Pavol Ivanyi

Nucleotide sequences of chimpanzee MHC class I alleles: evidence for trans-species mode of evolution.

To obtain an insight into the evolutionary origin of the major histocompatibility complex (MHC) class I polymorphism, a cDNA library was prepared from a heterozygous chimpanzee cell line expressing MHC class I molecules crossreacting with allele‐specific HLA‐A11 antibodies. The library was screened with human class I locus‐specific DNA probes, and clones encoding both alleles at the A and B loci have been identified and sequenced. In addition, the sequences of two HLA‐A11 subtypes differing by a single nucleotide substitution have been obtained. The comparison of chimpanzee and human sequences revealed a close similarity (up to 98.5%). The chimpanzee A locus alleles showed greatest similarity to the human HLA‐A11/A3 family of alleles, one of them being very close to HLA‐A11. Similarly, segments of the ChLA‐B alleles displayed greatest similarity to certain HLA‐B alleles. The calculated evolutionary branch point for the A11‐like alleles is 7 x 10(6) to 9 x 10(6) years, whereas the other A locus alleles diverged between 12 x 10(6) and 17 x 10(6) years ago. Since the human and chimpanzee lineages separated 5 x 10(6) to 7 x 10(6) years ago, our data support the notion that during evolution, MHC alleles are transmitted from one species to the next.

An improved biochemical method for the analysis of HLA-class I antigens. Definition of new HLA-class I subtypes

A simple method is described for the biochemical analysis of HLA class I antigens. It is a modification of a previously published procedure for one-dimensional isoelectric focusing (1D-IEF), giving improved resolution and offering larger sample capacity. One million viable cells suffice for analysis, and no more than 25 muCi of radioisotope (35S-methionine) are required. The usefulness of the method is illustrated by the characterization of a total of four biochemically distinct subtypes of HLA-B27, three subtypes of HLA-A24, two subtypes of HLA-A11, three subtypes of HLA-A2, two subtypes of HLA-Bw60, two subtypes of HLA-Bw62, and four subtypes of HLA-B35 in a panel of 24 cells selected for the expression of HLA-B27. We envision that this technique will allow a rigorous classification of HLA-A,B antigens into novel subtypes. Populations of distinct ethnic origin are especially of interest in this regard. This technique might also be used as an additional criterion for the official classification of HLA-A,B antigens.

Subtypes of HLA-B27 detected by cytotoxic T lymphocytes and their role in self-recognition

In the present study cytotoxic T lymphocytes were generated in MLC of lymphocytes from two unrelated HLA-A, B, C-identical, B27-positive, but D/DR-different, individuals. These CTL were shown to detect subtypes of HLA-B27. CTL specific for influenza virus lysed infected target cells matched for HLA-B27 only when they shared the same subtype. This indicates that the two subtypes of HLA-B27 detected by CTL function also as distinct elements in a self-restricted CTL response. Both subtypes were found among patients with ankylosing spondylitis.

Germ-free mice do not develop ankylosing enthesopathy, a spontaneous joint disease

Ankylosing enthesopathy (ANKENT) is a naturally occurring joint disease in mice with numerous parallels to human ankylosing spondylitis (AS). Similarities between AS and ANKENT include not only affected tissue (joint entheses) but also association of the disease with genetic background, including MHC genes, gender, and age. Young males with the C57Bl/10 background have been described to suffer from ANKENT and, among H-2 congenic strains, high frequency of afflicted joints has been recorded in B10.BR (H-2(k)) males. Interestingly, the incidence of ANKENT is higher in conventional (CV) males that in their specific-pathogen-free (SPF) counterparts. The latter finding suggests that microbes could play a role as an ANKENT-triggering agent. To further examine this hypothesis we have established a germ-free (GF) colony of B10.BR mice and observed ANKENT incidence in both GF males and their conventionalized (ex-GF) male littermates; 20% of ex-GF males developed ANKENT before 1 year of age. In contrast, no joint disease was observed under GF conditions (p < 0.0001). Our results show that live microflora is required in ANKENT pathogenesis.

Preservation of immunological and colony-forming capacities of long-term (15 years) cryopreserved cord blood cells

BACKGROUND Cryopreserved cord blood may be stored for decades before being used for allogeneic stem cell transplantation. Little is known about the effect of long-term cryopreservation in liquid nitrogen on the viability and function of cord blood cells. We examined the recovery, viability, clonogenic capacity, and T-cell reactivity to HLA alloantigens of cord blood samples cryopreserved up to 15 years. METHODS Progenitor cell recoveries were studied by (colony-forming unit-granulocyte-macrophage) clonogenic assays from 18 cord blood samples short-term frozen for 2-8 weeks and from 8 samples cryopreserved for 15 years. Proliferative and cytotoxic responses against HLA antigens of thawed cord blood mononuclear cells after short-term or long-term cryopreservation were tested in standard mixed lymphocyte cultures and cell-mediated lympholysis assays. RESULTS After thawing, the mononuclear cell recovery from long-term frozen cord blood low-density fractions averaged 80% (range, 64% to 92%). The presented data show that long-term frozen cord blood cells keep their clonogenic potential. No damaging effect was seen on the proliferative and cytotoxic capacities of long-term frozen cord blood T cells. CONCLUSIONS The results support the possibility of long-term storage of progenitor cells from umbilical cord blood for future bone marrow reconstitution.

Identification of new B27 subtypes (B27C and B27D) prevalent in oriental populations

With the aid of alloreactive cytotoxic T lymphocytes, three subtypes of HLA-B27 can be defined: B27W, B27K, and B27C (non-B27W and non-B27K). The B27C subtype can be distinguished by 1D-IEF, is not recognized by B27W- or B27K-restricted influenza-A-specific cytotoxic T lymphocytes, and is prevalent in Oriental populations. Preliminary data indicate that the various B27 subtypes (W, K, C) are in different linkage disequilibria with HLA-C antigens. A fourth subtype of B27 (non-B27W and non-B27K), designated as B27D, was detected by 1D-IEF. (See accompanying paper by Neefjes et al.) The finding of four B27 subtypes indicates that the serologically defined B27 antigen comprises a family of various, strongly cross-reactive class-I molecules.

Relations of HL‐A and Rh Systems to Immune Reactivity

HL-A, ABO, and Rh antigens, as well as different kinds of humoral and cell-mediated immune responses, were determined in 133 volunteers. These were selected from 463 subjects immunized with U ( D ) , or staphylococcus, or pertussis antigens according to their low and high immune responsiveness. HL-A and blood group antigens were correlated by a computer program to categorized immune parameters (low, medium, high values). The latter were correlated with each other. The only notable association of the immune parameters with HL-A antigens was found between HL-A 3 and 7 and spontaneous lymphocytotoxic activity in mouse fibroblast monolayer. A striking correlation was, however, found between R h antigens and a number of antibacterial antibody levels and parameters of cell-mediated immune responses. These correlations indicate that Rho(D)-positive subjects had rather high ‘natural‘ antibody levels and PHA-reactive lymphocytes. The latter were stimulated, however, only with high doses of PHA (PHA hyposensitivity) and they lacked spontaneous lymphocytotoxic activity. A reverse correlation was found in Rho(D)-negative subjects, the ability to produce anti-Rh,(D) antibodies being negatively associated with the level of some antibacterial antibodies. Natural antibody, immunoglobulin level and lymphocyte activity were significantly lower in males. Correlation was seldom found between categorized values of the individual immune parameters such as antibacterial antibodies, immunoglobulins, complement, and cellmediated immunity factors. Negative correlations were obtained between PHA hyposensitivity and a low antibacterial antibody level, and a positive one between IgA, complement level and lymphocyte stimulation. These results the Rh system may be associated with a gene or genes involved in immune response regulation.

Anti-H-2 antibodies induced by syngeneic immunization

Alloreactive cytotoxic antibodies were induced in BALB/c mice by syngeneic immunization with normal lymphoid cells. Sixteen out of 41 mice produced antibodies with distinct anti-H-2 specificity. Anti-Kk antibodies were present in all positive sera, but the individual sera produced different reactivity patterns when tested on a panel ofH-2 haplotypes. Absorption and immunoprecipitation experiments confirmed the H-2 specificity of the syngeneic sera.We hypothesize that virus-modified H-2d structures have triggered alloreactive B-cell clones to produce anti-H-2 antibodies.

Monomorphic anti-HLA monoclonal antibody (W6/32) recognizes polymorphic H-2 heavy-chain determinants exposed by association with bovine or human but not murine ß2-microglobulin

W6/32 is a mouse anti-HLA class I monoclonal antibody (MoAb) of BALB/c (H-2d) origin with a monomorphic reaction pattern on human cells. In this study, we explain that the previously reported (Ivanyi D et al., Immunogenetics 20:6gg, 1984) cross-reactions of W6/32 with the H-2Db antigen are completely dependent on the formation of a complex between the H-2Db heavy chain with bovine beta 2-microglobulin (beta 2m) from the culture medium. M0Ab W6/32 cross-reacted with various H-2 class I antigens only in the presence of bovine or human beta 2m but not in the presence of beta 2m from other species (goat, sheep, rabbit) or syngeneic mouse beta 2m. The exposure of the W6/32 determinant on mouse cells was dependent on the concentration of human or bovine beta 2m and was influenced by the temperature and time of incubation. The reaction pattern of W6/32 on a large panel of mouse strains showed that the binding is due to at least two critical factors: (i) the H-2 haplotype of the target cells; and (ii) the substitution of murine beta 2m for bovine or human beta 2m. These results show that exposure of a polymorphic class I determinant is dependent on the species origin of beta 2m with which the heavy chain is complexed. Comparison of beta 2m amino acid sequences from various species does not give a clear answer about the shared quality of human and bovine beta 2m. One amino acid position (89) was identified at which human and bovine beta 2m are identical but differ from all other known beta 2m sequences.

Grouped caging predisposes male mice to ankylosing enthesopathy.

OBJECTIVE: To evaluate the number of males per cage as a possible risk factor for murine ankylosing enthesopathy (ANKENT)--a spontaneous joint disease with parallels to human seronegative spondylarthropathies--since ANKENT shows incomplete penetrance of genetic susceptibility factors among individuals living in a stable environment. METHODS: Frequency of ANKENT was compared among males housed with females, with other males, or alone. RESULTS: In three independent cohorts, a trend was observed that males housed with females rarely develop the disease, in contrast to males housed with other males (P < 0.25, P < 0.05, and P < 0.01). Furthermore, no males caged alone developed ANKENT, whereas disease did occur in males grouped together (P < 0.01). When healthy males (retired breeders) were recaged either alone or with other males, ANKENT developed among the grouped males only (P < 0.005). CONCLUSIONS: Caging males together is a relative risk factor for ANKENT. Grouped caging may perturb the immune system through endocrine pathways or modify microbiological load through behaviour (for example, infection due to biting).