Secondary mouse mixed lymphocyte reaction (MLR) in vitro: Correlation between primed cells reactivity and serological typing for Ia specificities

B10.M (H-2^f) spleen lymphocytes were cultured for 14 days with X-irradiated B10.P (H-2 ^p ) lymphocytes. These primed cells were then tested for their capacity to respond to a secondary stimulation induced by a panel of mouse cells carrying differentH-2 haplotypes. The third-party cells induced various degrees of proliferation which could be expressed as a percentage of the proliferation induced by the primary stimulating strain (relative response = RR) and could then be classified according to the RR. Anti-Ia antibodies present in a B10.M anti-B10.P serum were studied by the dye exclusion lymphocytotoxicity technique (LCT) against the same panel, after absorption of H-2K, D antibodies on B10.P platelets. The strain panel could then be classified according to the LCT titers of the absorbed immune serum. A significant correlation (r=0.96,P<0.01) was found between both classifications. According to the Ia chart, the public specificities involved were Ia.6, 7, and 13, but these did not fully explain either the primed cell reactivity or the LCT results. An unexpected crossreactivity was observed between B10.P and B10. Absorption-elution experiments with A.TH anti-A.TL serum demonstrated that B10 and B10.P share the Ia.3 antigen. These results indicate that the structure(s) recognized by the primed lymphocytes is the Ia antigen(s).Abbreviations used in this paper RR Relative response - LCT Dye exclusion lymphocytotoxicity technique - MLR Mixed lymphocyte reaction - PC Primed cell - MHC Major histocompatibility complex - PLT Primed lymphocytes typing test     

Sources of bovine lymphocyte antigen (BoLA) typing reagents*

Sera from about 1000 cows were tested for cytotoxicity against a panel of up to 100 lymphocyte samples. Cytotoxic antibodies presumably resulting from trans-placental immunization of the cow by her calf were found in about 45% of these sera. The antibody titers of sera from parous cows rarely exceed 4², some persisted for over one year, but decreased notably at calving. Thirty-five immune sera were also produced by alloimmunization with lymphocytes. They usually reached peak titers of up to 4⁴ at 2 or 3 weeks after the initial immunization. Subsequent immunizations produced sera with very high titers but they were much more polyspecific. High-titered antibodies were also produced by skin graft recipients. Useful cytotoxic antibodies were found in 19 of 111 colostrum whey samples. Studies on 13 dam-calf pairs showed that the newborn calf may acquire cytotoxic antibodies from its mother's colostrum, but the only cytotoxic antibodies detectable in this calf s serum are those not directed against its own lymphocyte antigens. It is concluded that efficient lymphocyte typing requires antibodies from a variety of sources.     

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     

Simplified typing of mouse hemoglobin (Hbb) phenotypes using cystamine

Cellulose acetate electrophoresis of mouse hemoglobins modified with the disulfide reagent cystamine permits rapid, unequivocal discrimination of all combinations of the codominant mouse hemoglobin single (Hbb ^s ) and diffuse (Hbb ^d and Hbb ^p ) alleles. The single, diffuse major, diffuse d-minor, and diffuse p-minor adult hemoglobins are all resolved by this method, which depends on the presence of a cysteine in the chains of diffuse mice which is not found in the chain of single mice.This work was supported by research grants ACS-VC58 and NIH CA-01074. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

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.     

Serological typing of HLA-D: Predictive value in mixed lymphocyte cultures (MLC)

Locally produced antisera and antisera received through the Seventh International Histocompatibility Workshop exchange were investigated for specific B-cell cytotoxic activity in a panel of 95 unrelated HLA-D-typed donors. A number of sera formed clusters defining eight B-cell specificities which were strongly associated (p<0.001) to the HLA-D determinants Dw1–8. In panel investigations, only four triplets occurred. In five HLA recombinant families, these B-cell specificities followed the HLA-B-D chromosomal region, and in one —B/D recombination, DRw1 traveled with —Dw1. In MLCs between panel donors sharing zero, one, or two HLA-D-related B-cell specificities, significantly weaker MLC stimulation was observed with increasing compatibility, the median relative responses being 100, 52, and 17 percent, respectively. It is concluded that B cell-specificities HLA-DRw1–7 and WIA8 are probably coded for by HLA-D; they are excellent markers for the HLA-D determinants, which can thus be typed for by serological means; and serological typing for HLA-D has great value in predicting the outcome of MLCs.     

Evaluation of arbitrarily primed PCR for typing of Desulfovibrio desulfuricans strains

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of 15 (soil and intestinal) Desulfovibrio desulfuricans strains. The primer M 13, which is a core sequence of phage M 13, was found to be appropriate for the differentiation of isolates of this species. The analysis revealed characteristic band patterns for all of the examined strains of which two soil strains (DV-7 and DV-8) showed identical DNA fingerprints. According to Jaccard's coefficient, the soil bacterial group as well as intestinal bacterial group formed two different clusters. Furthermore, the soil strains showed greater variability than the intestinal isolates. Based on the AP-PCR fingerprints D. desulfuricans strains were differentiated depending on their origin. This study demonstrates that the typing method AP-PCR can be useful in epidemiologic investigations as a rapid and valuable tool for differentiation of the strains of D. desulfuricans species.     

The mixed lymphocyte reaction (MLR) stimulatory capacity of human lymphocyte subpopulations.

Using various cell separation techniques and combinations of these, suspensions were obtained highly enriched or depleted with respect to their content of E-rosette-forming T cells, Ig-bearing B cells, Fc-receptor-bearing cells, or monocytes. These purified populations were tested for their capacity to stimulate allogeneic cells in a mixed lymphocyte reaction (MLR). It could be demonstrated that the Ig-bearing B cells provide the strongest stimulus in the MLR.     

The dynamic expression pattern of B lymphocyte induced maturation protein-1 (Blimp-1) during mouse embryonic development

We have analyzed the spatial and temporal patterns of B lymphocyte-induced maturation protein-1 (Blimp-1) expression during mouse embryonic development. Blimp-1 expression is induced early in the anterior definitive endoderm, mesoderm of head process, and prechordal plate. In ectoderm-derived tissues at later stages, Blimp-1 expression is found in the primitive photoreceptors of neural retina, in differentiated epithelial cells of epidermis, tongue, oral and nasal cavities, and in the precursors of internal root sheaths of hair follicles. In mesoderm-derived tissues, Blimp-1 expression is observed in splanchnopleure, a subset of somatopleure-derived cells in limb buds, and myotomes of somites. Blimp-1 is also expressed in mesenchyme of developing hand plates, digits, branchial arches, nasal processes, and external genitalia. Blimp-1 is present in mesenchyme-derived chondroblasts, supporting cells of taste buds, and papilla of teeth, hair follicles and taste buds. In endoderm-derived tissues, Blimp-1 expression in the foregut region is restricted to a subset of epithelial cells at the headfold stage while expression in the endodermal epithelium of midgut and hindgut persists from the headfold stage to birth. Finally, Blimp-1 is expressed in the migrating primordial germ cells.     

Alternol inhibits proliferation and induces apoptosis in mouse lymphocyte leukemia (L1210) cells

Germline mutations of the serine/threonine kinase LKB1 (also known as STK11) lead to Peutz–Jeghers syndrome (PJS) that is associated with increased incidence of malignant cancers. However, the tumor suppressor function of LKB1 has not been fully elucidated. We applied yeast two-hybrid screening and identified that a novel WD-repeat protein WDR6 was able to interact with LKB1. Immunofluorescence staining revealed that WDR6 was localized in cytoplasm, similar to the localization of LKB1. Expression of LKB1 was able to inhibit colony formation of Hela cells. Interestingly, coexpression of WDR6 with LKB1 enhanced the inhibitory effect of LKB1 on Hela cell proliferation. Consistently, WDR6 was able to synergize with LKB1 in cell cycle G1 arrest in Hela cells. Coexpression of WDR6 and LKB1 was able to induce a cyclin-dependent kinase (CDK) inhibitor p27^Kip1. Furthermore, the stimulatory effect of LKB1 on p27^Kip1 promoter activity was significantly elevated by coexpression with WDR6. Collectively, these results provided initial evidence that WDR6 is implicated in the cell growth inhibitory pathway of LKB1 via regulation of p27^Kip1.     

Hybridization of a mouse T-cell lymphocyte line (HB1) with a polyoma virus-transformed mouse fibroblast line

The establishment of permanent T-lymphocyte cell lines by transformation with DNA viruses has not yet been achieved. This paper reports the successful transfer of polyoma virus genome into T-lymphocyte cells by somatic hybridization. A T-lymphocyte clone, HB₁, derived from (DBA/ 2J×AKR) spleen cells, isolated in vitro by cloning in semi-solid agar, was fused with a polyoma (Py) virus-transformed fibroblast C3HPy, clone 1. The authenticity of the hybrid C3H/HB was established by chromosome and histocompatibility antigen studies. This initial population and the various clones retained T-lymphocyte characteristics such as morphological appearance, growth properties (suspension culture) and differentiation antigen (Thy 1–2). The hybrid cell line and the various clones presented all the characteristics of Py transformation. Namely, they carried the Py genome originating from the fibroblastic parent and maintained Py virus tumour-associated antigens (TSTA, TSSA and T antigens). In most respects, this hybrid population resembled the C3HPy/C11 parent and exhibited the same tumorigenicity.