LD Typing Research Articles

Clonal analysis of T lymphocyte response to an isolated class I disparity

A bulk primed lymphocyte reagent generated in a class II identical class I (HLA-B) disparate sibling combination demonstrated both cytotoxic {cell mediated lympholysis (CML)} and proliferative {i.e., primed LD (lymphocyte) typing (PLT)} reactivity associated with the class I antigen, Bw62. Cells from this bulk population were plated by limiting dilution and cloned by micromanipulation. Three functional groups of clones were isolated. Some clones derived were found specifically to proliferate to and lyse cells bearing the Bw62 antigen. Based on such reactivities, these clones were analogous to the class of antigen-driven, helper cell independent cytotoxic (HITc) clones previously reported from our laboratories. Other clones responsed proliferatively to stimulation by Bw62 positive cells but were not cytotoxic, thus fitting characteristics of Th, although it will be necessary to test such clones for their ability to produce Interleukin 2 (IL-2). In addition, conventional cytotoxic clones which did not proliferate to, but did lyse cells bearing the Bw62 antigen were isolated. The results were consistent with the existence of both HITc and Tc mediated cytotoxicity generated against this isolated class I disparity.

Phenotyping by the mixed lymphocyte reaction in goats (LD typing)

The LD-system of the goat, as demonstrated by the mixed lymphocyte culture (MLC) was studied in related and unrelated goats. Suggestive evidence was obtained, indicating that LD determinants are under the control of gene(s) which may be in the same chromosomal region as those coding for the SD determinants.

Human primed LD typing lymphocytes (PLTs) grown and cryopreserved as continued cultured T-cells (CTC)

Human primed LD typing lymphocytes (PLTs) grown and cryopreserved as continued cultured T-cells (CTC)

The major histocompatibility complex (HLA) as a genetic marker in human craniofacial anomalies.

Study of the incidence and segregation of the serologically detectable A and B products of the HLA complex in 140 family units in which one or more offspring was afflicted with a developmental craniofacial anomaly has uncovered no evidence of an association between HLA-A or B antigens or haplotypes and the malformations under study. Further analysis of HLA-D products in the same family units by the mixed leukocyte culture (MLC) technique has, however, uncovered a relatively high incidence of non-reactivity between the cells of one (or both) parent(s) and cells of some offspring in 41 of the 140 families included in this study. The parent couples involved in this finding were unrelated and generally did not share any HLA-SD haplotypes. When this finding was studied further by Primed LD Typing techniques, the results in six families suggested that such MLC non-reactivity is a consequence of the sharing of LD alleles by each pair of parents in these families. The known polymorphism of the HLA-D locus (or loci) and the low incidence of comparable findings in the normal population suggest that LD allele sharing in this particular population may be related to the selection of certain particular HLA-D products in families afflicted with developmental craniofacial anomalies. This result may be relevant to the possible existence in man of an analogue of the murine T/t complex which may occur in linkage with the HLA complex, in the same manner as the linkage disequilibrium which is been documented between the t complex and H-2 in chromosome 17 of the mouse.

Human T lymphocyte clones reactive in primed lymphocyte typing and cytotoxicity

The human HLA-D region, the presumed evolutionary homologue of the H–2 I region in mouse and similar genetic regions in other species1, codes for multiple antigenic determinants, a finding consistent with the existence of multiple loci2,3. One method developed to define D region encoded determinants is primed LD typing (PLT) wherein cells of one individual are primed in vitro in a mixed leukocyte culture (MLC) to allogeneic HLA-D encoded determinants associated with one haplotype4,5. The PLT cells give a rapid proliferative response when restimulated in secondary culture with either the priming cells or third party cells that carry the priming antigen(s). However, as in many cases a PLT cell is probably primed to several determinants, different restimulating cells from unrelated individuals will engender a range of responses varying from very high to very low, presumably depending on the number of determinants shared (or cross-reactive) between the priming and restimulating cells. Such results are difficult to interpret in terms of defining antigenic determinants on the cells of various individuals. Fathman and Hengartner6 have circumvented this problem in mouse by cloning murine T lymphocytes from multiply restimulated PLT cells; the progeny of any one clone give highly specific proliferative responses. We report here the use of the supernatant of phytohaemagglutinin (PHA)-stimulated mixtures of human allogeneic cells7,8 (designated T-cell growth factor or TCGF) to grow ‘clones’ of human T lymphocytes following alloactivation in MLC. Clones that give PLT-type responses do so in a highly antigen-specific manner. Following cloning, cells in many cases have undergone 25 or more divisions, thus yielding in excess of 1×108 progeny.

Backstimulation by Blastogenic Factor in the Primed LD Typing Test: Possible Role in Anomalous Responses

The anomalous false-positive results often found in the primed lymphocyte typing (PLT) test seem to be partially due to the release of blastogenic factor (BF) from the stimulating cell, resulting in back stimulation of the primed lymphocyte. These anomalous responses can be greatly reduced by treating the stimulator cell with high doses of irradiation or with puromycin.

Summary of the first international workshop on human primed LD typing.

Summary of the first international workshop on human primed LD typing.

Effect of Ultraviolet Light on Dinitrochlorobenzenespecific Antigen-Presenting Function

Abstract HLA-A and -B antigens are resistant to ultraviolet (UV) light whereas HLA-D, as defined by the mixed leukocyte culture reaction (MLR), is sensitive to UV light in initial or first culture. In primed LD typing, the HLA-D responsible for stimulation is relatively resistant to UV light in second culture. We have developed a method for evaluating a specific proliferative response to DNP in dinitrochlorobenzene (DNCB)-sensitized human subjects and found a requirement for a component of autologous leukocytes to activate DNP-responsive lymphocyte clones. Autologous DNBSO3-coupled leukocytes with and without UV were used to stimulate lymphocytes in DNCB-sensitized subjects. 3HTdR incorporation was measured after 4 or 5 dys in 1st or “primary” culture. After 9 to 14 days, blast-derived lymphocytes were set up in 2nd or “secondary” cultures and exposed to the same autologous DNBSO3-treated leukocytes with and without UV. Accelerated 3HTdR incorporation was measured in these secondary clonally primed cultures after 20 to 44 hr. The results showed that UV light abrogated the antigen-presenting or stimulator function in the 1st culture and diminished, but did not abrogate, the antigen-presenter function in the 2nd culture. These findings indicate that the DNP-antigen presenting function is behaving in a manner similar to that observed for mixed leukocyte culture determinants in 1st and 2nd cultures.

Detection of HLA-D clusters and segregation studies using primed LD typing.

By testing a group of PLT cells over a panel of unrelated restimulating cells, the PLT's could be grouped into clusters according to their ability to discriminate antigen(s) in unrelated cells. The PLT clusters broadly correlated with the homozygous typing cell-defined HLA-D clusters represented on the panel. The PLTs grouped together clearly segregate with a particular HLA haplotype when tested in both unrelated families not possessing the sensitizing haplotype and in the family with the sensitizing haplotype. No influence of HLA SD antigens could be observed in PLT restimulation in the segregation studies.

Detection of HLA-D clusters using primed LD typing.

Using a set of 17 primed LD typing (PLT) cells tested on a panel of 35 unrelated cells, we showed that certain groups of PLT cells tended to detect similar unrelated cells. The PLT cells were grouped into seven clusters and these tended to correlate with the seven HLA-D specificities represented on the panel, as determined by HTC testing. These data suggest that the antigens that cause restimulation in PLT are similar to those HLA-D antigens detected by the homozygous typing cell (HTC) test or, alternatively but more unlikely, that the two typing methods are detecting genes in close linkage disequilibrium with the HLA-D region.

Typing an unrelated panel with PLT cells: association with DW clusters.

Primed LD typing (PLT) cells prepared in one Laboratory (Madison) were shipped in the frozen state and tested in Tübingen on a separate panel that had been typed with homozygous typing cells. Those PLT cells that had been grouped, on the basis of their reaction with test cells of the Madison panel, as defining an HLA PL antigen showed identical or nearly identical patterns of reactivity with the Tübingen panel. Clear association between certain PL antigens and DW clusters as defined with homozygous typing cells could be demonstrated. Of particular interest may be combinations of certain PLT reactions with D-locus-typed cells, where the primed cells do not react as expected from the target's HLA-D type.

Response of primed LD typing cells to homozygous typing cells.

At least two different methods using cellular responses have been described for defining the determinants of the HLA-D region: typing with HLA-D homozygous cells and primed LD typing. Primed LD typing cells were generated in one-haplotype-different combinations and grouped on the basis of two or more cells appearing to define the same HLA-D-region-determined PL antigen. Such cells were restimulated with homozygous typing cells for several of the presently known HLA DW clusters. A very strong correlation was noted: PLT cells defining the antigen PL1 were restimulated with homozygous typing cells for DW3, those PLT cells defining the antigen PL2 were restimulated by homozygous typing cells for DW2, and those defining PL5 were restimulated by homozygous typing cells for DW1.

Primed LD typing: reagent preparation and definition of the HLA-D-region antigens.

Primed LD typing cells were prepared against single HLA haplotypes within families and used to type a random panel of 48 individuals. PLT cells could be grouped on the basis of highly correlated responses with the panel test cells; the 21 different PLT cells could be used to define 6 different PL antigens. One of these, PL 3, was split by the responses of two of the 21 PLT cells which measured an antigen called PL 3.1.

Polygenic control of mixed lymphocyte reaction in dogs.

The genetics of the MLR of the dog are studied using LD typing cells. The four new homozygous reference cells described bring the total number of defined LD specificities in this species to 11. In families, LD specificities generally segregate en block with the SD specificities of the DLA complex. The recombination frequency between the SD and LD loci is provisionally estimated to be 0.007. A slight but significant stimulation between two SD identical LD typed siblings could not be explained on the basis of a single recombination between the SD loci and one LD locus and therefore suggested the existence of an additional LD system. Unrelated SD different individuals with two different LD determinants in common frequently stimulate each other in MLR, but low responses are also encountered. The results described are compatible with a hypothesis of two segregant series of alleles for the genetic control of the MLR and linkage disequilibrium between certain alleles of these series. More conclusive evidence of the validity of this hypothesis and further knowledge of other factors which determine the response in the MLR are required before a reliable estimate of the importance of LD structures in histocompatibility can be made.

LD typing and the lack of involvement of SD antigens in the priming process.

Peripheral blood lymphocytes with known HLA-SD were typed for LD determinants using a panel of primed cells prepared from SD identical lymphocytes. Then these lymphocytes (with known SD and LD) were primed by x-irradiated cells from the same SD and LD group and their secondary reactions were evaluated. Disparity in SD antigens of responder and stimulator cells did not produce primed cells and lymphocytes were primed only for LD differences. This finding allows LD typing using primed cells from a panel of randomly selected lymphocytes regardless of their HLA-SD.

Typing for HLA‐D: Primed LD Typing and Homozygous Typing Cells

Typing for HLA‐D: Primed LD Typing and Homozygous Typing Cells

Three HLA-D region antigens defined by primed LD typing.

We have recently described a new method, primed LD typing or PLT, for specific identification of HLA-D antigens. Highly discriminatory PLT cells have been developed which clearly differentiate between cells of individuals that restimulate strongly and those that restimulate weakly. Seven such discriminatory PLT cells have been used to define three antigens called PL1, PL2, and PL3; two more PLT cells may define antigen(s) PL4.

Genetic and cellular aspects of xenogeneic mixed leukocyte culture reaction.

The nature of the antigens stimulating xenogeneic lymphocytes was studied using "primed LD typing". Human lymphocytes were sensitized in vitro against mouse spleen cells and restimulated with spleen cells of mouse strains sharing non-H-2 antigens or various regions of H-2 with the initial stimulating strain. The largest thymidine uptake was caused by restimulation with cells from the specific primary stimulator or an H-2-identical strain. Species-specific antigens or strain-specific antigens carried in the C57BL/10 background account for less than 15% of the total stimulation; a non-H-2 antigen associated with the Mlsalpha genotype caused moderate restimulation, amounting to 25% of the average H-2 response. Within H-2, the strongest restimulation was caused by antigens controlled by the I-A subregion; the K and D regions caused moderate, the I-C and S regions very weak, and the I-B subregion no restimulation. Thus, the genetic control of antigens stimulating xenogeneic and allogeneic MLC responses requires T cells and adherent cells, but in the human-mouse MLC, both cell types must come from the human responder; the majority of the proliferating cells are T cells. It is suggested that allograft and xenograft reactions are fundamentally identical processes, and that the relative vigor of alloaggression may be explained by secondary potentiating mechanisms depending on species-specific interactions between aggressor and target cells.

Typing for HLA‐D: Primed LD Typing and Homozygous Typing Cells

Typing for HLA‐D: Primed LD Typing and Homozygous Typing Cells

EFFECTS OF SOME IMMUNOSUPPRESSIVE PROCEDURES ON MYASTHENIA GRAVIS*

Clinical Observations. A total of 53 MG patients have been treated with different immunosuppressive methods (alone or combined) with the following effects: Thymectomy was performed in 38 patients. The improvement was excellent in 15, and moderate or uncertain in 20. In three patients severe long-lasting deterioration followed the operation. ACTH treatment (n=32): Initial deterioration during the 5-7 days of heavy ACTH treatment (1000 IU) was followed by an improvement lasting on an average 4 months. The improvement was good or moderate in 78% of the patients. Betamethazone treatment has been tried in six patients where ACTH and azathioprine was ineffective. In four of these patients the results were excellent. Azathioprine treatment has been given to 26 patients for periods up to 7 years. An improvement is measurable after 6-12 weeks and it seems maximal after about 1 year. Of the 26, 80% responded favorable with reduction in the need for cholinesterase inhibitors. Severe complications were seen in three patients with one death. Drainage of thoracic duct lymph was initiated in 14 patients up to 4 weeks with rapid improvement lasting as long as drainage was performed. Long-termed effects of the drainage may be present, however. Retransfusion of homologous cell-free lymph precipitated a return of the myasthenic symptoms. Biochemical Studies on Myasthenic Lymph. Using a membrane preparation from the electric organ from Torpedo marmorata and tritiated Naja naja siamensis neurotoxin we demonstrated a decreasing binding of toxin to the receptor in the presence of MG lymph gamma-globulin fraction. Gammaglobulins from controls showed almost no inhibition of the neurotoxin binding. Immunological Studies. An increased frequency of HL-A1 and 8 was found in female patients. LD typing was also performed. During a period of three weeks of thoracic duct drainage 130X10(9) or about 10% of total number of lymphocytes in the body were removed. In the lymph an initial decrease in the proportion of thymus-derived lymphocytes (T cells) occurred, which was accompanied by a sequent increase in the proportion of bone-marrow-derived lymphocytes (B cells). Towards the end of drainage this effect was reverted. Mitogenic stimulation using lymphocytes from thoracic duct drainage revealed no differences as compared to normal cells. The proportions of T and B cells was studied in the peripheral blood in nine patients treated with ACTH. During treatment there was an initial decrease in the proportion of T cells accompanied by a subsequent rise in the proportion of B cells, which was maximal after 3-10 days. These proportions were reverted to normal 1-5 days after the maximal change. The effect of azathioprine on T and B cells has also been studied.