Heterogeneity ofHLA defined in PLT: A cellular assay detects differences not seen using HLA-DRw serology

The primed lymphocyte typing test (PLT) is used to detect the gene products of theHLA-D region which are responsible for secondary restimulation of cells primed in MLC. Alternatively, products of theHLA-D region may be detected serologically using antisera directed against a subpopulation of lymphocytes; these are the so-called DRw determinants. The PLT was used to see if it were possible to detect heterogeneity within a given serologically defined group using a cellular test. As priming combinations, we used family members identical for one haplotype and differing in theHLA-A, B andC regions, but not theD region of the second haplotype. Our results indicated that it was possible to prime against this second haplotype and that the segregation of the difference followedHLA. Therefore, using a cellular test it was possible to detect differences among cells belonging to a given DRw group. This suggests that PLT can be a useful tool to identify those serological groups which are composed of heterogenous determinants. In addition, it points out the problem in using any one test to establish identity of theHLA-D region, especially for clinical purposes.     

Linkage between C2 deficiency and theHLA-A10,B18,Dw2/BfS haplotype in a French family

A linkage between C2 deficiency and the HLA-A10,B18/BfS antigens has been found in a French family from the Strasbourg area. The propositus, suffering from a chronic glomerulonephritis, is homozygous forHLA-A10,B18/BfS and totally C2-deficient. The parents and the brother are heterozygous for C2 deficiency and share theHLA-A10,B18/BfS haplotype. MLC tests and HLA-D typing revealed that the homozygous C2-deficient patient is also homozygous at theHLA-D locus for the w2 specificity. Evidence was obtained for a heterogeneity of the HLA-Dw2 specificity. This observation confirms the remarkable association between C2 deficiency and theHLA-A10,B18,Dw2 haplotype.     

Intra-H-2 recombination inH-2b/H-2t1 heterozygotes in the mouse

Four cases of intra-H-2 recombination were detected during serological screening of 1066 backcross animals produced fromH-2^b/H-2^t1 heterozygous mice. Three of the intra-H-2 recombinants received theK region fromH-2^t1 and theD region from theH-2^b parental chromosome. The remaining recombinant received theK region from theH-2^b parental chromosome and theD region fromH-2^t1. Three of the four recombinants have been developed into inbred lines TBR2, TBR3, and TBR4 and were assigned the haplotype designations at², at³, and at⁴. Ss typing revealed that TBR2 and TBR3 originated fromK- S interval crossover events, while the remaining two recombinants resulted from crossing over in theS- D interval.     

Demonstration of a new genetic locus in the major histocompatibility system of the rat

A new recombination within the major histocompatibility complex (RT1) of the rat has been detected. The recombination occurred between a wild-derived haplotype, provisionally designated p1, and the RT1 haplotype of the BN strain. The recombinant haplotype, designated p3, carries the RT1.A locus (classical histocompatibility antigens) of the BN strain, a locus from the BN strain that codes for the expression of an Ia antigen and strong mixed lymphocyte response (MLR), and a second locus derived from the p1 haplotype that controls the expression of a second Ia antigen, the ability to elicit a strong MLR and the immune response to poly(G1u⁵²Lys³³Tyr¹⁵). This recombinant therefore demonstrates the division of the RT1.B region into two loci, tentatively designated RT1.B and RT1.D, and provides evidence for the existence of at least four loci in the MHC of the rat.     

The location ofC2, C4, andBF relative toHLA-B andHLA-D

The loci forHLA-A, B, C, D, andDR are known to be closely linked to the structural loci for the complement components C2, BF, and the duplicated loci for C4, C4A and C4B. Conflicting evidence has been presented for the order of these genes. However, new techniques have made possible identification of markers in theHLA-D andC4 region for nearly all identified haplotypes. In our population we have confirmed fiveHLA-B-D crossovers and in each case informative allotypes of C2, BF, or C4A and C4B segregated withHLA-D orDR suggesting that the loci for these proteins lie close toHLA-D andDR. These findings may be of importance for resolving problems encountered in the assignment ofHLA-D alleles.     

Multi-Locus control of MLC reactions in rhesus monkeys

The genetic basis for MLC reactivity in rhesus monkeys was further investigated. Cells from anintra-RhLA recombinant monkey were MLC reactive with those of an SD-identical sib and with each of two sets of homozygous typing cells carrying either paternal haplotype. Also, cells from a pair ofRhLA- identical sibs reacted in MLC with each other, as well as with three genotypically different siblings. This excludes control of MLC reactivity by the conventionalD locus only. Thus the results selected for presentation provide formal evidence for the existence of at least one additional MLC locus, namedD. The possibility thatD exerts its influence on MLC reactivity only after alloimmunization is discussed.     

The HLA-D system: At least two loci and four distinct phenotypic traits per haplotype. Introduction to component typing in families and population by primed lymphocyte typing

Using a number of intrafamilial PLTs raised against identical HLA haplotypes it has been possible to construct a model in an informative family defining the HLA-D region as a genetic system. This system consists of at least two regions separated by a recombination between HLA-D and GLO. In relation to the site of recombination, a minimum of one centromeric and three telomeric components can be identified per haplotype.—Fourteen PLTs raised and defined within the family were subsequently tested in a Caucasian population (n=84) and in 13 unrelated, complete families.—It is concluded that the hypothetical model proposed for the HLA-D region as a genetic system of linked loci, coding at the cell surface for associated but distinct components (at least four per haplotype), allows for typing of the components of the HLA-D system of any given haplotype. Serological typing of HLA-D components should, in the near future, provide a more convenient way of establishing component phenotypes than the present use of primed lymphocyte typing reagents. Among the components isolated, some have a high association with the classic alleles defined either by homozygous typing cells or DR serology. Others form the basis of cross-reactivity but their presence does not interfere with standard typing. Others, however, seem by their mere presence to be responsible for false assignments.—The concept of HLA-D as a genetic system clarifies many of the inconsistencies observed with a one-locus system.Research scientists from INSERM.Research Fellow from the Danish Medical Research Council.Central Blood Bank — Marseille     

Immunogenetic aspects of a canine breeding colony

A colony of dogs was expanded by selective breeding to study the immunogenetic determinants coded for by the major histocompatibility complex (DLA). Polymorphic determinants were identified by alloantisera specific for DLA-A and B loci antigens and by the mixed lymphocyte culture (MLC) which defined alleles at the D locus. Thirteen families totaling 58 offspring were produced and typed for allelic determinants coded for by each of the three gene loci. Allelic segregation in a codominant manner occurred as expected and a recombinant between the A and B loci was detected. A number of animals were homozygous at one or more loci, thus providing genetically standardized animals as a source of typing cells, antigens, and sera to further study the immunogenetic details of DLA and for in vivo studies in transplantation biology.     

The HLA-D determinants

Stimulating cells assumed to be homozygous at theHLA-D locus were used to identify nine different MLC-activating HLA-D determinants in a population of unrelated Norwegians. The results suggest that the HLA-D determinants typed for, which account for a combined gene frequency of approximately 0.80, are controlled by mutually exclusive codominant alleles at one locus. Some of the HLA-D determinants showed highly significant association to certain HLA-B antigens.     

Restimulation properties of human alloreactive cloned t-cell lines. Dissection ofHLA-D-Region alleles in population studies and in family segregation analysis

Alloactivated human lymphocytes were cloned by limiting dilution. After 1 month in culture with T-cell growth factor several clones incorporated tritiated thymidine when stimulated with the appropriate allogeneic cells. Specificity of restimulation of two primed lymphocyte clones, designated 12-2 and 12-8, was studied in detail after varying periods of culture (up to 50 days). Clone 12-2 cells were stimulated only by cells expressing the HLA-Dw antigens of the original priming cells (Dw3); furthermore, this primed lymphocyte reagent specifically recognized antigens associated with only one of the three distinct Dw3-bearing haplotypes from an informative family (KOH). Clone 12-8 cells, on the other hand, failed to recognize Dw3 antigens in the random panel or on homozygous typing cells (including the original priming cell), but were strongly restimulated by certain cells expressing Dw4 antigens. In addition, within family KOH, these restimulating products segregated with another one of the three Dw3-bearing haplotypes but with none of the three Dw4-bearing haplotypes. These two clones exemplify a hitherto unknown precision in cellular typing of theHLA-D region. Clone 12-2 allows the discrimination of a probably rare and as yet undetected HLA-Dw3 subtypic specificity. Clone 12-8, on the other hand, apparently identifies an allelic system segregating withHLA but distinct from the HLA-D determinants definable by HTC-typing.Abbreviations used in this paper MHC major histocompatibility complex - HLA human leukocyte antigens - PBL peripheral blood leukocytes - HTC homozygous typing cells - MLC mixed leukocyte culture - PLT primed lymphocyte testing - TCGF T-cell growth factor - CTC cultured T cells - Tdr tritiated thymidine     

Genetic and immunological characterization of naturally occurring recombinant B3 rats

The B-stock population of rats was bred for homozygosity at the loci controlling coat color. In this process, theAg-B1 andAg-B3 haplotypes became fixed in Hardy-Weinberg equilibrium. Extensive immunization and absorption studies showed that the specificities in the B-stock rats homozygous for theAg-B1 haplotype were the same as those found in the inbred F344 strain (Ag-B1), and that the specificities in the rats homozygous for theAg-B3 haplotype were the same as those found in the inbred BN (Ag-B3) strain. A homozygous line derived from the rats carrying theAg-B3 haplotype (B3) has the mixed lymphocyte reactivity and antibody responsiveness to poly (Glu⁵²Lys³³Tyr¹⁵) characteristic of the inbred strains in theAg-B4 group. Thus, it represents a naturally occurring recombination between the loci controlling MLR and immune responsiveness, on the one hand, and those controlling the Ag-B antigens on the other. Antibody responsiveness segregated with theAg-B3 haplotype in crosses between the B3 homozygotes and the low responder BUF and M520 strains; hence, this recombination is a stable one. There was no linkage of antibody formation or haplotype to coat color. The finding of a strain with a naturally occurring recombination in the major histocompatibility complex between the loci controlling mixed lymphocyte reactivity and the Ag-B histocompatibility antigens provides evidence for the separateness of these loci. Since the portion of the genetically determined mechanism controlling antibody responsiveness which is linked to the MHC was that characteristic of the MLR type, it too must lie outside the region defined by the serological specificities of theAg-B haplotype.     

The dynamics of interlocus associations in the three-locus hitchhiking model

The hitchhiking effects of a selected locus upon the dynamics of the pairwise association,D _nn between two neutral loci is examined analytically for the special case where at least one of the neutral loci is in linkage equilibrium with the selected locus. The results apply approximately whenever the product of the pairwise associations between the selected locus and each neutral locus is negligible with respect to the three-way linkage disequilibrium. It is shown that precisely four broad classes of trajectories are possible, whether the selected locus is between (nsn) or to one side (snn) of the neutral loci, and whatever the mode of selection operating.D _nn may: (1) decay rapidly to zero, at a rate faster in each generation than that expected for two isolated neutral loci; (2) monotonically decay to zero at a rate which is slower in every generation than under the usual neutral regime; (3) increase initially and/or in intermediate periods before eventually slowly decaying to zero; or (4) exhibit type 1 behavior in the first segment of the trajectory and either type 2 or 3 behavior in the subsequent generations, with the transition marked by a change in sign. The nature of a given trajectory is largely determined by the direction of gene frequency change at the selected locus, and the initial signs of bothD _nn and the three-way linkage disequilibrium.The single most important consequence of these results is that there is no simple relation between the amount of pairwise association between two neutral markers and the recombination fraction between them. Several factors influencing the magnitude of the hitchhiking effect are also examined. It is shown that, all else being equal, the greater the three-way linkage disequilibrium, the greater the departure ofD _nn from the expected neutral dynamic. Increased recombination among the loci reduces the hitchhiking effect onD _nn . The dependence of the behavior upon the exact position of the selected locus is also determined both within and betweennsn andsnn chromosomal systems. An interesting discovery is that given equivalentnsn andsnn systems, with each having the same recombination between their two neutral loci,D _nn will deviate more from the standard neutral dynamic in thesnn system if its selected locus is sufficiently tightly linked to the neutral loci.     

Lymphokine production in mouse mixed lymphocyte reaction (MLR)

We have investigated alloantigen differences which stimulate lymphokine release and³H-TdR uptake in primary ‘one-way’ MLC among allogeneic mice. When mice differing at the wholeH-2 region were tested, MIF and immune IF release was observed, along with a marked³H-TdR uptake. Differences atK, D, orI-S-G regions stimulate both lymphokine release and³H-TdR uptake, though stronger immune IF and³H-TdR responses were observed with differences atI-S-G regions. On the other hand, when mice differing in their minor histocompatibility antigens, and notably at theMls locus, were tested, lymphokine release took place even in the absence of proliferation. Lastly, in MLC between mice differing at multiple minor loci, butH-2 andMls matched, MIF release only, and not immune IF and³H-TdR responses were observed in a few combinations. These findings show that T lymphocytes can recognize alloantigens by releasing lymphokines even without going through proliferation. Moreover, different levels of T-lymphocyte activation exist, depending on the kind of stimulating alloantigens present.     

The complex nature of humanD-locus determinants: Heterogeneity within Dw3

The HLA-A and B homozygous cell, ABR, was found to be mutually nonstimulatory with Dw3-homozygous test cell 16001 (EB), and so was considered to be Dw3-homozygous. However, family studies revealed a difference in theD-locus determinant(s) inherited from the father and mother. The cells carrying the determinants derived from the paternal1,8 haplotype (a _f ) consistently stimulated the lymphocytes bearing the determinants from the maternal1,8 haplotype (ainm), but thea _m haplotype products could not stimulate those of haplotypea _f . Sincea _m is included ina _f , cell ABR behaves in population studies as a Dw3-homozygous cell. Typing forD-locus determinants showed the father to be Dw3-positive, the mother Dw3-negative. The Dw3 determinant seems not homogeneous and this should be taken into account in explaining the results withD-locus homozygous typing cells.     

Genetic Characterization of the Chromosomal Rearrangements That Accompany the Transgene Insertion in theChakragatiMouse Mutant

We have previously reported that the circling phenotype of thechakragatimouse segregates with the transgene integration event as an autosomal recessive trait. It was unclear, however, whether the phenotype was linked to the transgene integration point nearD16Ros1or to a potential disruption atD16Ros2,10 cM away. We report here that animals recombinant betweenD16Ros1andD16Ros2,homozygous for the transgene insertion atD16Ros1,but wildtype forD16Ros2,do indeed show the phenotype. We conclude that any potential disruption at theD16Ros2locus is not responsible for the circling phenotype. We further show that recombination betweenD16Ros1andD16Ros2occurs at a greatly reduced level in thechakragatimouse compared to wildtype strains. Detailed genetic analysis of recombinants indicates that the proximal-most 4.5 cM shows no recombination in over 1400 meioses. We propose that this is due to an inversion in this region, and we genetically define the proposed distal inversion break point to a 1.3-cM region betweenD16Mit63andD16Mit169.     

Linear density gradient separation of human lymphocyte subsets. II. Characterization of cells responding in secondary MLC and CML.

Lymphocytes were separated on linear density gradients (LDG) after they had been sensitized in vitro against allogeneic cells and had reverted to small cells. Cells from individual density fractions were restimulated with autologous, specific, or third-party cells and assayed 48 hr later for their response in secondary mixed leukocyte culture (MLC) and cell-mediated lympholysis (CML). Memory cells capable of responding in secondary MLC were broadly distributed and found in both heavy and light fractions. The various density classes of memory cells differed with respect to the degree of their specificity for the restimulating cells. In secondary MLC the greatest specificity for the originally sensitizing cells and the least cross-reactivity for third-party cells were primarily features of light- and medium-density cells. Memory killer cells for CML were fairly homogeneously grouped. Following restimulation, killers were enriched in light to medium fractions also, as was previously seen at the peak of the response on Day 6.     

Mapping of theLyb-4 gene to different chromosomes in DBA/2J and C3H/HeJ mice

Linkage has been established between the Lyb-4 alloantigen locus and the chromosome 4 markersLyb- 2 andMup- 1 using recombinant inbred (RI) strains. Only 2 of 24 BXD RI strains possess recombinant genotypes with respect to the B cell alloantigen lociLyb- 4 andLyb- 2, for an estimated recombination frequency of 0.024 ±0.019. One additional BXD RI strain was a recombinant with respect toLyb- 4 andMup- 1 (major urinary protein locus) for an estimated recombination frequency of 0.039 ± 0.026. These linkages were confirmed and further quantitated in a (C57BL/6J × DBA/2J)F₁ × C57BL/6J backcross population, in which the recombination frequency betweenLyb- 4 andMup- 1 was 0.049 ± 0.019. No recombination between the expression of Lyb-4.1 antigen and the ability of anti-Lyb-4.1 serum to suppress MLC reactivity was found, indicating that the genes controlling the antigenic determinant which is recognized with cytotoxic antibodies in anti-Lyb-4.1 serum is the same as, or is very closely linked to, the gene which is responsible for augmentation of the MLC response. In contrast, no linkage was observed between the gene controlling the Lyb-4.1 determinant andMup- 1 in RI strain and backcross mice derived from the cross of C3H/HeJ and C57BL/6J. Again, there was complete concordance between the serologically recognized determinant and the ability of anti-Lyb-4.1 serum to suppress the MLC response. Absorption of anti-Lyb-4.1 serum with C3H/HeJ, DBA/2J, and C57BL/6J lymphocytes, followed by the cytotoxic assay of the absorbed sera on lymphocytes of each of these three strains showed that serologically the Lyb-4.1 antigenic determinant on DBA/2 mice was indistinguishable from that on C3H/HeJ mice. Thus, both traits appear to be under the control of single genes in both DBA/2J and C3H/HeJ, but the C3H/HeJ gene appears to be nonallelic and unlinked to the DBA/2J gene.Abbreviations used in this paper LAD lymphocyte activating determinants - LPS lipopolysaccharide - MLC mixed lymphocyte culture - RI recombinant inbred     

Differential behavior of cytotoxic effector cells against HLA antigens in strong genetic linkage disequilibrium

Five sets of cytotoxic effector cells were generated, using haploidentical, first degree relatives in five different families, against the HLA-A3; B7 serological determinants combined with different DR antigens. When tested against a panel of cells bearing combinations of the HLA-A, -B and -DR antigens it was shown that the HLA-B7 antigen was as strong a CML target determinant alone as it was in the presence of HLA-A3. The strength of the HLA-A3 antigen as target determinant varied. With effector cells primed to the HLA-A3; B7; DR2 haplotype, the A3 antigen alone behaved as a weak target determinant. When the same target cells were tested with the effector cells generated against HLA-A3; B7 without DR2, the A3 antigen behaved as a strong target determinant. A number of target cells lacking the serologically detectable HLA determinants present on the sensitizing HLA haplotype were identified as being killed by specific effector cells. These data suggest either a number of new CML target determinants controlled by different loci or the presence of a single, new locus with multiple alleles controlling CML targets.     

The lymphocyte restimulation system: Evaluation by intra-HLA-D group priming

Homozygous typing cells (HTC) were primed, using responding and stimulating lymphocytes of the same HLA-D groups. These intra-HLA-D group primings showed strong specific responses. Restimulation by HLA-D heterozygous and homozygous cell panels showed no correlation between the restimulating determinant and HLA-D. On the other hand, an unrelated individual, not carrying Dw4 and primed to Dw4 HTC, is restimulated by three of four Dw4-HTC. Thus, one non-HLA-D-associated restimulating determinant and another HLA-D-associated determinant could be identified. The differences among the four Dw4 HTC recognized in secondary MLC could reflect either recognition of separate gene products or recognition of separate determinants on the same gene product.     

Complotype genetic loci segregate more frequently with HLA-DR than with HLA-B

The loci for BF, C2, C4A, and C4B are very closely linked to each other so that alleles of these plasma protein markers occur in populations in linkage disequilibrium and are inherited as single genetic units called complotypes. These complotypes are coded by a DNA region of the short arm of chromosome 6 embracing approximately 100 kilobases, which serve as a marker of the major histocompatibility complex. We have studied the complotypes of nine families with known HLA-B/DR crossovers. In seven families, the complotypes were inherited with HLA-DR, including in one family with a double recombination. The haplotype HLA-A28, Cw1, B27, FC3, 20, DR4 of JTr resulted from two recombinations between HLA-A2, Cwl, B27, SC42, DR7 and HLA-A28, Cwx or Cw1, B37, FC3, 20, DR4. In the remaining two families (Ro and Lo) the complotypes were inherited with HLA-B. The haplotype A2, Cw5, Bw44, SC30, DR3 of StLo resulted from paternal recombination between the haplotypes A2, Cw5, Bw44, SC30, DR4 and A24, B8, SC01, DR3, and the haplotype A24, Cw4, Bw35, SC31, DR3 of NaRo resulted from maternal recombination between A24, Cw4, Bw35, SC31, DR4 and A26, Bw41, FC31, DR3. Our data suggest that the complotype region maps closer to HLA-D than to HLA-B.