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Participation of Different Cell Populations in Antigen- and Mitogen-Induced Lymphocyte Proliferation. Evidence That Blastogenic Factor-Sensitive Lymphocytes Are Inactivated by 5-Bromo-2'-Deoxyuridine¹

From the Division of Immunology and Microbiology, American Dental Association Research Institute, 211 East Chicago Avenue, Chicago, Illinois 60611

Abstract

The nature of the subpopulations of lymphocytes which respond by blast transformation to stimulation by specific antigens and phytohemagglutinin (PHA) was investigated. Leukocytes from rhesus monkeys sensitized to keyhole limpet hemocyanin (KLH) and purified protein derivative from Mycobacterium tuberculosis culture filtrates (PPD) were stimulated with KLH or PPD in culture. The replacement of medium and the readdition of the same antigen on day 4 did not initiate a second round of DNA synthesis. The addition of a second antigen, however, markedly increased the ³H-thymidine (³H-T) incorporation within 1 day. However, no additive stimulatory effect occurred after simultaneous stimulation with KLH and PPD, possibly indicating competition for a limited cell population.

The lymphocyte proliferative response to KLH and PPD stimulation could be inhibited as much as 95% by treatment from days 2 to 4 with 5 x 10^-6 M 5-bromo-2'-deoxyuridine (BrdU) and exposure to visible light. Such treatment also markedly reduced the expected response of the cultures to restimulation with a second antigen, suggesting that the observed response to both antigens was dependent upon the same BrdU-sensitive cells. BrdU-light inactivated cultures lost most of their capacity to respond to blastogenic factor. These results were interpreted as indicating that: 1) a large proportion of the BrdU-sensitive cells are blastogenic factor-reactive and 2) distinct subpopulation of cells is not recruited by blastogenic factor produced by specific antigen-sensitive lymphocytes.

Peak incorporation of ³H-T in antigen-pretreated cultures, when stimulated with PHA, occurred later than in cultures stimulated by a second antigen, suggesting that PHA was stimulating a new population of cells. Moreover, an additive stimulatory effect occurred when PHA and KLH or PHA and PPD were added to cultures simultaneously. BrdU-light treatment of antigen-stimulated lymphocytes had little effect on subsequent stimulation by PHA. Antigen and PHA stimulation, therefore, may involve different cell populations.

Footnotes

¹ This work was supported by United States Public Health Service Grant AI 09126 from the National Institutes of Health. This work was presented in part at the 1973 FASEB meeting, Atlantic City, New Jersey.

² Recipient of United States Public Health Service Career Development Award DE-70123-01 from the National Institute of Dental Research.

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