- Email: [email protected]
Relevance of NKCF to NK cells mediated cytolysis
Abstracts
109
adherent cells. This phenotypic difference was stable since it was detectable for up to 92 hr in culture. While the most effective APC expressed high levels of D Q and DR, they did not express higher amounts of other surface antigens such as Leu M3 (monocyte marker) and LFA-3. These results suggest that quantitative expression of D Q / D R molecules on APC may be critical in determining their ability to activate T cell responses to nominal antigens.
ANTIGEN-SPECIFIC FUNCTIONS OF CD1 1 +/CD4 ~ HUMAN T LYMPHOCYTES WITH GRANULAR MORPHOLOGY. Yoshihisa Morishita, Paul J. Martin, Michael A. Bean, Hironori Yamada, and John A. Hansen; Fred Hutchinson Cancer Research Center, Seattle, WA We have previously shown that the C D 4 + subset of human lymphocytes can be divided into reciprocal, nonoverlapping populations of cells that express either C D 11 or a marker recognized by antibody 9.3 and designated Tp44. The present investigation was undertaken in order to determine the phenotypic and functional characteristics of C D 11 +/CD4 + cells. Cells of the C D 11 ÷/CD4 + population had the h o m o g e n e o u s m o r p h o l o g y of granular lymphocytes with scattered cytoplasmic granules and a moderately developed Golgi apparatus. Despite their granular morphology, C D 11 +/CD4 + cells had no spontaneous cytotoxic activity against K562 cells. T h e C D 11 ÷/CD4 ÷ cells uniformly expressed CD3 and could be induced to express IL-2 receptors by activation with P H A or anti-CD3 antibody. C o m p a r e d to unfractionated T cells, however, the C D 11 ÷/CD4 ÷ granular lymphocytes had a reduced ability to produce IL-2 after stimulation with P H A or anti-CD3 antibody. T h e CD11 ÷/CD4 ÷ population showed specific allocytotoxicity after stimulation with allogeneic cells and culture in IL-2. The CD11 +/CD4 ÷ cells also responded to tetanus toxoid and PPD. These findings suggest that some cells of the C D 11 +/CD4 ÷ population are capable of antigen-specific responses. We conclude that certain CD11 * cells with granular morphology have functions closely similar to those of bona fide T lymphocytes.
RELEVANCE OF NKCF TO NK CELL MEDIATED CYTOLYSIS. Zacharie Brahmi and Grace Hommel; Indiana University School of Medicine, Indianapolis, IN Natural killer cells (NK) are a heterogeneous population of lymphoid cells, usually large granular lymphocytes (LGL), that are able to lyse certain t u m o r target cells (TC) and virally infected cells through a mechanism apparently unrestricted by the m a j o r histocompatibility complex. Several mechanisms involving soluble factors released from N K cells such as N K C F , polyporphorins, and cytolysins from granules have been implicated in the lytic event but the relevance of these factors to N K - m e d i a t e d cytotoxicity ( N K - C M C ) remains unclear. W h e n comparing the ability of two NK-sensitive TC to induce the release of N K C F , greater amounts of N K C F were produced in response to K562 than to U937, although N K C F generated by these two T C was consistently m o r e potent against U937 than K562. C o n A and two NK-insensitive cell lines (Sp2/0 and MLA) were also able to stimulate the release of N K C F , although both TC do not bind N K cells. Surprisingly, ab-coated T C did not generate m o r e N K C F and N K C F was as potent against uncoated as ab-coated TC. Furthermore, contrary to previous reports, Ca 2 + is not required for the release of N K C F but is required for the lyric activity mediated by N K C F . Moreover, when N K cells were incubated in the presence of K562, U937, or C o n A in complete or serum-free medium for up to 24 hr, the N K cells were no longer able to lyse K562 or U937, suggesting that N K cells, following an interaction with a sensitive TC, "exhaust" their lytic potential. This loss of activity is not due to cell death, lack of binding, or loss of N K markers
110
Annual AACHT Meeting, 1985 and the inactivated N K cells regain their full activity following treatment with IL-2. It is notable that the loss of N K - C M C was concomitant with the loss ot A D C C , confirming earlier reports from our laboratory and those o f others that N K - C M C and ADCC are mediated by overlapping cell populations. In summary. we showed that: (1) N K C F can be released by a variety of TC in a calcium-free medium although NKCF-mediated lysis is calcium dependent, (2) following interaction with a sensitive TC but not a resistant one, N K exhaust their lytic potential although both types of TC lead to the production of NKCF; (3) tht: production of N K C F is not accompanied by degranulation of LGL suggesting that N K C F is probably different from cytolysin of granules.
IDENTIFICATION OF ALLOANTIGEN-SPECIFIC CTL 1N HUMAN PERIPHERAL BLOOD BY LIMITING DILUTION ANALYSIS. Patrick W. Adams, Eva A. Pagel, and Charles G. Orosz: The Ohio State University, Columbus, OH Several qualitative techniques (MLR, PLT, CML) are widely used in clinical histocampatibility testing laboratories for analysis of cellular alloreactivity. However, quantitative analytic techniques such as limiting dilution analysis (LDA), have been lacking in human transplant immunology. We have developed LDA culture conditions suitable for the study of peripheral blood mononuclear cells (PBMNC) which can develop specific cytolytic activity against sensitizing ceils bearing defined HLA alloantigens. For LDA, various numbers of responding P B M N C (ranging from 20,000 to 300 cells/well) are added to groups ,~f 2.i replicate U-bottom microtiter wells containing a constant number ¢2.4 x 10 ~ cells/welt) of allogeneic, irradiated P B M N C and an optimal concentrati~on of commercially available interleukin-2 (IL-2). Microcultures are fed at ~-day lw tervals with additional IL-2-containing medium. After 7 days, the microculturc contents are transferred to V-bottom microtiter wells along with 5 x 1()~ ~Crlabeled, PHA-activated allogeneic P B M N C targets. The 5~Cr released in each well over the 4-hr incubation period is assessed and minimal estimates of CTL precursor frequencies are calculated according to the Poisson distribution e q u a tions as the slope of a line relating the number of responding cells/well and the percentage o f microwells which failed to develop cytolytic activity. Using thi~ technique we observed that approximately one cell/2000 P B M N C from normal healthy donors could develop cytotoxicity against fully HLA mismatched PBMNC targets. Optimal detection of cytolytic T cells in limiting dilution microculturus required the presence of both stimulators and exogenous IL-2 during the " days o f incubation. Cytolytic activity was specific for stimulator HLA antigens sinct ~ there was little or no lytic activity directed at autologous or nonrelated P B M N C targets. Further, the frequency of precursor cells determined for a particular responder against the same stimulator performed on three different occasions over an interval of 6 weeks yielded essentially the same frequency. This LDA technique appears to be a sensitive, reproducible method for quantitating T celI alloreactivity. As a clinical application, this LDA technique should also allow the' quantitative monitoring of donor-reactive CTL in the peripheral blood o f organ transplant recipients. DEVELOPMENT OF THE DONOR-REACTIVE CTL POPULATION IN SPONGE ALL()GRAFTS MONITORED BY LIMITING DILUTION ANALYSIS. Charles G. Orosz, Nancy E. Zinn, Lawrence P. Sirinek, and Ronald M. Ferguson; The Ohio State Unizersity, Columbu.i, Ott We have used sponge matrix allografts to study the dynamics of cytotoxic T cell (CTL) activity at sites of engraftment. C57BL/6 (H-2 b) mice were implanted with polyurethane sponges, some of which were injected with C57BL/6 or DBA/2 (H-2 a) splenocytes. These mice reject D B A / 2 skin grafts within 10 days. Sponge~
109
adherent cells. This phenotypic difference was stable since it was detectable for up to 92 hr in culture. While the most effective APC expressed high levels of D Q and DR, they did not express higher amounts of other surface antigens such as Leu M3 (monocyte marker) and LFA-3. These results suggest that quantitative expression of D Q / D R molecules on APC may be critical in determining their ability to activate T cell responses to nominal antigens.
ANTIGEN-SPECIFIC FUNCTIONS OF CD1 1 +/CD4 ~ HUMAN T LYMPHOCYTES WITH GRANULAR MORPHOLOGY. Yoshihisa Morishita, Paul J. Martin, Michael A. Bean, Hironori Yamada, and John A. Hansen; Fred Hutchinson Cancer Research Center, Seattle, WA We have previously shown that the C D 4 + subset of human lymphocytes can be divided into reciprocal, nonoverlapping populations of cells that express either C D 11 or a marker recognized by antibody 9.3 and designated Tp44. The present investigation was undertaken in order to determine the phenotypic and functional characteristics of C D 11 +/CD4 + cells. Cells of the C D 11 ÷/CD4 + population had the h o m o g e n e o u s m o r p h o l o g y of granular lymphocytes with scattered cytoplasmic granules and a moderately developed Golgi apparatus. Despite their granular morphology, C D 11 +/CD4 + cells had no spontaneous cytotoxic activity against K562 cells. T h e C D 11 ÷/CD4 ÷ cells uniformly expressed CD3 and could be induced to express IL-2 receptors by activation with P H A or anti-CD3 antibody. C o m p a r e d to unfractionated T cells, however, the C D 11 ÷/CD4 ÷ granular lymphocytes had a reduced ability to produce IL-2 after stimulation with P H A or anti-CD3 antibody. T h e CD11 ÷/CD4 ÷ population showed specific allocytotoxicity after stimulation with allogeneic cells and culture in IL-2. The CD11 +/CD4 ÷ cells also responded to tetanus toxoid and PPD. These findings suggest that some cells of the C D 11 +/CD4 ÷ population are capable of antigen-specific responses. We conclude that certain CD11 * cells with granular morphology have functions closely similar to those of bona fide T lymphocytes.
RELEVANCE OF NKCF TO NK CELL MEDIATED CYTOLYSIS. Zacharie Brahmi and Grace Hommel; Indiana University School of Medicine, Indianapolis, IN Natural killer cells (NK) are a heterogeneous population of lymphoid cells, usually large granular lymphocytes (LGL), that are able to lyse certain t u m o r target cells (TC) and virally infected cells through a mechanism apparently unrestricted by the m a j o r histocompatibility complex. Several mechanisms involving soluble factors released from N K cells such as N K C F , polyporphorins, and cytolysins from granules have been implicated in the lytic event but the relevance of these factors to N K - m e d i a t e d cytotoxicity ( N K - C M C ) remains unclear. W h e n comparing the ability of two NK-sensitive TC to induce the release of N K C F , greater amounts of N K C F were produced in response to K562 than to U937, although N K C F generated by these two T C was consistently m o r e potent against U937 than K562. C o n A and two NK-insensitive cell lines (Sp2/0 and MLA) were also able to stimulate the release of N K C F , although both TC do not bind N K cells. Surprisingly, ab-coated T C did not generate m o r e N K C F and N K C F was as potent against uncoated as ab-coated TC. Furthermore, contrary to previous reports, Ca 2 + is not required for the release of N K C F but is required for the lyric activity mediated by N K C F . Moreover, when N K cells were incubated in the presence of K562, U937, or C o n A in complete or serum-free medium for up to 24 hr, the N K cells were no longer able to lyse K562 or U937, suggesting that N K cells, following an interaction with a sensitive TC, "exhaust" their lytic potential. This loss of activity is not due to cell death, lack of binding, or loss of N K markers
110
Annual AACHT Meeting, 1985 and the inactivated N K cells regain their full activity following treatment with IL-2. It is notable that the loss of N K - C M C was concomitant with the loss ot A D C C , confirming earlier reports from our laboratory and those o f others that N K - C M C and ADCC are mediated by overlapping cell populations. In summary. we showed that: (1) N K C F can be released by a variety of TC in a calcium-free medium although NKCF-mediated lysis is calcium dependent, (2) following interaction with a sensitive TC but not a resistant one, N K exhaust their lytic potential although both types of TC lead to the production of NKCF; (3) tht: production of N K C F is not accompanied by degranulation of LGL suggesting that N K C F is probably different from cytolysin of granules.
IDENTIFICATION OF ALLOANTIGEN-SPECIFIC CTL 1N HUMAN PERIPHERAL BLOOD BY LIMITING DILUTION ANALYSIS. Patrick W. Adams, Eva A. Pagel, and Charles G. Orosz: The Ohio State University, Columbus, OH Several qualitative techniques (MLR, PLT, CML) are widely used in clinical histocampatibility testing laboratories for analysis of cellular alloreactivity. However, quantitative analytic techniques such as limiting dilution analysis (LDA), have been lacking in human transplant immunology. We have developed LDA culture conditions suitable for the study of peripheral blood mononuclear cells (PBMNC) which can develop specific cytolytic activity against sensitizing ceils bearing defined HLA alloantigens. For LDA, various numbers of responding P B M N C (ranging from 20,000 to 300 cells/well) are added to groups ,~f 2.i replicate U-bottom microtiter wells containing a constant number ¢2.4 x 10 ~ cells/welt) of allogeneic, irradiated P B M N C and an optimal concentrati~on of commercially available interleukin-2 (IL-2). Microcultures are fed at ~-day lw tervals with additional IL-2-containing medium. After 7 days, the microculturc contents are transferred to V-bottom microtiter wells along with 5 x 1()~ ~Crlabeled, PHA-activated allogeneic P B M N C targets. The 5~Cr released in each well over the 4-hr incubation period is assessed and minimal estimates of CTL precursor frequencies are calculated according to the Poisson distribution e q u a tions as the slope of a line relating the number of responding cells/well and the percentage o f microwells which failed to develop cytolytic activity. Using thi~ technique we observed that approximately one cell/2000 P B M N C from normal healthy donors could develop cytotoxicity against fully HLA mismatched PBMNC targets. Optimal detection of cytolytic T cells in limiting dilution microculturus required the presence of both stimulators and exogenous IL-2 during the " days o f incubation. Cytolytic activity was specific for stimulator HLA antigens sinct ~ there was little or no lytic activity directed at autologous or nonrelated P B M N C targets. Further, the frequency of precursor cells determined for a particular responder against the same stimulator performed on three different occasions over an interval of 6 weeks yielded essentially the same frequency. This LDA technique appears to be a sensitive, reproducible method for quantitating T celI alloreactivity. As a clinical application, this LDA technique should also allow the' quantitative monitoring of donor-reactive CTL in the peripheral blood o f organ transplant recipients. DEVELOPMENT OF THE DONOR-REACTIVE CTL POPULATION IN SPONGE ALL()GRAFTS MONITORED BY LIMITING DILUTION ANALYSIS. Charles G. Orosz, Nancy E. Zinn, Lawrence P. Sirinek, and Ronald M. Ferguson; The Ohio State Unizersity, Columbu.i, Ott We have used sponge matrix allografts to study the dynamics of cytotoxic T cell (CTL) activity at sites of engraftment. C57BL/6 (H-2 b) mice were implanted with polyurethane sponges, some of which were injected with C57BL/6 or DBA/2 (H-2 a) splenocytes. These mice reject D B A / 2 skin grafts within 10 days. Sponge~