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Family studies of lymphocyte proliferative responses to synthetic polypeptides GT, GAT, and (T,G)-A--L
294
Annual American Association for Clinical Histocompatibility Testing Meeting, 1980
COMPUTATIONAL PROBLEMS IN SCREENING SERA BY MAXIMUM LIKELIHOOD WHEN RANDOM ERRORS OCCUR. Max A. Woodbury, and David P. Stinson, Division of Biomathematics, Duke University Medical Center, Duke University, Durham, North Carolina, 27710. It has been demonstrated (Nau et al., "A Mathematical Analysis of Human Leukocyte Antigen Serology", Mathematical Biosciences 40: 243-270, 1978) that the combinatorial problem of assigning HLA specificities in 'error free' serum screening is a difficult computational problem (NP hard), i.e., p~l known algorithms for solution require an exponentia. .mount of time according to the size of the input. Here we show that the statistical problem is of equivalent difficulty because of the imbedded combinatorial problem. Practically, we find that the imbedded problem is much reduced in size and relates most to those specificities which appear only on reacting cells. The remainder of the estimation problem, including determination of unreactive specificities and weak/short specificities, can be done in polynomial (less than exponential) time. An algorithm for defining these specificity classes (100% reactive, weak/short, unre4ctive) and for solving the remaining polynomial time problems is presented, along with an experimental computer program that implements this procedure for serum screening. The FORTRAN computer program is available to interested investigators. (Research supported under NIH grant AI-15513-01.)
FAMILY STUDIES OF LYMPHOCYTE PROLIFERATIVE RESPONSES TO SYNTHETIC POLYPEPTIDES GT, GAT, AND (T,G)-A--L. Young, E., and En@leman, E.G. Stanford University School of Medicine, Stanford, Ca. Despite indirect evidence that immune response (Ir) genes in humans are similar to those found in other species, direct demonstration of such gener is lacking. We have studied the human lymphocyte proliferative responses to synthetic polypeptides in 47 members of 9 families to determine if response patterns could be correlated with H L A h a p l o t y p e and thereby yield direct evidence of the existence of the putative Ir genes of humans. Peripheral blood leucocytes (PBL) were incubated with 1 to 1,000 ug/ml (T,G)-A--L (L-tyrosine, glutamic acid coupled to D,L-alanine on a poly-L-lysine backbone), GAT (copolymer of glutamic acid, alanine and tyrosine), or GT (glutamic acid, tyrosine). After 6 days iD 5% CO 2 at 37Oc, the cultures were pulsed with 3H-thymxdine and harvested 18 hours later. Results indicate familial clustering and suggest a pattern of inheritance because the response patterns of children generally correlated
Abstracts
295
with those of their parents. Clear-cut evidence for a single dominant Ir gene in the HLA complex was not found, but interpretation of the data was difficult because of high frequencies of responder status in the population and the possibility of incomplete penetrance of the putative Ir gene(s).
FDNCTIONAL DIFFERENCES BETWEEN CELLS FROM MLA COLONIES GROWN IN SOFT AGAR CULTURE. A. Zeevi, K.M. Chiu, R.J. Duquesncy. The Blood Center of Southeastern Wisconsin, Milwaukee, Wis. The adaptation of the soft agar technique for growing human T cells has provided the opportunity to examine the initial events of the allogeneic response. We have developed a method of growing soft agar colonies of human T lymphocytes activated in MLC. Two types of colonies were demonstrated. Type I-lower colonies grew within the agar layer and Type II-upper colonies grew on the surface of the agar layer. Three-day priming in MLC and the use of supernatants of 3-day MLC cultures to provide T cell colony growth factor were necessary for optimal colon~ formation. Lymphocytes obtained from colonies were grown in long-term (2-4weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from upper colonies exhibited significantly more PLT activity than cells from lower colonies. (Mean PLT stimulation index: 125 ÷ 50 vs 56 + 36; p < 0.01.) Also showed considerab[~[y h i ~ e r C[iL activity in cells from the upper colonies than cells from lower colonies. (Mean % cytotoxic3ty 36 + 13 vs 6 + 8; p < 0 . 0 0 1 . ) Cells from both types of colonies c ~ t a i n e d radiosensitive suppressor cell activity in primary MLC. The suppressive effect of lower colony cells was specific for the original stimulator. These data suggest that the soft agar colony assays offers a promising approach to separate subpopulations of lymphocytes activated in a MLC. Experiments are in progress to assess functional differences of cells from single colonies. (Supported by Grant AI-12507.)
Annual American Association for Clinical Histocompatibility Testing Meeting, 1980
COMPUTATIONAL PROBLEMS IN SCREENING SERA BY MAXIMUM LIKELIHOOD WHEN RANDOM ERRORS OCCUR. Max A. Woodbury, and David P. Stinson, Division of Biomathematics, Duke University Medical Center, Duke University, Durham, North Carolina, 27710. It has been demonstrated (Nau et al., "A Mathematical Analysis of Human Leukocyte Antigen Serology", Mathematical Biosciences 40: 243-270, 1978) that the combinatorial problem of assigning HLA specificities in 'error free' serum screening is a difficult computational problem (NP hard), i.e., p~l known algorithms for solution require an exponentia. .mount of time according to the size of the input. Here we show that the statistical problem is of equivalent difficulty because of the imbedded combinatorial problem. Practically, we find that the imbedded problem is much reduced in size and relates most to those specificities which appear only on reacting cells. The remainder of the estimation problem, including determination of unreactive specificities and weak/short specificities, can be done in polynomial (less than exponential) time. An algorithm for defining these specificity classes (100% reactive, weak/short, unre4ctive) and for solving the remaining polynomial time problems is presented, along with an experimental computer program that implements this procedure for serum screening. The FORTRAN computer program is available to interested investigators. (Research supported under NIH grant AI-15513-01.)
FAMILY STUDIES OF LYMPHOCYTE PROLIFERATIVE RESPONSES TO SYNTHETIC POLYPEPTIDES GT, GAT, AND (T,G)-A--L. Young, E., and En@leman, E.G. Stanford University School of Medicine, Stanford, Ca. Despite indirect evidence that immune response (Ir) genes in humans are similar to those found in other species, direct demonstration of such gener is lacking. We have studied the human lymphocyte proliferative responses to synthetic polypeptides in 47 members of 9 families to determine if response patterns could be correlated with H L A h a p l o t y p e and thereby yield direct evidence of the existence of the putative Ir genes of humans. Peripheral blood leucocytes (PBL) were incubated with 1 to 1,000 ug/ml (T,G)-A--L (L-tyrosine, glutamic acid coupled to D,L-alanine on a poly-L-lysine backbone), GAT (copolymer of glutamic acid, alanine and tyrosine), or GT (glutamic acid, tyrosine). After 6 days iD 5% CO 2 at 37Oc, the cultures were pulsed with 3H-thymxdine and harvested 18 hours later. Results indicate familial clustering and suggest a pattern of inheritance because the response patterns of children generally correlated
Abstracts
295
with those of their parents. Clear-cut evidence for a single dominant Ir gene in the HLA complex was not found, but interpretation of the data was difficult because of high frequencies of responder status in the population and the possibility of incomplete penetrance of the putative Ir gene(s).
FDNCTIONAL DIFFERENCES BETWEEN CELLS FROM MLA COLONIES GROWN IN SOFT AGAR CULTURE. A. Zeevi, K.M. Chiu, R.J. Duquesncy. The Blood Center of Southeastern Wisconsin, Milwaukee, Wis. The adaptation of the soft agar technique for growing human T cells has provided the opportunity to examine the initial events of the allogeneic response. We have developed a method of growing soft agar colonies of human T lymphocytes activated in MLC. Two types of colonies were demonstrated. Type I-lower colonies grew within the agar layer and Type II-upper colonies grew on the surface of the agar layer. Three-day priming in MLC and the use of supernatants of 3-day MLC cultures to provide T cell colony growth factor were necessary for optimal colon~ formation. Lymphocytes obtained from colonies were grown in long-term (2-4weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from upper colonies exhibited significantly more PLT activity than cells from lower colonies. (Mean PLT stimulation index: 125 ÷ 50 vs 56 + 36; p < 0.01.) Also showed considerab[~[y h i ~ e r C[iL activity in cells from the upper colonies than cells from lower colonies. (Mean % cytotoxic3ty 36 + 13 vs 6 + 8; p < 0 . 0 0 1 . ) Cells from both types of colonies c ~ t a i n e d radiosensitive suppressor cell activity in primary MLC. The suppressive effect of lower colony cells was specific for the original stimulator. These data suggest that the soft agar colony assays offers a promising approach to separate subpopulations of lymphocytes activated in a MLC. Experiments are in progress to assess functional differences of cells from single colonies. (Supported by Grant AI-12507.)