Typing for HLA-D Determinants. Comparison of Typing Results using Homozygous Stimulating Cells and Primed Cultures

Typing for HLA-D determinants, using primed lymphocyte typing (PLT), has been compared to the results obtained using conventional homozygous stimulating cell primary MLC tests. The HLA-Dwl, -Dw2 and -Dw3 specificities were studied. Preliminary results indicate that these two methods essentially type for the HLA-D determinants and that reproducible results can be obtained employing the PLT technique after 24 hours of incubation in the secondary cultures.     

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.     

Population studies of HLA-linked SB antigens and their relative importance in primary MLC typing. Analysis of HLA-D homozygous typing cells and normal heterozygous populations

SB phenotyping was undertaken on 96 HLA-D homozygous typing cells (HTCs) and 129 normal unselected heterozygous donors in the German population, using Interleukin-2-propagated primed lymphocyte typing (PLT) reagents. The results showed that the SB antigens in the normal population behave as a system of alleles at a single locus in Hardy-Weinberg equilibrium (p approximately equal to 0.20). Estimated gene frequencies in the German population appeared to be significantly different (p less than 0.002) from the North American Caucasian population: the principal differences were increased frequencies of the specificities SB1 and SB4, and decreased frequencies of blanks. Of HLA heterozygous donors 41% typed for two distinct SB specificities; 57% typed for one; and 2% were blank. In the HTC group, 20% typed for two specificities; 68% typed for one; and 12% were blank. Thus, a significant proportion of HLA-D homozygous test cells were, nonetheless, heterozygous for HLA-linked SB antigens. Performance of checkerboard mixed leukocyte cultures (MLCs) between 16 SB typed HLA-Dw3 HTCs, however, did not indicate that the observed mutual or one-way responses were influenced in any simple way by SB antigens; neither heterozygosity nor assumed homozygosity for SB antigens appeared to influence the frequency of MLC typing responses of HLA-Dw3-positive donors on these HTCs. These results add further confirmation of the genetic and functional independence of the SB gene product(s) and the HLA-D/DR gene product(s).     

Generation of HLA-D specific primed lymphocyte typing (PLT) cells and cross-reactions of PLT-cells primed with homozygous typing cells

An approach for the selection of HLA-D specific primed lymphocyte typing (PLT) cells is described. The responder cells were primed with homozygous typing cells. Reproducible extra reactions were found and were analyzed in relation to HLA-D antigens defined by homozygous in cells (HTC's). The secondary response of 105 different PLT-cell combinations generated by 29 different primary responders against 19 different homozygous typing cells of the specificifies Dw1 to Dw8 and the local specificity "H" were tested in secondary PLT toward 17 different homozygous typing cells and 10 heterozygous cells. Cross-reactions were defined as reactions equal to or higher than the lowest HLA-D specific reaction observed. The entire experimental design and data analysis gave rise to a conservative definition of cross-reactivity. Two main groups of cross-reacting HLA-D determinants seem to exist: (i) Dwl, 3, 4, 7, and the local specificity "H", and (ii) Dw2, 5, 6, 8, and "H". The primary pairwise cross reactions were in group (i): Dw1-3, Dw1-"H", Dw3-4, Dw3-7, Dw7-"H", and in group (ii): Dw2-6, Dw2-8, Dw5-8, and Dw5-"H". The existence of such cross-reactions is likely to interfere with the results of PLT-typing and should be taken into account when attempts are made to develop HLA-D specific PLT-cells.     

Quantitation of HLA-D Differences

The predictability of MLC non-reactivity by HTC typing was tested in a single checkerboard experiment which involved 39 unrelated individuals belonging to 12 different groups of HLA-D identical phenotypes. The strength of one-way MLC reactions in all possible responder-stimulator combinations (39 × 39) was quantitated by the Linear Clustering Analysis Program. Individuals who, by HTC typing, were identical for two HLA-D antigens gave 51% negative, 36% intermediate and 13% strong positive MLC responses. Identity for only one HLA-D antigen resulted in 3% negative, 40% intermediate and 57% strong MLC reactions. When no HLA-D antigen was shared, 88% of the reactions were strongly positive. HLA-D antigens behaved as equipotent stimulators in MLC between half-identical pairs. The only exception consisted of the higher frequency of weak responses displayed by the Dw5 positive individuals against stimulators differing by LD107. The same pattern was observed when LD107 homozygous cells were used as stimulators, suggesting that determinants of this specificity might be partially included in Dw5.     

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.     

HLA–D and –DR antigens on human amniotic fluid cells

Human amniotic fluid cells, known to express HLA-A, -B, and -C antigens, were tested for the presence of lymphocyte-stimulating antigens (LD or HLA-D) using modifications of the mixed lymphocyte culture (MLC) and primed lymphocyte typing (PLT) tests. Peripheral blood lymphocytes were co-cultured with various concentrations of allogeneic amniotic fluid cells, either growing as a monolayer culture in microtiter plates or suspended in medium following treatment with trypsin. The kinetics of such mixed lymphocyte amniotic fluid cell culture (MLAC) reactions were followed during days 3 to 8. Under none of these conditions did amniotic fluid cells significantly stimulate allogeneic lymphocytes, even after lymphocytes were specifically primed in the PLT assay to the HLA-D antigens segregating in the family of the amniotic fluid cell donor. Furthermore, in three-cell experiments, amniotic fluid cells failed to inhibit an ongoing MLC reaction, indicating that the absence of proliferative response to amniotic fluid cells is not due to active suppression. Taken together, these data strongly suggest that amniotic fluid cells either do not express HLA-D antigens or do not express them in a form that is detectable in either primary or secondary MLC.     

Detection of HLA-D Clusters Using Primed LD Typing

Using a set of 17 primed LD typing (PLT) cells tested on a panel of 35 unrelated cells, we showed that certain groups of PLT cells tended to detect similar unrelated cells. The PLT cells were grouped into seven clusters and these tended to correlate with the seven HLA-D specificities represented on the panel, as determined by HTC testing. These data suggest that the antigens that cause restimulation in PLT are similar to those HLA-D antigens detected by the homozygous typing cell (HTC) test or, alternatively but more unlikely, that the two typing methods are detecting genes in close linkage disequilibrium with the HLA-D region.     

Response of Primed LD Typing Cells to Homozygous Typing Cells

At least two different methods using cellular responses have been described for defining the determinants of the HLA-D region: typing with HLA-D homozygous cells and primed LD typing. Primed LD typing cells were generated in one-haplotype-different combinations and grouped on the basis of two or more cells appearing to define the same HLA-D-region-determined PL antigen. Such cells were restimulated with homozygous typing cells for several of the presently known HLA DW clusters. A very strong correlation was noted: PLT cells defining the antigen PL1 were restimulated with homozygous typing cells for DW3, those PLT cells defining the antigen PL2 were restimulated by homozygous typing cells for DW2, and those defining PL5 were restimulated by homozygous typing cells for DW1.     

Typing an Unrelated Panel with PLT Cells: Association with DW Clusters

Primed LD typing (PLT) cells prepared in one Laboratory (Madison) were shipped in the frozen state and tested in Tübingen on a separate panel that had been typed with homozygous typing cells. Those PLT cells that had been grouped, on the basis of their reaction with test cells of the Madison panel, as defining an HLA PL antigen showed identical or nearly identical patterns of reactivity with the Tübingen panel. Clear association between certain PL antigens and DW clusters as defined with homozygous typing cells could be demonstrated. Of particular interest may be combinations of certain PLT reactions with D-locus-typed cells, where the primed cells do not react as expected from the target's HLA-D type.     

A new homozygous typing cell with HLA-D"H" (DB6) specificity

A new homozygous typing cell (HTC), SK is described. Mixed leukocyte culture (MLC) showed that SK is homozygous for HLA-D"H" (DB6) defined by the HTC, Herluf, and that the HLA-D typing results obtained by Herluf and SK are highly significantly correlated (Kendall's R = 0.46, p = 2.5 X 10(-5)). Both Herluf and SK are also homozygous for a new class II determinant, DN-1, defined by the monoclonal anti-B-lymphocyte antibody, 9w925, developed by Aizawa. The corresponding DN-1 antigen was present in 2.2% of 136 random, unrelated Danes and in all of six unrelated HLA-D"H" positive but in none of 20 HLA-D"H" negative individuals. Thus, there is an absolute and highly significant (p = 4 X 10(-6)) association between the cellularly defined HLA-D"H" determinant and the serologically defined DN-1 antigen, which strongly suggests that HLA-D"H" can now be detected serologically. DN-1 may be identical to DRw12 which is, however, poorly defined. The HTC-donor SK was immunized by pregnancy, and her serum contains anti-HLA-B7, which can easily be absorbed, and anti-B-lymphocyte antibodies which reacted with cells from 87.7% of 536 unrelated Danes. The reaction pattern of this serum is negatively associated with DR3, w6, and w8. This serum may define a new broad, cross-reacting antigen belonging to the same group as DRw52 and DRw53 or DQw1-w3, but it is clearly different from each of these antigens.     

Confounding factors in HLA-DW 2 typing of human leucocytes.

Three healthy HLA-B7 homozygous subjects were found with similar but not identical HLA-D antigens; one was DW 2 homozygous according to independent typing results. This could be an expression of "long" and "short" HLA-D antigens or be due to differences in weak antigens outside the HLA-D region. Two further healthy HLA-B7 homozygous subjects were studied; one was apparently heterozygous for DW 2, the other apparently carried no DW 2 antigen. Both could discriminate between different DW 2 homozygous test cells. Two such test cells--one from a patient with multiple sclerosis (MS) and the other from a man with two children with MS--gave variable and absurb reactions with cells from the two subjects in question. It is tentatively suggested that genes exist which, when present in both moities in a mixed leucocyte reaction (MLR), can impair the MLR and give false "typing" reactions. This might be more common among patients with MS and perhaps also some other diseases (certain arthritides, e.g. rheumatoid arthritis) than among healthy subjects and can complicate or make impossible the interpretation of HLA-D typing data. It could also explain the previously-described impaired MLR between cells from patients with these diseases.     

Occurrence of paternal and maternal HLA-B/D recombinants in a single family

Two B/D recombinant offspring in one family were identified by HLA-A,B,C,D, and DR typing with the Eighth International Workshop sera and cells, intrafamily MLC and PLT test.

Restimulation of cells primed against the four parental haplotypes showed good discrimination between family members who shared the D alleles and those who did not. When the B/D recombinant haplotypes were primed, accelerated proliferative response was observed only with cells sharing the HLA-D region. Cells that shared with the priming cells the HLA-A to B interval but differed for the D region did not restimulate.

These results demonstrated the role of HLA-D disparity in evoking secondary proliferative response and showed that the determinant(s) mapping between HLA-A and B did not restimulate.     

MLC (HLA-D) Typing: A Family Study

The genetics of five HLA-D specificities (Dw1, Dw2, Dw3, Dw4 and Dw6) have been assessed in 21 normal families with four or more children. The HLA-D traits, as defined by typing response against homozygous typing cells, normally behave as dominant characters. The data support the concept of allelic factors. The locial flaw in the basic algorithm of MLC typing (HLA-D typing), i.e. to draw positive conclusions from negative observations, has been amply reinforced in the following studies. Five assignments could not be verified genetically under the assumption of dominant traits. Homozygous lack of specific response genes is among the mechanisms proposed as a cause for the phenomenon which has not yet been fully explained. The estimated magnitude of the frequency of false assignments is approximately 10%.     

The Influence of Matching for SB on MLC Typing Is Significant but Marginal

To investigate the role of SB in MLC typing responses, reactions of lymphocytes from 23 DW3-positive, HLA-D-heterozygous individuals against 9 Dw3 homozygous typing cells (HTCs) were evaluated. Significantly more clear typing reactions were observed in those combinations that were matched for SB as compared with those that were mismatched. Nevertheless, MLC responses towards HTCs that were HLA-D/DR- and SB-compatible could be very strong. An additional analysis of the influence of HLA-B and the newly defined determinants LB-Q1 and LB-Q2 demonstrated that in combinations that were matched for these markers as well, stabilized relative responses could still be over 100%.     

HLA-D typing with homozygous cells identified in an American indigenous isolate: II. Family studies and D/DR relationship

Twelve American Indian nuclear families with 2-5 siblings have been HLA-D typed using mixed lymphocyte cultures and clusters of homozygous typing cells (HTC) of Caucasoid origin to detect DW1-DW7 and typing cells of American Indian origin to detect LDSA, LD15A and LDl5B antigens. Results obtained demonstrate complete absence of DWl-DW7 in these families and illustrate the inheritance and segregation of LDSA, LDl5A and LD15B. DR typing results obtained with the 8th International Histocompatibility Workshop genetic set of antisera indicate inheritance in coupling of DR2 with LD5A, of DR6.2 (DR3+6, MTl negative, MT2 positive) with LD15A, and of DRW8 with LDlSB. The existence of MLC activating antigen(s) different to DW4, yet associated to DRW4 in this population is postulated. The D/DR relationship present in this American Indian isolate demonstrate once more that DR2 can be inherited in combination with an HLA-D antigen different to DW2, and that LD15A HTC define a second sub-cluster of the broad DW6 specificity group, which is inherited with DRW6.2 and BW62 antigens in the Warao population.     

Role of the mixed lymphocyte culture (MLC) reaction in marrow donor selection: Matching for transplants from related haploidentical donors

Abstract: The utility of the MLC assay as a test of HLA-D region matching and predictor of acute graft-versus-host disease (GvHD) was evaluated in 157 patients receiving marrow grafts from HLA-A, B identical related haploidentical donors. All donors and recipients were tested by HLA-DR serology, by Dw phenotyping with homozygous typing cells (HTC) and by standard MLC. Ninety-nine of the donor-recipient pairs were mismatched for a serologically defined HLA-DR antigen while 109 pairs were mismatched for the HLA-D region by HTC typing. Donor anti-recipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from -4% to 100%, with a median of 25%. A comparison of reactivity in MLC with presence or absence of matching by Dw phenotyping, however, showed a significant overlap in the distribution of RRs from HLA-Dw matched versus Dw mismatched pairs, suggesting that the MLC was not a reliable predictor of HLA-Dw matching. Using an optimally-defined cutoff of 3% RR, the MLC was correlated with risk of developing clinically significant grades II–IV acute GvHD (p = 0.03) but not with risk of developing severe grades III-IV GvHD (p = 0.18). In contrast, matching by Dw phenotype was a significant predictor of GvHD, with Dw-compatible transplant recipients less likely to develop either grades II-IV (p = 0.004) or III-IV (p = 0.036) GvHD than Dw-incompatible transplant recipients. Overall, these results underscore the difficulty in using the MLC to measure HLA-D region compatibility and predict the risk of severe graft-versus-host disease among patients receiving related haploidentical marrow grafts. HLA-D (HTC) typing results correlate primarily with DRB compatibility, and with the advent of DRB1 allele matching by sequence-specific oligonucleotide probes (SSOP) or by direct sequencing, the precision in donor matching achievable with these methods is far greater than with either HLA-D typing or direct MLC testing.     

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.     

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.