Primed LD Typing Research Articles

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.

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