Heated lymphocytes express HLA-DR antigens despite their inability to stimulate in MLC

We have utilized serological techniques and mixed lymphocyte culture (MLC) reactions to examine HLA-DR and HLA-D expression by heated (45 degrees C for 1 h) lymphocytes in order to study the functional relationship of these antigens. Heated lymphocytes do not stimulate proliferation of allogeneic lymphocytes in MLC, yet they express HLA-DR antigens. The fraction of peripheral blood lymphocytes (PBL) expressing DR is not altered by heating, nor is the staining intensity altered as detected by fluorescence microscopy. Alloantisera to "B cell alloantigens" recognize HLA-DR determinants on heated cells without any detectable change in either specificity or quantitative cytotoxic effects. Flow cytometry with monoclonal antibody demonstrates only minimal decrease in HLA-DR expression after heating. Thus stimulation in MLC requires more of the stimulating cell than the mere expression of HLA-DR.     

HLA-D region products are expressed in endothelial cells

The mixed lymphocyte endothelial cell culture was studied by the primed lymphocyte typing (PLT) technique. By comparing the HLA-D/DR specificity of the secondary response when using either peripheral blood mononuclear cells (PBM) or endothelial cells from umbilical cords for priming or restimulation of lymphocytes, it was found that PBM from newborn would induce a clear-cut specificity for HLA-D/DR when used for priming a well a for restimulation. No HLA-D/DR specificity was seen, however, when endothelial cells were used for restimulation of lymphocytes primed to HLA-D/DR on PBM. On the other hand, lymphocytes primed to endothelial cells showed significant, albeit not very strong specificity for HLA-D/DR when restimulated with PBM. Our experiments suggest that HLA-D region products are present on endotheial cells, and thus confirm and extend serological studies using anti DR antisera.     

Lack of shared lymphocyte-stimulating determinants between H-2I and HLA-D/DR using mouse primed lymphocyte typing cells

A number of anti-H-2 alloantisera containing antibody reactive with I region gene products (Ia) of the major histocompatibility complex cross-react with determinants expressed by human peripheral blood B lymphocytes. Such data have led to the conclusion that Ia and DR antigens share cross-reacting determinants. We have attempted to generate mouse primed T lymphocyte populations specific for defined I region gene product determinants which concomitantly recognize DR determinants on human peripheral blood leukocytes in primed lymphocyte typing (PLT) analysis. Mouse PLT cells were generated in primary MLC using strain combinations identical to those in which positive mouse/human cross-reacting antisera have been obtained. The resulting PLT cells exhibited strong, yet specific, secondary MLC responses against mouse cells expressing the Ia determinants used as the primary stimulus. In contrast, when examined on panels of human peripheral blood leukocytes, no reactivity was detected. This lack of cross-reactivity suggests that mouse T cells primed toward Ia determinants do not regularly recognize cross-reacting determinants of DR or D-associated antigens expressed on human PBLs. Consequently, mPLT cells are not a useful reagent in defining HLA-D region polymorphism.     

Differential antigen presentation by heat-treated peripheral blood mononuclear cells and Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL): heated EBV-LCL present alloantigen and soluble antigen but are deficient in the stimulation of autologous EBV-LCL primed T cells

Heat-treated PBM (1 hr at 45 degrees C) cannot present soluble Candida albicans antigens (CAN) or stimulate in the mixed lymphocyte culture (MLC) reaction. This is despite their continued expression of serologically defined class II MHC antigens. In contrast, heat-treated EBV-LCL present soluble CAN and stimulate allogeneic T cells in the MLC. Heated EBV-LCL stimulate strong secondary responses from allogeneic alloprimed T-cell lines in the primed lymphocyte test (PLT), while heated PBM stimulate only weak secondary allogeneic responses. To test whether this difference was due to a subtle difference in the thermal stability of the functional expression of class II MHC antigens on PBM and EBV-LCL cells, the EBV-LCL cells were heated for 1 hr at temperatures from 45 degrees C to 60 degrees C. Even after treatment at 60 degrees C, the heated EBV-LCL strongly stimulated alloreactive T cells in MLC and PLT reactions. Heated EBV-LCL are not nonspecifically mitogenic, as they do not stimulate autologous T-cell lines primed to alloantigens. However, the weak response of alloprimed T-cell lines to heated allogeneic PBM can be augmented by coculturing with autologous heated EBV-LCL, suggesting heated EBV-LCL maintain a metabolic activity necessary for allogeneic stimulation that is deficient in heated PBM. While heated EBV-LCL stimulate allogeneic alloprimed T-cell lines, they no longer stimulate autologous EBV-LCL primed T-cell lines; irradiated EBV-LCL stimulate both strongly. This suggests the involvement of a heat labile antigenic or metabolic factor in the T-cell recognition of autologous but not allogenic EBV-LCL.     

HLA-D-DR relationships: PLT studies of HLA-D specificities associated with DR1, DR2, and DR4

The primed lymphocyte test (PLT) detects gene products of the HLA-D-DR region which activate the secondary (memory) response of MLC stimulated T cells. In the present study attempts were made to determine whether different HLA-D alleles associated with the same DR, such as DR1, 2, and 4 can be discriminated by PLT typing. PLTs were generated by using, as responders and primary MLC stimulators, HLA-D different HTCs which shared all DR groups (major DR, supertypic MT and second locus MB) or only the MT or MB groups. As secondary stimulators, lymphocytes from an HLA-D selected panel of 72 individuals were used. PLTs raised in DR identical responder-primary stimulator combinations were able to discriminate between the different HLA-D antigens associated with the same DR. In contrast, when priming was performed in combinations differing for the major DR group, the restimulation response was highly associated with the DR specificity of the primary stimulator, regardless of whether or not this was compatible with the responding HTC for the MT or MB groups.

This data indicate that the specificity of primed lymphocytes largely depends on the combinations used for priming and that the memory response can be activated by both HLA-D and DR antigens. The dissociation of HLA-D from DR by PLT typing might provide a useful tool for further analysis of this HLA region.     

Stimulation in primary MLR caused by a PLT defined non-HLA-D/DR determinant, EP1

The influence in primary mixed lymphocyte culture reaction (MLR) of a primed lymphocyte typing (PLT) defined non-HLA-D/DR determinant, EP1, was investigated. In primary MLR between HLA-D/DR compatible lymphocytes, the response of the lymphocytes from 14 EP1-negative HLA-D/DR heterozygous individuals towards two EP1-positive homozygous typing cells (HTCs) was on an average approximately 35% higher than the response towards two EP1-negative HTCs ( P < 0.01). The strength of the MLR between lymphocytes from 25 EP1-negative and 10 EP1-positive individuals matched for two HLA-D/DR antigens was investigated. The average responses of EP1-negative lymphocytes against EP1-positive lymphocytes were approximately 40% higher than the average responses against EP1-negative lymphocytes ( P < 0.01). These data indicate that the PLT defined determinant EP1 causes stimulation in primary MLR.     

Alloreactive T cells: Expression of HLA-D antigens, stimulation of autologous MLR, and possible immunoregulatory function

Primed in MLC with allogeneic stimulators T cells acquire the capacity of expressing HLA-D and DR antigens and of stimulating the MLC response of autologous lymphocytes When primed T cells from HTCs are used as stimulators, a bimodal distribution of response with clear-cut “typing responses” and no significant “back stimulation” is observed This pattern may be due to the expansion during priming of a population of HLA-D restricted suppressors since irradiated primed T cells inhibit the MLC responsiveness of HLA-D “compatible” lymphocytes. The development and size of such a population is not dependent, however, on the strength of the antigenic stimulus used for priming since no differences were seen between the pattern of reactions induced by T cells primed against HLA-D identical or HLA-D different cells Primed OKT4⁺ and OKT8⁺ T cells share the capacity of expressing Ia antigens and of inducing “HLA-D restricted suppression.” We suspected that a similar phenomena accounted for the behavior of two HLA-D heterozygous cells as if they were HTCs Although no suppression was found, the fact that these cells typed for their “silent” antigen when tested as responders, yet failed to express it when tested as stimulation, supports the theory that different genes control the MLC-responding and stimulating capacities.     

Genetic Specificity of Primary and Secondary Proliferative and Cytotoxic Responses of Human Lymphocytes Grown in Continued Culture

Human T peripheral blood lymphocytes were grown in continued culture using conditioned medium obtained from phytohaemagglutinin-stimulated, pooled human leucocytes. These cultured T cells (CTC) were tested in mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) assays to determine the genetic specificity of their proliferate and cytotoxic responses. Primary responses were measured after initial in vitro stimulation by allogeneic cells, and secondary responses were measured after a second in vitro stimulation by allogeneic cells. Doth primary and secondary proliferative responses were found to be stimulated by alloantigens controlled by the HLA region and, more specifically, by antigens of the HLA-D region, in accordance with the responses of normal peripheral blood T lymphocytes, When CTC KW established from unsensitized PBL and then stimulated with allogeneic cells, they could respond by proliferation in MLC, but, in contrast to PBL, they did not show subsequent cytotoxic responses. On the other hand, CTC established from PBL that had been stimulated first with allogeneic cells in either primary or secondary MLC displayed high levels of cytotoxic reactivity in CML. The strongest cytotoxicity was directed against allospecificites controlled by the HLA region and specific for the MLC-stimulating cells, but lower levels of cross-reactive cytotoxicity were also observed.     

Further studies on psoriasis-associated HLA-Dw/DR7 using PLT cells primed against a familial psoriasis-linked HLA haplotype

The products of the HLA-D region and their correlation to psoriasis vulgaris was studied using the primed lymphocyte typing (PLT) assay. In families with one healthy and one psoriatic parent and one psoriatic child primary MLC (mixed lymphocyte culture) stimulation was carried out between responder cells from the healthy parent and stimulator cells from the psoriatic child. In this way 21 PLT reagents directed against putative psoriasis-associated lymphocyte activating determinants were produced. Three reagents that gave clear bimodal stimulation patterns against lymphocytes of 51 psoriasis patients were further tested against 78 controls. Specificity of these reagents was studied using homozygous typing cells (HTC's) as stimulators. Compiled data show that cells from DR7 positive psoriatic patients give higher restimulation of these PLT reagents than do cells from healthy DR7 positive controls. In addition, a higher frequency of psoriasis patients compared to controls gave significant restimulation. Therefore we concluded that these PLT reagents recognized at least partly different DR7 associated determinants in the psoriasis patients and in controls. The reason is either that psoriasis patients carry a different lymphocyte activating DR associated specificity or, alternatively, that restimulation was caused by products of a distinct psoriasis associated locus in linkage disequilibrium with D/DR7 or of a determinant recognized together with D/DR7 as a restriction element. These data further support the notion that psoriasis is a disease with primary HLA associations to both class I and class II MHC genes.     

Allogeneic Responses In Vitro Induced by Fetomaternal Alloimmunization*

ABSTRACT: The present study was undertaken in order to determine what type(s) of pregnancy-induced allogeneic reaction could alter MLC (mixed lymphocyte culture) reactivity in routine HLA-D typing of lymphocytes in multi-parous women (MW) possessing antibodies against paternal HLA-DR antigens. Unresponsiveness to homozygous typing cells (HTC) representing a paternal and probably fetal HLA-DR determinant was frequently observed. Kinetics experiments ruled out an early secondary proliferative response to HTC representing the paternal HLA-D determinant, which would be missed in a classical long-term mixed lymphocyte culture. Direct cytotoxicity against paternal or panel target cells was not always associated with inhibition of proliferative response to the same stimulator cell. Specific anti-HLA-DR blocking activity (antibodies?) in the supernates of restimulation reactions of lymphocytes from MW could be responsible for this inhibitory effect. Moreover, the study points to the existence of suppressor cells in the immunized MW acting independently of specific restimulation. The in vitro suppression appeared to be selective, restricted to cells sharing HLA-D linked structures with the suppressor cells, and suggests that auto-regulator mechanisms could be induced in pregnancy in order to modulate antibody production.     

Functional study and detection of HLA-D products on fractionated human bone marrow cells

Bone marrow cells from nine normal human volunteers obtained from the Iliac crest, were used in this work for antigen determination and functional studies. The bone marrow aspirated cells were sequentially separated: elimination of erythrocyte, granulocytes and monocytes achieved by Ficoll-Isopaque centrifugation followed by plastic adherence. Purified bone marrow cells were finally separated by size using velocity sedimentation. The slow sedimenting small cells were shown to be mainly T lymphocytes, probably of blood origin. The medium sized bone marrow cells were shown to contain myeloid precursors (CFu-c). Large immature cells were in cycle actively synthesizing DNA molecules. HLA-D and HLA-DR detections on the fractionated cells were performed using three techniques: fluorescence with specific anti HLA-DR allo and xeno antisera; primed lymphocyte typing (PLT) with anti HLA-DR monospecific in vitro primed lymphocytes and detection of the HLA-D stimulating product using the bone marrow fractionated cells as stimulators in a mixed leukocyte culture. Concordant results were obtained with the three techniques. Lymphocytes in the bone marrow express HLA-D products a peripheral lymphocytes. Bone marrow fractions depleted of lymphocytes and monocytes also contain approximately 20% of cells expressing HLA-D products The meaning of the expression of HLA-D products on immature precursors non-lymphoid cells is discussed.     

Secondary MLC Responses of Primed Lymphocytes after Selective Sensitization to Non-HLA-D Determinants

Two HLA-B,D-identical siblings, who differed only for the HLA-A region because of a maternal recombinational event, were studied in primary (1 degrees) and secondary (2 degrees) mixed lymphocyte culture (MLC). The HLA-A:B recombinant child did not respond to its HLA-B,D-identical sibling in either 1 degrees or 2 degrees MLC. In the reciprocal combination the non-recombinant child responded only weakly in 1 degrees MLC but responded significantly in 2 degrees MLC to the HLA-A:B recombinant child. Thus, it was possible to selectively prime to a non-HLA-D determinant, which is controlled by a gene located distal to HLA-B. Because this determinant was not present on T-cells, it could be distinguished from the serologically defined antigen controlled by the HLA-A locus. Such primed lymphocytes, as well as lymphocytes primed between HLA-identical siblings, revealed high autologous control responses which were not observed when using lymphocytes primed in conventional one-haplotype combinations. The significant 2 degrees MLC response to autologous cells after sensitization to allogeneic cells may reflect recognition of self antigens and raises the question to what extent genetic similarity between responding and stimulating cells is required in the priming phase to elicit a 2 degrees response to autologous cells.     

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.     

Function of alloproliferative T lymphocyte clones correlates better with their class II recognitive specificity than with their cell surface phenotype

Alloproliferative primed lymphocyte typing (PLT) clones recognizing determinants associated with HLA-DR/Dw, SB, MB, or novel "SB-like" gene products were screened for their ability to suppress lymphoproliferative responses in primary and secondary mixed lymphocyte cultures (MLC), and for their surface marker phenotypes. Two nonsuppressive HLA-D specific PLT clones were OKT4-, OKT8+ whereas all others possessed an OKT4+, OKT8-, Leu-8- phenotype. All clones secreted interleukin 2 (IL2) after specific stimulation. The eight PLT clones specific for "SB-like" antigens strongly suppressed MLC, whereas only one of 35 DR/Dw-specific, and none of 20 SB-specific PLT clones did so. Suppressive activity of such PLT clones was not restricted by major histocompatibility complex products, was radioresistant (20 Gy), and was not caused by absorption of IL2 or by cytotoxicity of the cloned cells. Suppressive clones exerted their effects directly on proliferating T cells, as assessed by their ability to prevent growth of cloned PLT cells stimulated by B cell lines, and their ability to block primary MLC even when added 96 h after the start of the 144-h culture. Culture supernatants from suppressive, but not from nonsuppressive, PLT clones also strongly and nonspecifically inhibited lymphoproliferative responses. The suppressive factor(s) was not dialyzable, not sensitive to pH 2 or heat treatment and not cytotoxic. Thus, all T cell clones proliferating against novel "SB-like" but not SB antigens, as well as rare clones specific for D region determinants, possess powerful nonspecific suppressive activities dissociated from their "helper-related" OKT4+, OKT8-, Leu 8-, IL2-secreting phenotypes.     

Restimulation in Secondary MLC by Autologous Non-T Cells

Lymphocytes sensitized in mixed leucocyte culture (MLC) were restimulated with non-T or complete cells. Cells primed against autologous non-T cells and restimulated with autologous cells gave responses with the kinetics of a secondary response. In contrast, cells primed against either type of autologous cell responded to restimulation by allogeneic cells with the kinetics of a primary MLC response. Following priming against alloantigens good secondary responses against both complete and non-T allogeneic restimulators were seen. Restimulation with autologous non-T cells, but not autologous complete cells, also led to secondary responses. The observation of different responses to antigens expressed on non-T autologous lymphocytes, in comparison to complete cells, implies that the secondary MLC involves more than response against alloantigens. Allogeneic and autologous MLC may be qualitatively different phenomena, the latter representing the recognition of identifying structures between interacting populations of cooperating cells.     

The Specificity of Secondary MLC Assayed by Titration of Primed Lymphocyte Populations

The specificity of responses in secondary MLC was studied by titration (100 x 10(3) to 5 x 10(3)) of responder primed lymphocytes. In all instances, significant proliferative responses of responder primed lymphocytes to the specific stimulator occurred using lower responding cell numbers. When tested against allogeneic stimulating donors, three patterns of responses were observed: no significant responses, responses only at higher (100 x 10(3)) responding cell densities, and responses at lower responding cell densities, similar to those with the priming donor. In instances where primed lymphocytes responded significantly to allogeneic stimulating cells at lower cell densities, the responses were considered positive and the stimulating cells positive for the PL determinant. In several instances, primed lymphocytes responded significantly to allogeneic stimulators negative for the specific HLA-D and/or HLA-DR specificities. On the other hand, in experiments where allogeneic stimulating donors shared either HLA-D or DR specificity with the priming donor, a significant response was always observed. Thus, the PL determinant was present on stimulating allogeneic cells negative for specific HLA-D and DR specificities. The present data suggest that an analysis of the specificity of primed populations could be profoundly affected by the responder cell density at which the assay is performed. Also, the data suggest that other MHC determinants, including non-HLA loci may be important in secondary MLC.     

The major histocompatibility complex and its relationship to allergic disease.

Two tests, the mixed leukocyte culture (MLC) and cell-mediated lympholysis (CML) tests, have been used as in vitro models of the in vivo allograft reaction. These tests have been applied to histocompatibility testing for transplantation as well as to assay of immune function. They involve the use of peripheral blood lymphocytes as "responding" cells in mixed leukocyte culture or as "stimulating" cells in the generation of a proliferative or a cytotoxic response. The proliferative events in MLC are primarily in response to LD antigens of the major histocompatibility complex; the cytotoxic cells use as their targets the SD antigens of that complex. A new method for defining the LD antigens, the primed LD typing (PLT) test, is based on in vitro sensitization of lymphocytes to certain LD antigens of the major histocompatibility complex and their subsequent restimulation with test cells.     

Primed lymphocyte typing predicts MLC reactivity between unrelated individuals

The present study was carried out to evaluate the ability of primed lymphocyte typing (PLT) to predict mixed lymphocyte culture (MLC) reactivity between unrelated individuals. Eleven PLT cells generated against independent familial haplotypes, and one PLT cell generated against a homozygous typing cell (HTC), were tested for their response to cells of a random panel of 53 unrelated individuals. From Chi-square analysis of the response patterns of these 12 cells, nine "PLT specificities" could be defined. Most panel members (81.2%) types for 1 or 2 of these specificities; equal numbers (9.4%) types for 3 or none, respectively. Four of these 9 specificities were shown to be significantly correlated with HLA-Dw antigens defined by HTCs. Typing for these nine PLT specificities was found to be predictive of subsequent primary MLC reactivity between panel members: a) pairs of panel members sharing PLT specificities produced three-fold lower MLC results on the average than pairs of panel members disparate for PLT specificities (p less than 0.0001), and b) in MLC combinations, an increase in number of foreign PLT specificities presented by the stimulator cell was paralleled by a statistically significant increase in MLC reactivity. To the extent that low MLC reactivity is correlated with improved graft survival, the PLT method could have significant value in selecting histocompatible donors for organ transplantation.     

Anomalous Mixed Lymphocyte Culture Reactivity between HLA—A, —B, —C, —DR Identical Siblings

Complete HLA typing including HLA--A, --B, --C, --DR (D related B cell typing), --D, mixed lymphocyte culture (MLC), and primed lymphocyte testing (PLT), together with complete red blood cell (RBC), glyoxalase (GLO), GBG (Factor B), and phosphoglucomutase 3 (PGM3) typings were performed on a informative family. The five siblings inherited the four possible combinations of parental HLA haplotypes, and two of the siblings were HLA--A, --B, --C and --DR identical. Repeated MLC testing of the family revealed positive mixed lymphocyte reactivity in all combinations. B cell typing for the DR specificities demonstrated no variation from the expected inheritance pattern and specifically no recombination event. GBG and GLO typings militated against a recombination involving the paternal chromosome. HLA--D testing revealed that only one of the HLA--A, --B, --C, and --DR identical siblings gave typing responses to the HLA--Dw3 specificity present on that maternal haplotype. Utilizing HLA haploidentical combinations, lymphocytes were primed against the four parental haplotypes and the non-Dw3 haplotype of interest (Aw24--B8--DRw3--LDY) in the PLT. The sibling inheriting this haplo-type did not restimulate cells primed against the A2--B40--DRW6--LDY specificity. Furthermore, no discrimination was observed in the restimulation of lymphocytes primed against this haplo-type. Possible interpretations of these family data include: a spontaneous mutation, non-major histocompatibility locus (MHC) stimulation, and HLA--DR/D recombination.