Robert J. Hartzman

Human helper T-cell clones that recognize different influenza hemagglutinin determinants are restricted by different HLA-D region epitopes

Human T-lymphocyte clones (TLCs) were generated against the hemagglutinin (HA) of A/Texas/1/77 influenza virus by limiting dilution. TLCs were then screened for antigen specificity on chemically synthesized peptides representing the HA1 molecule. It has been hypothesized that different T cells that recognize the identical antigenic determinant are controlled by (restricted by) the same class II epitope. Two TLCs, HA1.4 and HA1.7, both recognized the same HA peptide and in proliferation studies exhibited identical restriction patterns. Two other clones, HA 1.9 and HA 2.43, recognized different HA determinants and also had distinct restriction patterns. Proliferation inhibition studies with monoclonal antibodies against human class II molecules demonstrated three unique patterns of blocking with the clones, suggesting that clones may be restricted to a unique class II epitope depending on the HA determinant recognized. These data can be interpreted as supporting the argument that human immune responses to influenza hemagglutinin are under Ir gene control exerted at the level of the viral antigenic determinant recognized in association with particular D-region restricting elements. The determinant selection and clonal deletion theories are compared for their capacity to best explain these findings.

The DR3(w18),DQw4 haplotype differs from DR3(w17),DQw2 haplotypes at multiple class II loci

The polymorphism of HLA class II molecules in man is particularly evident when comparisons between population groups are made. This study describes a DR3 haplotype commonly present in the American black population. Unlike the Northern European population, in which almost all DR3 individuals are DQw2, approximately 50% of DR3-positive American blacks express a DQw4 allelic product. This study characterizes the DR subregion of that haplotype. cDNA sequence analysis has revealed a DR beta gene which differs at several positions from previously described DR3 beta 1 genes. It is postulated that a gene-conversion-like event with a DRw52 beta gene as donor has generated some of these differences. The haplotype carries a DRw52a allele as defined by oligonucleotide hybridization studies. DNA restriction fragment analysis using a family and several unrelated individuals has allowed us to identify DR alpha and beta fragments associated with the DR3(w18),DQw4 haplotype. The most striking observation is that the DR3(w18),DQw4 haplotype differs from DR3(w17),DQw2 haplotypes at multiple class II loci. Several genetic mechanisms including reciprocal recombination, gene conversion, and point mutation were involved in generating the differences between these haplotypes. Once established, the DR3(w18),DQw4 haplotype appears to be relatively stable in the population.

Diversity associated with the second expressed HLA-DRB locus in the human population.

Abstract Although diversity within the HLA-DRB region is predominantly focused in the DRB1 gene, the second expressed DRB loci, DRB3, DRB4, and DRB5, also exhibit variation. Within DRB1*15 or DRB1*16 haplotypes, four new variants were identified: 1) two new DRB5 alleles, DRB5*0104 and DRB5*0204, 2) a haplotype carrying a DRB1*15 or *16 allele without the usual accompanying DRB5 allele, and 3) a haplotype carrying a DRB5*0101 allele without a DRB1*15 or *16 allele. The evolutionary origins of these haplotypes were postulated based on their associations with the DRB6 pseudogene. Within HLA haplotypes which carry DRB3, a new DRB3*0205 allele and one unusual DRB3 association were identified. Finally, two new null DRB4 alleles are described: DRB4*0201N, which exhibits a deletion in the second exon, and a second allele, DRB4*null, which lacks the second exon completely. Gene conversion-like events and variation in the number of functional genes through reciprocal recombination and inactivation contribute to the diversity observed in the second expressed HLA-DRB loci.

HLA-B alleles associated with the B15 serologically defined antigens.

Cells expressing HLA molecules in the B15 family were identified by serologic typing in routine testing of volunteer donors of various ethnic backgrounds for a bone marrow registry. DNA sequencing was used to identify HLA-B15 alleles associated with each serologic type and to examine the diversity within the B15 antigen family. Alleles which appeared predominantly in each B15 serologic cluster included: B15 with no defined serologic subdivision (B*1501), B62 (B*1501), B63 (B*1516, B*1517), B75 (B*1502, B*1521), and B76/77 (B*1513). Other B*15 alleles were also found associated with the serotypes and some of these alleles (e.g., B*1501 and B*1516) were found in two or more serologic clusters illustrating the complexity of this family. The B15 unsplit and B75 groups were the most complex exhibiting 16 and 7 alleles, respectively, within each serotype. Five new B*15 alleles (B*1530, B*1531, B*1533, B*1534, B*1535) and 5 other new HLA-B alleles (B*38022, B*3910, B*4010, B*51012, and B*5108) were also identified.

Human allospecific TLCs generated against HLA antigens associated with DR1 through DRw8 - I. Growth and specificity analysis

To study the fine specificity of the HLA-D region, a panel of human T-lymphocyte clones (TLCs) was generated against alloantigens associated with HLA-DR1 through DRw8. HLA-DR-homozygous peripheral blood lymphocytes (PBLs) were stimulated with DR-heterozygous PBLs in primary mixed lymphocyte cultures for 4 days. Blasts were cloned by limiting dilution at 0.3 cells/well in the presence of 20% T-cell growth factor and irradiated stimulator cells. Viable clones were subsequently tested in proliferation assays against the original stimulator and a limited panel of stimulators bearing relevant DR specificities. Initial primings produced approximately 800 clones; some recognized DR-associated antigens, 70 recognized only their original stimulator, and approximately 50% were nonresponsive. Analysis on extended stimulator panels revealed alloantigenic complexity within similar DR-associated antigens as recognized by TLCs. The data are consistent with evidence that extreme heterogeneity exists within the HLA-D region.

HLA-A*28 ALLELE FREQUENCIES IN THE FIVE MAJOR U.S. ETHNIC GROUPS

The frequency of each A*28 allele was determined by PCR-SSOP typing in 5 major U.S. ethnic populations: Caucasians, African Americans, Asians/Pacific Islanders, Hispanics, and Native Americans. The percent of serologically defined A28-positive individuals in the 5 populations ranged from 2.7-17.9%. Fifty-nine individuals who were previously serologically typed as A28, A68 or A69 were randomly chosen for allele-level typing from each ethnic group from a database of 82,979 consecutively typed unrelated individuals. The most common A*28 allele for Caucasians, Asians/Pacific Islanders, Hispanics, and Native Americans was A*68012, while A*6802 was found in the majority of African Americans. Only four and three A*28 alleles were seen in Caucasians and African Americans, respectively, while five to six A*28 alleles were seen in the other population groups. The A*6804 and A*6806 alleles were not observed in any of the five ethnic groups.

Localization of C4 genes within the HLA complex by molecular genotyping

Segregation of the complement component, C4, was analyzed in six families that each included an individual who inherited an HLA haplotype where a crossover event had occurred in the region between HLA-B and HLA-DR. Two cDNA clones corresponding to the C4 gene were utilized as probes in Southern blot analysis of DNA from members of each family. Restriction fragment length polymorphisms (RFLP) were observed and were assigned to haplotypes. In one family RFLP, hybridizing with the C4 probes, segregrated with HLA-B, and in four families RFLP segregated with HLA-DR; one family was not informative in this respect. These analyses have made it possible to localize the genes for C4 between HLA-B and HLA-DR by molecular genotyping and to characterize three different genomic configurations of C4 genes by limited restriction mapping.

Human allospecific TLCs generated against HLA antigens associated with DR1 through DRw8 - II. Population analyses and blocking studies with monoclonal antibodies

Serologic, cellular, and molecular evidence supports the concept of extreme complexity within the HLA-D region. To study the complexity and fine specificity of the HLA-D region at the level of T -cell recognition, a panel of T-cell clones was generated against alloantigens associated with HLA-DRI through -DRw8. After initial screening of more than 800 clones, 89 representative lines were selected for extensive testing against 204 unrelated stimulator cells. Clone-by-clone correlation analyses were performed to test whether any clones recognized similar or identical epitopes. In addition, clonal reactivity patterns were correlated with known HLA specificities. Twelve clusters of clones were identified with similar reactivity patterns using clone-by-clone correlation analysis. Some groups were significantly correlated with specificities associated with various D-region haplotypes; others had no significant correlation with any defined D-region specificity. Five general types of clones obtained in our study can be categorized as follows: (1) Those recognizing epitopes clearly demonstrating a primary association with the classically defined D-region molecules against which the clones were primed. (2) Clones recognizing epitopes associated with one of the priming antigens and also with another unrelated D-region specificity. (3) Clones detecting epitopes which showed significant correlation with D-region molecules totally different from those against which they were originally primed. (4) Clones with limited reactivity in population studies and no correlation with defined D-region molecules. (5) Clones recognizing class I-associated epitopes.

Expression of T-lymphoblast-encoded HLA-DR, MT, and SB antigens on human T-B lymphoblast hybrids

The Ia-like antigens of hybrids of the B-lymphoblastoid cell line (B-LCL) WI-L2 and a subline of the T-lymphoblastoid cell line (T-LCL) HSB were examined. Antigens of the HLA-DR and MT series were analyzed by indirect immunofluorescence with chimpanzee anti-DR sera and monoclonal antibodies, and antigens of the SB series were analyzed by primed lymphocyte typing (PLT). The WI-L2 X HSB hybrids expressed antigens of each series not found on either parent cell. In each case, the novel antigens were indistinguishable from those found on SB, a B-LCL established from the same individual as HSB, and are thus presumably HSB encoded.

Measles virus-specific human T cell clones: Studies of alloreactivity and antigenic cross-reactivity

Cross-reactivity between altered self and foreign major histocompatibility complex (MHC) may be of etiologic importance in autoimmune disease. We have studied 29 measles virus-specific cloned and uncloned T cell lines from a patient with multiple sclerosis (MS) and from a normal subject. Two of the T cell clones derived from the normal subject reacted with foreign MHC determinants. No cross-reactivity between measles virus and either myelin basic protein (BP) or galactocerebroside (GC) was detected. T cell clones which are specific for nominal antigen and which also recognize alloantigen were detected with much smaller frequency than that reported in murine systems. Our data do not support a role for alloreactive measles-specific T cells, nor for cross-reactivity between measles virus and either BP or GC, in the pathogenesis of MS.