Identification of mouse H-2 antigens by mixed lymphocyte culture in the presence of PHA. II. Blastformation of mouse lymphocytes in culture according to the difference in H-2 antigens

In the mixed tissue culture of mouse lymphocytes with addition of PHA the rate of the appearance of large and intermediate cells increases markedly, but which side of the two cell groups have reacted stronger remains obscure. In order to solve this problem, mixed cultures were conducted in such a way that only one cell group of the two would react. Namely, one cell group was exposed to C0 6).irradiation (Table 2) prior to the culture and cultured with another viable cell group (FJ test group, Table 2) to see the percentage of the appearance of large and intermediate cells. Simultaneously, the skin homograft from respective donor mouse was transplanted to each other and the survival days of each skin graft were compared. As a result it has been shown that the percentage of blastformation and the survival time of the skin transplant in each group prove to be in an inverse relation. The results of these mixed cultures indicate that the extent of blast. formation reflects significantly the difference in B-2 histocompatibility antigens.</p

Identification of mouse H-2 antigens by mixed lymphocyte culture in the presence of PHA. 3. Blastformation of mouse lymphocytes according to the difference in non- H-2 antigens

It has been demonstrated that by the mixed cultures in the presence of PHA the combination of those cells whose H-2 antigens differ from each other shows a higher rate and more significant difference of blastformation than in the combination where non-H-2 antigens differ (Table 1). The blastformation observable in the combinations where non-H-2 histocompatibility antigens or sex.linked antigens are weaker, is not, so marked as the difference seen of the blastformation in the case with H-2 isoantigens. This in vitro lymphocyte stimulation test can be applied to the histocompatibility test in the combinations of strong H-2 antigens.</p

Identification of mouse H-2 antigens by mixed lymphocyte culture in the presence of PHA. I. Blastformation in the tissue culture of mouse lymph node cells in the presence of PHA

It is said blastformation can hardly be observed in the tissue culture of mouse lymphocytes. However, in our experiments of mouse lymphocytes (obtained either from axillary or cervical lymph nodes) mixed with various cells in combination of other cells as A+C3H, A+C57BL, or C3H+C57BL, it has been verified that these lymphocytes readily undergo blastformation in the presence of PHA (phytohemagglutinin M) as adjuvant. In the single tissue culture of these lymphocytes without PHA, the blastformation is observable in 6 per cent of the cells, while in the presence of PHA it is seen in 13. 7 per cent of the cells. In the cases of mixed cultures blastformation is observable in 14 per cent in the absence of PHA, whereas it is seen in 35.4 per cent in the presence of PHA. There is obviously a significant difference (p=O.OOI) in the blast. formation when cultured in the presence of PHA, and its reproducibility also proves to be quite high.</p

Correlation between morphological blastformation rate and functional 3H-thymidine uptake in mixed lymphocyte culture in the presence of PHA

By dividing at random 14 normal persons into 7 pairs of two individuals each, lymphocytes were isolated from their peripheral blood and taking one of the pairs as stimulating cells or antigens and the others as responding cells, mixed lymphocyte culture (MLC) was carried out. As for the method of MLC we used our MLC method of unidirectional mixed culture with a small amount of lymphocytes in additition of 1% (v /v) PHA.M and cultured for three days, and a widely used conventional method in which 3H.thymidine uptake was the parameter of the blastformation rate and cultured for seven days. In comparing the results of these two groups of MLC the data in six experiments out of the seven coincided. Namely, with 5x 104 cells each of stimulating cell group and responding cell group, it is possible to achieve satisfactory MLC, the culture can be done only for three days without requiring any special technique and the results can be readily evaluated. Therefore, MLC by our simple method would yield satisfactory results in clinics.</p

An Observation of the Stability : Statics and Dynamics

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