Biomedical subjects
Publications and source records attributed to F C Grumet.
A recent outbreak of bacillary dysentery in The Netherlands revealed that, despite the close association of HLA-B27 with post-dysenteric or reactive arthritis (ReA), not even in one family did all HLA-B27 positive patients infected by an arthritogenic bacterium, develop ReA. This dissociation shows that additional factors beside B27 may determine susceptibility to ReA.
A human-mouse cell line that is hypoxanthine-aminopterin-thymidine sensitive and ouabain resistant was derived from a fusion between human B lymphocytes and a mouse myeloma line. This new mutant, when fused to a relatively unstable EBV-transformed B cell secreting a human monoclonal anti-A (red blood cell antigen) antibody, resulted in stable hybridomas capable of long term production of the specific human monoclonal antibody. Furthermore, some of the hybrid clones secreted antibody in far greater titer than the original EBV cell line. We conclude that fusion to this human-mouse line is an efficient approach to the production of human monoclonal alloantibodies and an effective method of 'rescuing' secretion of desired antibody from EBV cell lines.
Anti-Epstein-Barr virus and antiinfluenza A cytotoxic T lymphocytes (CTL) have been used to study the restriction of human antiviral responses by HLA-B27 antigens. Three functional subgroups of HLA-B27 have been clearly distinguished by this "restriction-typing assay". No cross-reaction could be detected between the three subgroups either at the CTL level or at the level of antigen-presenting cells. The cells of subgroup 1 are always positive [M2(+)] when tested in immunofluorescence with a monoclonal B27-specific antibody which divides HLA-B27 into a major M2(+) and a minor M2(-) subgroup. These M2(+) group 1 cells are apparently also HLA-B27W as previously shown by Ivanyi and co-workers using anti-HLA-CTL. Subgroup 2 includes only M2(-) cells. A comparison between this group and the previously described HLA-B27K is not fully conclusive, since two typing cells which were clearly HLA-B27K apparently did not belong to group 2. Only two donors, both of Oriental origin, have been included in subgroup 3. Both of them were "M2 intermediate". These results demonstrate (1) the existence of several functional subgroups of HLA-B27 with an interesting correlation with the M2(+), M2(-), or M2 intermediate phenotypes, and (2) the possibility of using the restriction-typing assay to define such functional subgroups not detected by classical allosera.
Variants of a lymphoblastoid cell line, LCL 526 (SB3 MB1 DR1 B44 C5 A2/SB4 MT4 DR4 B27 C2 A24), which lost various HLA specificities were selected with monoclonal antibodies and complement using a method developed by Kavathas et al. (PNAS 77:4251, 1980). Using alpha B27 monoclonals, 8 B27 only loss mutants and 4 B27 haplotype multiple loss mutants were generated. The parental LCL 526 and two of the B27- mutants were used to select alpha B27 CTLs. The selection of six A2 loss, one A2-C5 loss, and 14 A2 haplotype multiple loss variants as well as secondary selection on haplotype loss variants to obtain A null, B null, DR null, and total A, B, C, null variants is also described. The usefulness of these mutants for the study of the relationship between B27 and disease and as two new haplotypes for immunologic, genetic, and molecular research is discussed. These mutants are available to other researchers.
A cytolytic human T cell (CTL) clone, designated F/M-F159, has been produced, the lytic specificity of which distinguishes subtypes of HLA-B27. This was demonstrated in cell-mediated lympholysis (CML) assays of: 1) a panel of target cells from unrelated donors, 75 B27 + and 36 B27-; 2) six families, including 20 B27 + and 14 B27- individuals; and 3) B27 + and B27- variants of a B27+ lymphoblastoid cell line (LCL). Specificity of F/M-F159 for HLA-B27 was confirmed by blocking studies with monoclonal antibodies. Lysis of B27 + targets reactive with the anti-B27 monoclonal antibody B27M2 was 30-104%, while lysis of B27 +, B27M2- targets was 4-22%. Lysis of B27- targets expressing HLA-Bw47, known to be cross-reactive with the B27M2 antibody, was 10 to 19%, while lysis of all other B27- targets was less than or equal to 10%. Clone F/M-F159 lysed B27 + targets, and failed to lyse B27- targets, irrespective of the clinical status of the cell donors. It is concluded that F/M-F159 recognizes an epitope present on the majority of serologically identified HLA-B27 molecules and that this epitope is closely related to, but not identical with, the epitope recognized by the antibody B27M2. These findings are interpreted as supporting a direct role for HLA-B27 in disease pathogenesis.
A trial of the killed Coccidioides immitis spherule vaccine was undertaken with 151 healthy skin test negative adult volunteers and controls to evaluate the safety of selected regimens, the induction of humoral and cell-mediated immune responses, and to determine if there were immunogenetic differences in these responses. The vaccine was given as three intra-deltoid doses over 8 weeks. No severe systemic symptoms were noted, although 3% of 3.5 mg doses (but no 1.75 mg doses) were associated with severe local reactions. Half the vaccinees had skin test conversions, which generally persisted greater than or equal to 6 months, two-thirds showed boosting of lymphocyte transformation in vitro, and 16% given three 3.5 mg doses developed antibody. There was an association between degree of local adverse vaccine reaction and immunostimulation, and a trend to immune response in persons of O blood type and with some HLA phenotypes. There was no evidence of deficient response to vaccination in subpopulations known to respond to coccidioidal infection poorly. A regimen of three 1.75 mg doses appears to be safe and without reduced immunogenicity, and there is no evidence dosage modification for certain subpopulations would be necessary in efficacy studies.
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Sensitivity of the carboxyfluorescein diacetate (C-FDA) thrombocytotoxicity technique for the detection of antiplatelet antibodies has been enhanced by the addition of an anti-Kappa light chain antibody facilitation step. This new technique, KC-FDA, was compared with the platelet suspension immunofluorescence test (PSIFT) by titering platelet-reactive allo-anti-PlAl and anti-HLA antibodies. The results show that compared to PSIFT, KC-FDA is more sensitive for detecting platelet specific antibodies (PlAl), is more or equally sensitive for detecting other antibodies (HLA), and is significantly faster and easier to perform.
A new modification of an HLA-DR typing technique is described which makes DR typing as rapid and simple as routine HLA-A,B,C typing. In this new method, designated the TM1 technique, carboxyfluoresceindiacetate labeled peripheral blood lymphocytes are added directly to DR typing trays. The T cells are then lysed by addition of TM1, a pan-T cytotoxic IgM monoclonal antibody, and residual B-cell reactivity with cytotoxic DR alloantibodies is read as in routine fluorochromasia microlymphocytotoxicity. HLA-DR typing by the TM1 technique compares favorably to typing by methods using B cells enriched by sheep red blood cell rosetting or by Degalan bead columns. The TM1 technique also works well with cells that have been cryopreserved as well as with cells that have been separated from whole blood drawn as much as 3 days earlier. Finally, because TM1 is so effective in lysing normal T lymphocytes, this antibody may prove useful in functional in vitro and in vivo studies requiring T-cell depletion.
The role of genetic susceptibility in the pathogenesis of Hodgkin's Disease has been considered relatively minor because of the rarity of familial disease, the absence of an identified pattern of inheritance, and the weakness of HLA associations in population studies. The availability of four prospectively ascertained HD families permitted reappraisal of the cosegregation of HLA and HD susceptibility by a new extended concordance analysis method. HLA haplotype concordance among patients was greater than that expected by chance alone for our four families (P less than 0.022) and also for these in combination with twelve informative families in the literature (P less than 0.0015). This study thus provides a new method, based on genotype concordance of affected relatives, for assessing linkage of HLA and disease susceptibility, and new evidence for the genetic control of susceptibility to HD. The model presented, as well as alternative and more complex models, points to the existence of an HD susceptibility gene in or near the HLA region, which, in the presence of a suitable etiologic agent or additional genetic susceptibility, leads to the induction of HD.
Because HLA-B27 is such an important genetic marker for susceptibility to spondyloarthropathies and other related diseases, study of this alloantigen was undertaken using murine monoclonal antibodies. The first monoclonal anti-B27 antibody, B27M1, is an IgG2a lymphocytotoxic antibody that reacts with all B27 antigens and cross-reacts to a lesser degree with the B7 antigen. Cross-reactivity with B7 is attributed to a B27-like epitope on the B7 antigen but which, as determined by cytotoxicity blocking studies, is distinct from the B7 allospecific epitope itself. The second B27 monoclonal antibody, B27M2, is a lymphocytotoxic IgM antibody reacting with most (greater than 85%) but not all of B27 antigens tested and having no cross-reactivity with B7. As determined by cytotoxicity blocking studies, the B27M2 epitope appears near the allospecific B27 epitope and the B27M1 epitope on the B27 antigen molecule. Immunochemical studies reveal isoelectric point and size differences between the B27M2+ and the B27M2- variants of HLA-B27. Based on these data, a model is proposed for the B27 antigen in which a B27M1 epitope is constant but the B27M2 epitope is present on the B27 antigen of some individuals and absent from that of others. B27M1 antibody reactivity does not appear to be associated with unusual disease susceptibility but preliminary data suggest the B27M2- variant of B27 may be more strongly associated with ankylosing spondylitis.
A DNA probe specific for the HLA-B locus has been isolated from a broadly cross-reactive HLA class I genomic clone. Locus specificity of the probe appears to be derived primarily from a stretch of approximately 180 nucleotides comprising the last (7th) intron of the original B7 gene. Use of the probe to analyze Southern blots of genomic DNA from unrelated individuals provides the first direct demonstration of intragenic localization of an HLA allele-specific restriction endonuclease site. Availability of this probe should make practicable the study of HLA-B locus restriction fragment length polymorphism as genetic markers of disease susceptibility, and should provide a model for developing probes specific for other HLA class I loci.
To explore the possibility that the HLA-B27 antigen may exist in more than one form, murine monoclonal antibodies were produced to B27 molecules. The first such antibody, anti-B27M1, reacts with 100% of B27 + cells and, therefore, does not distinguish B27 + healthy individuals from those with spondyloarthropathies. A second antibody, anti-B27M2, recognizes a variant of the B27 molecule that is present in most but not all Caucasian B27 + individuals. The frequency of B27M2(+) variants also appears to differ among different ethnic groups. Preliminary studies suggest that B27 + patients with ankylosing spondylitis are less likely than B27 + healthy individuals to express the B27M2 variant.
Detailed study of HLA-B27 was prompted by the extremely strong associations between this antigen and spondyloarthropathies. Despite the relative homogeneity of this antigen when defined by alloantisera, B27 reactivity with the monoclonal antibody B27M2 suggests previously unrecognized heterogeneity. To define and confirm this heterogeneity on a molecular level, detergent extracts were prepared from B cell lines derived from individuals reactive (+) or unreactive (-) with the B27M2 antibody. Extracts were immunoprecipitated by specific allogeneic or monoclonal antibodies and analyzed by two-dimensional polyacrylamide gel electrophoresis. By this method the B27M2+ and B27M2- variants of HLA-B27 had different isoelectric points (pl) and could be distinguished from each other and from a different (Bw44) control alloantigen. Blockade of glycosylation by pretreatment of cells with tunicamycin did not alter pl but did reduce HLA antigens by approximately 3000 daltons. These data demonstrate that B27 antigens can be subdivided into subsets with different molecular composition. The effects of this heterogeneity upon the associations of B27 and disease are not yet known.
The mechanism of the cytotoxic-negative, absorption-positive (CYNAP) phenomenon was studied using the model of the Bw49/Bw50 split of the BW21 antigen. Anti-Bw49 antibody bound 60% as well to Bw 50 as to Bw49 cells; however, even at a cytotoxic titer of 64 against Bw49 cells, the antibody was not cytotoxic to Bw50 Cells. At equal numbers of antibody molecules bound, the anti-Bw49 antibody activated C4 and C3, and induced lysis for Bw49 but not for the Bw50 cells. These data are consistent with a model in which different spatial orientations for shared epitopes can account for CYNAP reactivity for at least some selected Bw4/Bw6-associated splits of B locus antigens.
Human thymuses were examined by tissue section staining with antibodies specific for monomorphic and polymorphic HLA-A, B, C, and DR determinants. The principal cell type expressing high levels of HLA antigens has the distribution of epithelial cells. Immunoelectron microscopy confirmed their epithelial nature. As in the mouse, both medullary and cortical epithelial cells express high levels of class II (DR) antigens, a finding that is remarkable in that these antigens were originally thought to be restricted to lymphoid and accessory cells. Class I (A, B, and C) antigens are also present on thymic epithelial cells. They are easily detectable on medullary epithelial cells, but two distinct patterns of cortical staining were observed. One group of antibodies produced intense dendritic staining throughout the cortex; the other group produced only faint or no cortical dendritic staining at all. These different staining patterns do not correlate with known properties of the antibodies and thus appear to be due to intrinsic properties of the different A, B, and C antigens.