Restimulation properties of cloned alloactivated lymphocytes: Detection of a novel type of HLA-D/DR region determinant and allelic subtypes for HLA-Dw3 using cloned reagents

Lymphocytes alloactivated by Dw3 homozygous typing cells were cloned by the method of limiting dilution and cultured for prolonged periods using T-cell growth factor and irradiated pooled leukocytes (as feeder cells). Restimulation specificity of two clones functioning as primed lymphocyte typing reagents was investigated in panel and family studies. Cells from one of the clones (12-2) were always specifically stimulated by HLA-Dw3 antigens shared between the original priming cells and the stimulating panel cells. In an informative family KOH, however, cells from this clone seemed to detect a split in the Dw3 cluster. Cells from the other clone (12-8) failed to respond to Dw3 antigens as expressed by the original priming cells or by panel stimulating cells; rather, specificity of restimulation seemed to be associated with the expression of Dw4 antigens. Family segregation analysis did not support this conclusion however, since stimulating products segregated with one of the three Dw3-bearing haplotypes and with none of three Dw4-bearing haplotypes. This suggested that 12-8 cells may be responsive to antigens different from those detected by homozygous typing cells.     

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.     

Primary and secondary MLR characterization of three allelic variants of HLA-Dw7 segregating in a single family

Three of four parental haplotypes of a kindred from the Old Order Amish religious isolate carried HLA-DR7 and specificities of the HLA-Dw7 “cluster”. Intrafamily primary and secondary mixed lymphocyte responses clearly distinguished the three Dw7-related allelic specificities. Two of the specificities fall within the Dw11 crossreacting group, designated here as Dw11 “short” (Dw11S) and Dw11 “long” (Dw11L), while the third is more closely related to Dw7. Reaction patterns in this family illustrate the complexity of antigen recognition in primary and secondary mixed lymphoyte responses and the important role played by the responder cell in generating discriminatory primed lymphocyte typing reagents.     

Hemizygous HLA mutants of lymphoblastoid cells: A new cell type for histocompatibility testing

HLA variants that have lost expression of multiple cis-linked alleles as determined serologically and enzymatically were analyzed for expression of HLA-D PLT-stimulating (PL) determinants using the primed lymphocyte test. All 19 variants that had lost HLA-DRw3 expression simultaneously had lost expression of the HLA-D region-associated PL specificity. PLT cells made by priming to a variant that was hemizygous for HLA genes resulted in priming to HLA-D PL determinants encoded for by just one haplotype, analogous to priming to an HLA homozygous cell.     

Insulin-dependent diabetes--associated HLA-D region encoded determinants

We have studied the relative frequency of Dw specificities (defined with homozygous typing cells or primed LD (lymphocyte defined) typing reagents) associated with DR4 and DR2 in the normal and insulin-dependent diabetic population. Our findings demonstrate that there is a highly significantly increased frequency of Dw4 in DR4 positive diabetics as compared with normals and a significantly decreased frequency of Dw2 and Dw12 in the few DR2 positive insulin-dependent diabetics that we have found. In addition, we have used PLT reagents to define a new LD specificity, LD-MN2, that is associated with DR2 and is found significantly more frequently in DR2+ IDD patients than in DR2+ normals. These results suggest that determinants of import in the association between HLA-D and IDD may be more closely related to Dw than to DR.     

Identification of four SB antigens by cloned cells: Population studies of Norwegians

By priming in vitro with allogeneic HLA-DR compatible and also HLA-A,B mostly compatible lymphoid cells, PLT cells resulted in recognizing a group of non D/DR allelic antigens provisionally named K, L, M and N. To improve discrimination these bulk primed typing reagents were cloned and expanded. By typing of previously SB typed lymphoblastoid B cell lines (LCL) the provisional specificities could be identified as SB1, 4, 3 and 2, respectively. Typing of 186 unrelated Norwegians gave the following gene frequences: SB1: 0.05, SB2: 0.16, SB3: 0.13, SB4: 0.42 and SB blank: 0.24. No triplets were found, the calculated gene frequencies fit with Hardy-Weinberg equilibrium, and typing of a B-DR recombinant family confirmed that the SB locus is situated centromeric to B. Associations between SB and A, B, DR antigens in the same material were generally weak, the most significant associations found were between SB1-DR3 and SB4-DR2.     

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.     

High resolution HLA-C typing by PCR-SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology

Recent evidence indicates that HLA-C molecules are biologically relevant by eliciting T-cell responses and exerting control over NK cell function. In addition, HLA-C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA-C has however proved difficult because detection of HLA-C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA-C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA-C blank alleles. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable, accurate and rapid method for medium resolution HLA-C typing. We have now developed high resolution HLA-C typing by PCR-SSP utilizing allele and group-specific PCR-SSP reactions which can identify all HLA-C alleles (except non-coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA-C, PCR-SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA-C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High-resolution HLA-C typing of 112 International Histocompatibility Workshop cell lines has also been performed     

High resolution HLA-C typing by PCR-SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology

Recent evidence indicates that HLA-C molecules are biologically relevant by eliciting T-cell responses and exerting control over NK cell function. In addition, HLA-C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA-C has however proved difficult because detection of HLA-C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA-C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA-C blank alleles. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable, accurate and rapid method for medium resolution HLA-C typing. We have now developed high resolution HLA-C typing by PCR-SSP utilizing allele and group-specific PCR-SSP reactions which can identify all HLA-C alleles (except non-coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA-C, PCR-SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA-C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High-resolution HLA-C typing of 112 International Histocompatibility Workshop cell lines has also been performed.     

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).     

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.     

Differences between Primed Allogeneic T-Cell Responses and the Primary Mixed Leucocyte Reaction

Recent investigations have demonstrated that the primary mixed lymphocyte reaction (MLR) is dependent on certain accessory molecules, e.g. CD4 and LFA-1. We have compared the requirements of the primary MLR and the responses of alloreactive, primed lymphocytes (PL) by inhibition studies using monoclonal antibodies (MoAb) directed against (i) adhesion molecules belonging to the CD11 cluster of leucocyte antigens (CD11a, LFA-1; CD11b, MAC1 = CR3; and CD11c, p 150,95); (ii) various T cell-related antigens (CD2, CD4, CD5 and CD8); and (iii) recombinant IL-1 beta. The CD5-, CD11a- and CD11c-reactive MoAb significantly inhibited the primary MLR (inhibition = 25%, P less than or equal to 0.01; 48%, P less than or equal to 0.01 and 13%, P less than or equal to 0.05, respectively) but these MoAb did not inhibit the primed lymphocyte reaction (PLR). The CD11b-reactive MoAb had no significant influence on either of the responses. CD2- and CD4- reactive MoAb significantly inhibited both primary MLR (greater than 80%, P less than or equal to 0.01) and to a lesser extent the PLR (40-65%, P less than or equal to 0.01). A MoAb reactive with IL-1 beta inhibited the primary MLR (38%, P less than 0.01) and the purified protein derivative (PPD) induced lymphocyte transformation response (42%, P less than or equal to 0.01) of peripheral blood mononuclear cells (PBMC), whereas primed allogeneic responses to PBMC and Epstein-Barr virus (EBV) cell lines were unaffected by this MoAb. In addition, preliminary data indicated that PL seemed neither to bind exogenous IL-1 (as opposed to CD4+ PBMC) nor to possess membrane-bound IL-1. The differences between 'virgin' and primed, allogeneic T-cell responses indicate that profound changes in the functional capability of the responding T-cell population take place during the bulk expansion. The results indicate that during repeated priming with alloantigen and bulk expansion, the proliferative response of T lymphocytes becomes independent of (i) the interaction with the CD11 adhesion molecule(s), (ii) the CD5 molecule, and (iii) the cytokine IL-1 beta.     

Specificity of Human Lymphocytes Primed against Allogeneic Cells in vitro. II. Discrimination and Cross-Reactivity in Repeated Priming

When studying the specificity of human lymphocytes primed in vitro against HLA-D determinants on allogeneic cells, it was found that specific restimulation of the primed cells 10 days after the first priming did not influence their discriminatory power compared to cells primed only once. Likewise, priming to one HLA-D determinant and repeated priming against another HLA-D determinant did not change the discrimination for the first priming antigen. Neither was there any increased relative reactivity to the second priming determinant. On simultaneous priming against two HLA-D determinants carried by two different stimulating cells, a good discrimination for both antigens was obtained compared to third party cells, but if the stimulations were separated by 24 h or more, the second priming was mainly without effect. Antigeneic competition is thought to be the mechanism involved in this early restriction of specificity.     

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.     

Bovine Lymphocyte Antigens (BoLA): A Serologic, Genetic and Histocompatibility Analysis

Eleven lymphocyte antigens have been defined in cattle using 81 lymphocytotoxic sera. These sera (typing reagents) were selected from over 1,000 normal sera, 35 alloimmune sera, and 111 samples of colostrum whey. Absorptions revealed that nine of the 11 antigens detected by these reagents are serologically independent. Segregation of the genes controlling these antigens was observed among 470 calves from mating of 56 heterozygous sires and 360 dams, indicating that each of the 11 antigens is controlled by one of 11 alleles at a single autosomal co-dominant locus BoLA-A (Bovine Lymphocyte Antigen, locus A). To determine the relation between BoLA antigens and histocompatibility, skin allografts were exchanged among four unrelated adult cows. Incompatibilities for the SD antigens, but not MLR or blood group antigens, were predictive of early rejection of allografts. Furthermore, graft recipients produced antibodies against the SD antigens of incompatible donors. These results indicate that the BoLA-A locus is part of the major histocompatibility complex (MHC) of cattle.     

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.     

HLA-linked B cell alloantigens of a new segregant series: Population and family studies of the SB antigens

In order to define new human histocompatibility antigens, we have generated primed lymphocytes using responder and stimulator cells matched for all recognized HLA-linked histocompatibility antigens (A,B,C,D,DR,MB). Many such primed lymphocytes give highly discriminatory proliferative responses specific for antigens which differ between HLA-A,B,C,D,DR, and MB matched restimulating cells. Five distinct antigens have been defined which appear to be part of a single segregant series (designated “SB”). Studies in a DR/GLO recombinant family indicate that the antigens are coded by an HLA-linked gene telomeric to GLO. Family studies of 57 HLA haplotypes provide an estimate of genotype frequency which is 12% or less for four of the SB alleles but approximately 50% for the most common (SB4, which may be a “public” determinant); approximately 25% of haplotypes are black. Population studies of one of the SB antigens (SB1) suggest that it is in linkage disequilibrium with A1, B8, and DRw3. These results, together with results of other studies [1], indicate that the SB antigens are part of a highly polymorphic new segregant series of B cell alloantigens encoded by a gene that maps between HLA-B and GLO.     

Blood group molecular genotyping

DNA-based typing methods of red cell antigens are applied in several fields. Genotyping is used to clarify problems in patient serology. It is also increasingly applied for routine mass-scale typing of blood donors for minor red cell antigens and screening for donors with rare blood types. An advance in this field is the non-invasive fetal RHD diagnostics in pregnancies of D-negative women to determine the fetal RHD. Reference centres for immunohaematology commonly use molecular methods to clarify problems, discrepancies or unusual results in patient serology. It is often applied in individuals with variant RhD expression to detect the contributing weak D or partial D types. Genotyping is also used to identify the individual ABO and RhD blood group in patients after mismatched transfusion. In some patients with positive direct antiglobulin test (DAT) it is necessary to type for antigens by DNA techniques. Genotyping is also helpful when reagents are not available or only weakly reactive, or to confirm weakly expressed antigens. Blood establishments are constantly challenged with the blood supply for patients carrying irregular red cell antibodies. A high number of donors have to be typed to find compatible blood units. Increasingly, DNA-based methods are used alongside standard serological typing. The advantages of molecular methods are the wider range of different antigens available for typing and that some DNA methods are less expensive than phenotyping. Extensive donor antigen typing leads to short response times, from request to issuing blood units. Genotyping is also used for identifying donors with extremely weak RhD expression (DEL) to prevent mistyping as RhD-negative. Non-invasive fetal typing of RHD from maternal blood in pregnancies of D-negative women is implemented in many Caucasian blood establishments. It allows that D-negative fetuses can be identified at early stage. In these cases, no anti-D immunoglobulin is required, which provides a more efficient use of this human blood derivate. Furthermore, in anti-D alloimmunized women, the assay allows to accurately identify fetuses at risk for haemolytic disease.     

Sperm priming reveals specific autoreactive lymphocyte clones

Human spermatozoa from 18 donors were used to prime lymphocytes from 15 donors in a total of 60 different combinations. The sperm primed lymphocytes were tested in second culture against a total of 60 autologous and 180 allogeneic x-ray irradiated cells. We found that lymphocytes primed for 10–12 days in first culture with allogeneic spermatozoa showed highly significant responses in second culture to leukocytes autologous to the responder. The degree of autologous reactivity between specific subjects ranged from weak to significantly higher than the usual preferential primed lymphocyte typing reaction seen in a secondary mixed leukocyte culture reaction. These findings indicate that healthy human volunteers possess a set of self-reactive lymphocytes that can be detected by priming with allogeneic sperm in specific combinations.