T cell blasts activated in the mixed lymphocyte reaction (MLR) carry on their surface stimulator alloantigens which can be removed by treatment of the blasts with trypsin. After overnight incubation in trypsin-free medium, the treated cells exhibit specific alloantigen binding ability: they bind much more effectively cellular material, either obtained from the corresponding MLR supernatant or released by nitrogen cavitation from fresh cells from the stimulating strain, than that from an unrelated H-2 different strain. Trypsin-treated cells which have been incubated in the presence of low concentrations of puromycin are unable to bind stimulator cell fragments.

Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Attempts to control JD have proven inordinately difficult due to low levels of sensitivity by currently available diagnostic tests, which are also incapable of detecting prepatent MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for serological diagnosis of subclinical and clinical JD in cattle. The FCM was capable of distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Results of the FCM were compared to that of a commercially available ELISA using 82 serum samples from JD-positive and JD-negative dairy and beef cattle farms that were separated into the following groups: (1) sera from a JD-free farm; (2) sera from JD-positive farms that had tested negative by ELISA; and (3) sera from JD-positive farms that tested JD-positive by ELISA. The FCM found that groups 1-3 were 6.6%, 73.3%, and 97.3% positive for MAP infections, respectively. By using 30 fecal culture-negative samples from a JD-free farm and 21 fecal culture-positive samples from JD-positive farms, diagnostic sensitivity and specificity of the FCM were calculated to be 95.2% and 96.7%, respectively. A retrospective study of 10 JD-positive cows showed that the FCM detected MAP infections 6-44 months earlier than the fecal culture test. Further, the FCM specifically detected MAP infections in serum samples as early as 170 days after experimental inoculation of calves with MAP and did not react with calves inoculated with other mycobacteria. Production of IgG against MAP was detected by FCM in all the calves inoculated with MAP 240 days after inoculation, whereas positive anti-MAP IgG production was not detected in control calves or calves experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay might form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD.

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We have established 16 independent hybrid lines derived from a fusion between an AKR thymoma and mixed leukocyte reaction-stimulated C57BL/6 spleen cells. The hybrids express both parental H-2 alleles and show hybrid glucose-6-phosphate isomerase activity. Two of the lines showed Fc receptor activity not expressed in the parental thymoma. The AKR Thy-1.1 allele was expressed in all hybrid lines, but no Thy-1.2 could be detected. No killing activity was retained in the hybrid cells, probably because Sendai virus-mediated fusion of killer cells resulted in the lysis of the thymoma fusion partner.

The specificity of binding of stimulator-derived H-2 antigens by mixed lymphocyte culture (MLC)-activated T blasts was investigated under conditions of antigen excess. We have shown that the detectable proportion of alloantigen-binding blasts from primary MLC is a function of antigen concentration, and can represent up to more than 90 percent of total blasts, when the antigen is presented in the appropriate form (on mitomycin-treated viable stimulator cells, or membrane vesicles prepared from lipopolysaccharide blasts), and at nonlimiting concentration. Thus stimulator alloantigen-binding directly parallels the proliferative response and is not restricted to a subpopulation of T blasts. However, the marked dependence of the binding on antigen concentration indicates that cells with a wide range of receptor affinities for the stimulating determinants are involved. In view of this possibility, the specificity of binding by these cells was studied. We have demonstrated that stimulator K, I, and D region products are bound by nonoverlapping subpopulations of blasts, the sum of which may represent 93 percent of total blasts. Thus, specific distinction by these cells between different H-2 region products is not affected by the putative heterogeneity in terms of receptor affinities. However, specificity with respect to unrelated H-2 haplotypes is strictly dependent on antigen concentration. A preferential binding of stimulator membrane vesicles occurs at limiting concentrations; whereas the majority of blasts bind stimulator and third- party vesicles equally well at high vesicle concentrations. The binding of both vesicle types is specific in that it can be inhibited with the relevant anti-H-2 sera. Furthermore, stimulator and third-party vesicles seem to compete for binding sites on the same cells, as shown by cold antigen inhibition. From these results, we propose that there is an imperfect distinction between stimulator and third-party H-2 antigens by the majority of primary MLC blasts. In contrast, highly selected long-term MLC blasts do not bind third-party H-2 antigens at any concentration, and seem to have high affinity for the stimulating antigens. We conclude that large numbers of clones with low-affinity (cross- reactive) receptors are generated in primary MLC, most of which become eliminated during long-term selection. This implies that the frequency of cells strictly specific for nonshared stimulating determinants must be minute.