Genetic control of the immune response. A selective defect in immunologic (IgG) memory in nonresponder mice.

The kinetics of antibody formation after immunization with the synthetic polypeptide poly-L(Tyr, Glu)-poly-D, L-Ala--poly-L-Lys [(T, G)-A--L] in aqueous solution were studied in genetically high (H-2(b)) and low (H-2(k)) responder strains of mice. During the 1st wk after immunization both strains developed brisk primary responses consisting of IgM antibody. With subsequent antigen challenge, only the high responder mice showed immunological memory, producing high titers of IgG antibody. In contrast, the low responder mice continued to make a persistent low level of IgM antibody and appeared unreactive to secondary or tertiary antigen challenge. These data are consistent with the hypothesis that the immune response-1 gene [controlling response to (T, G)-A--L] exerts its effect on the immune response at the time of switchover from IgM to IgG antibody production.

Genetic control of the immune response. The effect of graft-versus-host reaction on the antibody response to poly-L(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys in nonresponder mice.

The transfer of parental (H-2(k/k)) nonresponder lymphoid cells into heterozygous (H-2(k/q)) nonresponder recipients at the time of primary challenge with aqueous poly-L(Tyr,Glu)-poly-D,L-Ala-poly-L-Lys [(T,G)-A--L] elicited the production of both IgM and IgG anti-(T,G)-A--L antibody. Normally, the production of IgG anti-(T,G)-A--L antibody is restricted to strains possessing the responder Ir-1 allele. The timing and intensity of the graft-versus-host (GVH) reaction required for this effect were found to be critical. Injection of H-2(k/k) cells into H-2(k/q) recipients 1 wk before antigen challenge did not elicit IgG anti-(T,G)-A--L antibody production, and markedly suppressed IgM anti-(T,G)-A--L antibody production. The transfer of alloimmune (H-2(q)-primed) H-2(k/k) cells at the time of antigen challenge was also associated with no IgG and little IgM anti-(T,G)-A--L antibody production. These data are consistent with the model that nonresponder thymus-derived lymphocytes (T cells) activated in a GVH reaction can substitute for (T,G)-A--L-reactive T cells to induce a shift from IgM to IgG anti-(T,G)-A--L antibody production.

Genetic control of the immune response. The effect of thymectomy on the primary and secondary antibody response of mice to poly-L(tyr, glu)-poly-D, L-ala--poly-L-lys.

The effect of thymectomy on the genetically controlled murine immune response-1 (Ir-1) to the synthetic polypeptide poly-L(Tyr, Glu)-poly-D, L-Ala--poly-L-Lys [(T, G)-A--L] was studied with both aqueous and adjuvant immunization regimens. Adult thymectomy (combined with irradiation and bone marrow transfusion) did not affect the aqueous antigen-induced (IgM) primary response of either high or low responder mice, but did ablate the (IgG) secondary or tertiary response, a response which is restricted to the high responder strains. Adult thymectomy also blocked the normal high response to (T,G)-A--L in Freund's adjuvant in high responder mice and the high response to methylated bovine serum albumin (MBSA)-(T,G)-A--L in low responder mice. Neonatal thymectomy was also effective in blocking the response to (T, G)-A--L in Freund's adjuvant in high responder mice. These data are consistent with the concept that the Ir-1 gene effect is mediated via thymus cell interaction with antigen and with "B"-cells during the time of induction of IgG antibody formation.

HLA DR15 (DR2) and DQB1*0602 typing studies in 188 narcoleptic patients with cataplexy.

Narcolepsy is considered a homogeneous clinical entity when excessive daytime sleepiness and cataplexy are present. Cataplexy is a polymorphic symptom that can be very mild and is thus subjectively defined. The Multiple Sleep Latency Test (MSLT) is widely used as a diagnostic test for narcolepsy. A short mean sleep latency and multiple sleep onset REM periods (SOREMPs) are typically observed in narcoleptic patients. The discovery of a tight association of narcolepsy with HLA class II antigens offers a unique opportunity to explore the respective value of the MSLT or of the presence of clear-cut cataplexy in defining an etiologically homogeneous group of narcoleptic patients. In this study, we carried out HLA typing for DR15(DR2) and DQB1*0602 in 188 narcoleptic patients with cataplexy in three ethnic groups (24 Asians, 61 Blacks, and 103 Caucasians). These results confirm the importance of DQB1*0602 typing rather than DR15 (DR2) typing in Black narcoleptic patients and demonstrate that the presence of clear-cut cataplexy is a better predictor for DQB1*0602 positivity than the presence of abnormal MSLT results.

Rescue of human monoclonal antibody production from an EBV-transformed B cell line by fusion to a human-mouse hybridoma.

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.

Evans' syndrome as a presenting manifestation of atypical paroxysmal cold hemoglobinuria.

Paroxysmal cold hemoglobinuria is a rare and potentially life-threatening acquired hemolytic anemia occurring either as an acute transient anemia following several different viral syndromes, or in a chronic idiopathic form. Episodic hemolysis in paroxysmal cold hemoglobinuria is usually associated with a biphasic (Donath-Landsteiner) IgG cold-reactive complement-fixing autohemolysin with anti-P specificity. Paroxysmal cold hemoglobinuria has not previously been associated with malignancy nor has it been clearly shown to be steroid-responsive. This report describes a patient with steroid-responsive autoimmune hemolytic anemia and immune thrombocytopenia (Evans' syndrome) associated with oat cell carcinoma of the lung and a unique biphasic anti-IgM autohemolysin. This case extends the spectrum of biphasic antibody-mediated immune cytopenias and widens both the clinical and the serologic definition of paroxysmal cold hemoglobinuria.

Platelet transfusion tolerization by liver membrane treatment.

A nontoxic murine model has been developed in which sensitization to allogeneic platelet transfusions in adults can be prevented. This model is based upon allogeneic liver membrane (LM) induction of specific humoral tolerance to major histocompatibility alloantigens. In normal mice of the C3H background, syngeneic and H-2-incompatible congeneic platelets had a t1/2 = 15 +/- 3 hr; for mice of the C57BL background, the comparable t1/2 was 33 +/- 6 hr. Platelets of C3H background transfused into C57BL background recipients, and vice versa, had t1/22 midway between, 24 +/- 3 and 24 +/- 2 hr, respectively. These data suggest genetically determined variation in normal platelet survival. In passively immunized C3H background mice, the t1/2 was decreased to 10 +/- 2 hr. In C57BL background mice actively immunized i.p. with allogeneic lymphoid cells, t1/2 was decreased to 18 +/- 4 hr. When allogeneic LM was given concomitantly with the allogeneic cells, however, sensitization to foreign H-2 antigens was blocked, and survival of both C3H and C57BL background allogeneic platelets remained normal. These data demonstrate that in this free cell allograft system LM treatment is a safe and effective method for preventing adult sensitization to allogeneic platelets.

Clinical transplantation tolerance twelve years after prospective withdrawal of immunosuppressive drugs: studies of chimerism and anti-donor reactivity.

BACKGROUND: Previous studies showed the feasibility of inducing transplantation tolerance to cadaveric renal allografts in patients given pretransplant total lymphoid irradiation (TLI). Microchimerism has been theorized to be an important or necessary factor in long-term graft acceptance and tolerance in humans. METHODS: A cadaveric renal transplant recipient given pretransplant total lymphoid irradiation and withdrawn from immunosuppressive drugs more than 12 years ago was tested for microchimerism using a sensitive nested polymerase chain reaction technique, and for anti-donor reactivity using the mixed leukocyte reaction and an ELISA screen for anti-HLA antibodies. Donor and recipient were mismatched for all HLA-A, B, and DR antigens. RESULTS: The "tolerant" recipient had good graft function, no detectable donor-type cells in the blood by polymerase chain reaction analysis, vigorous reactivity to donor stimulator cells in the mixed leukocyte reaction, and no detectable serum anti-HLA antibodies. CONCLUSIONS: Operational tolerance to HLA-A, B, and DR mismatched organ allografts can be induced prospectively in humans for at least 12 years after withdrawal of immunosuppressive drugs. The allograft can be maintained in the absence of detectable donor microchimerism and in the presence of anti-donor reactivity in the mixed leukocyte reaction, suggesting that neither chimerism nor clonal deletion or anergy of recipient T cells to alloantigens presented by donor Class II HLA molecules is required for persistence of the tolerant state using this total lymphoid irradiation protocol.

Familial coagulation factor V deficiency caused by a novel 4 base pair insertion in the factor V gene: factor V Stanford.

An index patient with pseudohomozygosity for factor V Leiden was identified. Each of his two children inherited a different paternal factor V allele; a daughter was heterozygous for factor V Leiden, with 100% factor V activity, and a son was heterozygous for factor V deficiency, with 50% factor V activity. Genomic DNA was obtained from family members, and the 25 factor V exons and flanking intronic regions were sequenced in the proband and confirmed in the children. Within exon 13 of factor V, a 4 base insertion was found at NT 2856 in the proband and son. but not the daughter. This mutation, here designated factor V Stanford, results in a frameshift with loss of a thrombin activation site (R1545V) and premature termination of translation at amino acid 1560.

Lack of serologically defined arthritogenic Shigella flexneri cell envelope antigens in post-dysenteric arthritis.

Post-dysenteric or reactive arthritis (ReA) is closely associated with HLA-B27. This histocompatibility antigen is heterogeneous and consists of 2 serologically defined variants: B27M1+M2+ and B27M1+M2-. This paper gives a qualitative evaluation of the antibodies present in the sera of 62 patients with dysentery due to Shigella flexneri 2a, a known arthritogenic bacterium. The patients were classified in 4 groups: B27M1+M2+ReA+ (n = 5), B27M1+M2+ReA- (n = 7); B27M1+M2-ReA- (n = 1); B27-ReA- (n = 49). The isolated infectant possessed cell envelope antigens with B27M2-like epitopes (Mr 20,000). Analysis of the spectrum of antibodies directed against the separated cell envelope antigens of S. flexneri in the sera of these patients revealed 7 main patterns of reactivity. The detectable immunogens encompassed protein stainable antigens (Mr 98, 78, 68, 54, 50, 44, 41, 35, 14 and 13 kDa), lipopolysaccharides and peptidoglycan. None of the sera possessed detectable antibodies to the B27M2-like antigen. Consequently, this antigen is unlikely to be associated with ReA, and this applies equally to other antigens or patterns of antigens. The arthritogenicity of S. flexneri may therefore not be determined by the presence or absence of detectable antibody titers to certain cell envelope antigens. We hypothesize that other properties of these antigens could be of significance.

HLA-B27M1M2 and high immune responsiveness to Shigella flexneri in post-dysenteric arthritis.

The heterogeneous HLA-B27 antigen is closely associated with post-infectious or reactive arthritis (ReA) and is comprised of two serologically defined variants: B27M1+M2+ and B27M1+M2-. An outbreak of dysentery (n = 120) caused by a Shigella flexneri 2a strain, which possessed cell envelope antigens with epitopes resembling B27M2, resulted in five B27M1+M2+ patients with ReA. The remaining seven B27M1+M2+, one B27M1+M2- and all but three B27-negative patients remained free of joint symptoms; the latter three displayed arthralgia. IgM, IgG and IgA serum titers were statistically raised in all patient groups, but were exceptionally and persistently high in the B27M1+M2+ patients with ReA, especially IgA, as determined in acute-phase sera and sera sampled 1 year after dysentery. B27M1+M2+ thus appears to be a marker for a subset of disease, characterized by a high immune response. It is concluded that the B27M2 epitope is not unequivocally disease-related to Shigella ReA, that B27M1+M2+ is not likely to be the only immune-response-regulating gene involved in this form of ReA and that cross-reactivity between bacterial antigenic epitopes and B27 can only be part of a multifactorial process leading to ReA and in itself not sufficient to produce ReA. The intensity of the immune response appears to be another important factor.

A new micromethod for the in vitro detection of antiplatelet antibodies: C-FDA thrombocytotoxicity.

A new microtechnique, C-FDA, for the in vitro detection of antiplatelet antibodies, is described. This technique is faster and simpler than either 51Cr thrombocytotoxicity or immunofluorescence (IF). C-FDA is more sensitive than 51Cr for all (anti-HLA, --P1A1, ABO, drug-related, and ITP-related) antibodies tested. Although IF was more sensitive for many types of antibodies, C-FDA was as good or better a clinical test method for all drug-related and isoimmune neonatal thrombocytopenia patient sera tested. Preliminary data also suggest that this method detects possible new non-HLA, non-ABO, nonP1A1 platelet antigens.

New polymorphisms of HLA-B27 and other B locus antigens detected by RFLP using a locus-specific probe.

Genomic DNA from 46 B27+ ankylosing spondylitis, Reiter's syndrome, or normal individuals was digested with Taq I and probed, in Southern blots, with the HLA-B locus specific probe, EI7. Four restriction fragment length polymorphisms (RFLP), 2.5, 3.4, 3.8 and 4.0 or 8.0 kb, were observed for the B27 gene. In Caucasians, one of the B27 variants (2.5 kb) was more frequent in normals and almost never appeared in patients, suggesting a trend that is not yet statistically significant. In the course of defining the B27 polymorphisms, three and two RFLP, respectively, were also found for the B18 and B44 genes.

Detection of platelet antibodies by anti-kappa light chain facilitation of C-FDA (KC-FDA) thrombocytotoxicity.

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.

Mechanisms of the CYNAP phenomenon: evidence in the Bw49/Bw50 model for epitopes with different spatial orientation of antibody.

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.

Strong linkage disequilibria among HLA-Bw50, BfS1, and HLA-DR3/DR7, and mapping of the Bf locus.

Based on genotypic and phenotypic studies we have found strong linkage disequilibria in Caucasians among the genes HLA-Bw50, BfS1, and HLA-DR3 and/or -DR7. The relative disequilibria, which are among the highest described in man, are delta r (BfS1, DR7) = 0.51, delta r (Bw50, BfS1, DR7) = 0.36, delta r (Bw50, DR3 or 7) = 0.72, delta r (BfS1, DR3 or 7) = 0.91, delta r (Bw50, BfS1, DR3 or 7) = 0.73. The previously described high delta r (Bw50, BfS1) and delta r (Bw50, DR7) have also been confirmed. A B parallel DR crossover family is also presented that, together with previously reported recombinant families, confirms that the Bf locus resides between HLA-B and HLA-DR. These data suggest the existence of a supergene complex of Bw50, BfS1, DR3/7 (or MB2), and hypotheses to account for the observed disequilibria are discussed.

A monoclonal antibody against HLA-A11 and A24.

A monoclonal IgG antibody was produced from a mouse immunized with an A11, A24; B27, B44 Epstein-Barr virus transformed B lymphoblastoid cell line. The antibody, A11.1M, by standard lymphocytotoxicity assay, reacts with all cells expressing HLA-A11 and -A24. Absorption studies with both A11+, A24- and A11-, A24+ platelets removed antibody reactivity against A11 and A24 lymphocytes. The shared antigenic determinant between A11 and A24, as defined by this antibody, A11.1M, represents a new "supertypic" determinant.

ST-16: a new Bw16 subtype present in Mexican-Americans.

Using a large battery of Bw16, w38, and w39 antisera, a new variant of Bw16 has been identified in four unrelated Mexican-American families. The serologic pattern obtained is distinct from that for Bw38, Bw39, and 8W57 antigens. Absorption studies confirm the existence of this new Bw16 subtype which we term ST-16. ST-16 is Bw6-associated, with antigen frequency estimated to be 2.5% in Mexican-Americans.

Expression of HLA antigens by human thymic epithelial cells.

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.

Secretion of genetically engineered human/mouse class I antigens.

Two soluble, secreted forms of HLA-B7 were engineered by the creation of hybrid human/mouse molecules containing the polymorphic 5' region of the HLA-B7 gene and the secretory 3' region of the mouse Q10d gene. The hybrid, designated F1, is the first construct with only human extracytoplasmic domains, consisting of exons for the leader peptide and the three extracellular domains (alpha 1, alpha 2, alpha 3) of B7 spliced to the exons for the Q10d truncated transmembrane and 3' untranslated (3'UT) sequences. The second construct, designated C2, is similar but has the human alpha 3 replaced by the Q10 alpha 3 domain. Protein product from each construct was best demonstrated after gene transfection into the J27.2 cell line. In particular, secretion of the F1 product proves that the Q10 alpha 3 domain is not necessary for secretion of class I/Q10 hybrids. Moreover, the two soluble B7 forms, which differ only in their alpha 3 domain, are similarly recognized by monoclonal antibodies W6/32 (anti-HLA-ABC), BBM.1 (anti-human beta 2 microglobulin), and allo-B7-antibody, but differentially recognized by monoclonal antibody Q1/28 (anti-HLA class I heavy chain). Production of such soluble hybrid class I molecules in large amounts should allow critical structural and functional studies of these proteins.