotyping for HL‐A determined antigens and reactivity in mixed lymphocyte culture (MLC) was demonstrated. In 42 one‐way MLC's between serotypically identical siblings no stimulation was found; the mean ratio between mixtures and unstimulated controls being 1.13 × 0.44 (s.d.), while the lowest ratio in mixtures between family members differing for one or two HL‐A haplotypes was 3.70. The possibility of an additive effect of non HL‐A antigens on the stimulation provoked by HL‐A antigens was investigated by comparing the variation obtained in stimulation of cells from a given individual by cells from two HL‐A identical siblings with the technical variation of the method. It was concluded that if non HL‐A antigens show an additive effect in MLC this effect is so small that it is undetectable by the present technique. This conclusion was supported by the finding of equal mean stimulation in 24 one‐way cultures between siblings differing for two HL‐A haplotypes and 40 one‐way cultures between unrelated persons.

Cytoplasmic Fibrils in Mixed Lymphocyte Cultures

Abstract Bundles of cytoplasmic fibrils are described in activated (increased cytoplasm and organelles) lymphocytes and blasts in human mixed lymphocyte cultures. Similar bundles were not observed in the transformed lymphocytes in PHA cultures. They were encountered in the activated lymphocytes in the control cultures, but with a lower incidence. The fibrils were usually perinuclear, were frequently in contact with the nuclear envelopes and occasionally penetrated through defects in the nuclear membrane into the nucleus. A less frequent and less convincing connection with mitochondria was also observed. These morphologic observations seem to indicate that the transformed cells observed in mixed lymphocyte and PHA cultures are not identical.