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Functional differences and adenosine deaminase activity in isolated OKT4+ and OKT8+ lymphocytes
145
ACTTVATTON ~V B-CET.L~ TNMYAgTHENIC THYMUR. A. I. Levinson, M.D.~ B. Zweiman~ M.D.~ R. P. Lisak~ M.D.~ A. Dziarski~ M.S. and A. Moskovitz~ B.S.~ Philadelphia~ Pennsylvania The thymus has been discussed as a possible site for autosensitization in myasthenia gravis (MG). Therefore, we studied immunoglobulin (IS) secretion by freshly isolated and pokeweed mitogen (PWM) stimulated thymus cells (Thy) and blood mononuclear cells (BMC) obtained from I) patients with MG and 2) control subjects undergoing elective cardiac surgery. We used a protein A reverse hemolytic plaque assay to enumerate cells secreting IgG, IgM, and IgA (IgSC) and an ELISA assay for measuring IgG secreted into culture supernatants. Significantly higher IgSC levels were present in fresh Thy of 26 MG patients than in 8 controls (means of 1000 vs. 161; p& 0.05). IgSC levels in Thy ~ those in corresponding BMC were found in 8/26 MG patients but in none of t~e controls. Sizable IgSC numbers ( 9 I000/i0 cultured cells) were generated by PWM stimulated cultures of 15/21 MG patients and 4/8 controls with corresponding increases in secreted IgG levels. Of note, IgSC in stimulated Thy cultures w e r e ~ IgSC in BMC cultures in 4 MG patients and 3 controls although B (surface Ig bearing) cells were much less frequent in Thy than BMC (A,_ 1% vs. 8%, respectively) at culture initiation. These findings indicate: i) Thy B cells~ though infrequent, differentiate extremely well into IgSC following stimulation; 2) MG thymus is a site of accentuated in vivo B-cell activation as evidenced by increased numbers of resident IgSC.
147
FUNCTIONAL DIFFERENCES . ~ kDENOSINE DEAMINASE ACTIVITY IN ISOLATED OKT4+ AND OKT8+ LYMPHOCYTES. D . R . Ownby, M.D. and J. A. McCullough, Detroit, Michigan The relationship between adenosine deaminase (ADA) activity and the function of T-lymphocytes (TL) subpopulations was investigated by separating peripheral blood lymphocytes from I0 normal donors into 4 populations. TL were separated from non-TL by En rosette depletion. The purified T L w e r e then separated into OKT4 depleted (T4D) and OKT8 depleted (T8D) subpopulations by reaction with antibody followed by complement lysis. ADA activity was determined for each population. The TL, T4D, and TSD cells were recombined with macrophages and their DNA synthetic responses to Con-A and tetanus toxoid determined. The data are not complete for all measurements. In contrast to previous report~, we found that the ADA activity of non-TL cells was significantly greater than TL (36.7 vs. 25.2, p = .02). Activity was not significantly different, however, between T4D and T8D (22.9 vs. 28.0). There was no evidence that the separation procedures altered the ADA activity. When the DNA synthetic responses are compared, the T8D cells respond significantly better to both Con-A and tetanus toxoid than the T4D cells (Con-A 55.8 vs. 13.6, TT 33.5 vs. 2.1, cpm x 10 -2 , p < .05). The T8D also tended to respond better than the unseparated or TL populations. These results agree with previous renorts of the functional uniqueness of T4D and TSD populations, but this difference is not reflected in the ADA levels in these cells.
146
HUMAN CORD BLOOD (CB) LYMPHOCYTE T HELPER AND SUPPRESSOR PHENOTYPES, IMMUNOGLOBULIN (IG) PRODUCTION, AND EFFECTS ON NORMAL ADULT IG PRODUCTION. P.R. LoGalbo, M.D.~ and R.H. Buckley, M.D., Durham, N.C. In investigating the .~munologic maturity of newborn B cells, 2xi0 blood mononuclear cells (MNC) from 10 normal adults and 10 CB samples were held in I ml cultures for 12 days with and without 10 .o~l pokeweed mitogen. Supernatant IgG was measured by double antibody radioimmunoassay. T-cell subsets were quantified by cytofluorography with monoclonal anti T cell antibodies T3 (pan-T), T4 (helper) and T8 (cytotoxic/suppressor). Geometric mean IgG production by CB samples was less than that by samples from the adults (25 vs 2318 ng/ml, respectively, p
148
NATURE OF THE IMMUNOGENEIC MOEITY RECOGNIZED BY THE HUMAN T CELL PROLIFERATING IN RESPONSE TO TRINITROPHENYL MODIFIED SELF ANTIGENS. R.S. Geha ~ M.D., E.J. Yunis, M.D. and R.L. Siesel, M.D., Boston, MA. Normal human T cells were primed in vitro for 7 days in the presence of TNP modified monocytes, washed then cultured for 48 hours in the presence of fresh TNP modified autologous monocytes (Mo.) and their proliferation was measured. Rabbit antiserum to TNP inhibited T cell proliferation to fresh TNP modified Mo. as well as to TNP modified Mo. which have been aged for 18 hours. This inhibition resulted from masking of TNP determinants and not from clearance of Mo. processed TNP in a bystander fashion nor from the masking of TNP derivatized DR determinants because the proliferative response of fresh T cells to TNP modified tetanus toxoid antigen pulsed Mo. was not affected by the addition of anti TNP. Turkey antiserum to human p 29, 34 but not to human B2 microglobulin inhibited the proliferative response of primed T cells to TNP modified Mo. Furthermore, a DR related gene dose effect was observed when TNP primed T cells were stimulated in the presence of TNP modified Mo. which shared none, one, or two of their DR antigens with T cells. The present data can be interpreted to indicate that the human T cells which proliferate in response to TNP modified self antigens recognize native TNP in association with products of the DR region. Unlike protein antigens such as TT the TNP hapten may not require processing by Mo. to elicit T cell proliferation.
129
ACTTVATTON ~V B-CET.L~ TNMYAgTHENIC THYMUR. A. I. Levinson, M.D.~ B. Zweiman~ M.D.~ R. P. Lisak~ M.D.~ A. Dziarski~ M.S. and A. Moskovitz~ B.S.~ Philadelphia~ Pennsylvania The thymus has been discussed as a possible site for autosensitization in myasthenia gravis (MG). Therefore, we studied immunoglobulin (IS) secretion by freshly isolated and pokeweed mitogen (PWM) stimulated thymus cells (Thy) and blood mononuclear cells (BMC) obtained from I) patients with MG and 2) control subjects undergoing elective cardiac surgery. We used a protein A reverse hemolytic plaque assay to enumerate cells secreting IgG, IgM, and IgA (IgSC) and an ELISA assay for measuring IgG secreted into culture supernatants. Significantly higher IgSC levels were present in fresh Thy of 26 MG patients than in 8 controls (means of 1000 vs. 161; p& 0.05). IgSC levels in Thy ~ those in corresponding BMC were found in 8/26 MG patients but in none of t~e controls. Sizable IgSC numbers ( 9 I000/i0 cultured cells) were generated by PWM stimulated cultures of 15/21 MG patients and 4/8 controls with corresponding increases in secreted IgG levels. Of note, IgSC in stimulated Thy cultures w e r e ~ IgSC in BMC cultures in 4 MG patients and 3 controls although B (surface Ig bearing) cells were much less frequent in Thy than BMC (A,_ 1% vs. 8%, respectively) at culture initiation. These findings indicate: i) Thy B cells~ though infrequent, differentiate extremely well into IgSC following stimulation; 2) MG thymus is a site of accentuated in vivo B-cell activation as evidenced by increased numbers of resident IgSC.
147
FUNCTIONAL DIFFERENCES . ~ kDENOSINE DEAMINASE ACTIVITY IN ISOLATED OKT4+ AND OKT8+ LYMPHOCYTES. D . R . Ownby, M.D. and J. A. McCullough, Detroit, Michigan The relationship between adenosine deaminase (ADA) activity and the function of T-lymphocytes (TL) subpopulations was investigated by separating peripheral blood lymphocytes from I0 normal donors into 4 populations. TL were separated from non-TL by En rosette depletion. The purified T L w e r e then separated into OKT4 depleted (T4D) and OKT8 depleted (T8D) subpopulations by reaction with antibody followed by complement lysis. ADA activity was determined for each population. The TL, T4D, and TSD cells were recombined with macrophages and their DNA synthetic responses to Con-A and tetanus toxoid determined. The data are not complete for all measurements. In contrast to previous report~, we found that the ADA activity of non-TL cells was significantly greater than TL (36.7 vs. 25.2, p = .02). Activity was not significantly different, however, between T4D and T8D (22.9 vs. 28.0). There was no evidence that the separation procedures altered the ADA activity. When the DNA synthetic responses are compared, the T8D cells respond significantly better to both Con-A and tetanus toxoid than the T4D cells (Con-A 55.8 vs. 13.6, TT 33.5 vs. 2.1, cpm x 10 -2 , p < .05). The T8D also tended to respond better than the unseparated or TL populations. These results agree with previous renorts of the functional uniqueness of T4D and TSD populations, but this difference is not reflected in the ADA levels in these cells.
146
HUMAN CORD BLOOD (CB) LYMPHOCYTE T HELPER AND SUPPRESSOR PHENOTYPES, IMMUNOGLOBULIN (IG) PRODUCTION, AND EFFECTS ON NORMAL ADULT IG PRODUCTION. P.R. LoGalbo, M.D.~ and R.H. Buckley, M.D., Durham, N.C. In investigating the .~munologic maturity of newborn B cells, 2xi0 blood mononuclear cells (MNC) from 10 normal adults and 10 CB samples were held in I ml cultures for 12 days with and without 10 .o~l pokeweed mitogen. Supernatant IgG was measured by double antibody radioimmunoassay. T-cell subsets were quantified by cytofluorography with monoclonal anti T cell antibodies T3 (pan-T), T4 (helper) and T8 (cytotoxic/suppressor). Geometric mean IgG production by CB samples was less than that by samples from the adults (25 vs 2318 ng/ml, respectively, p
148
NATURE OF THE IMMUNOGENEIC MOEITY RECOGNIZED BY THE HUMAN T CELL PROLIFERATING IN RESPONSE TO TRINITROPHENYL MODIFIED SELF ANTIGENS. R.S. Geha ~ M.D., E.J. Yunis, M.D. and R.L. Siesel, M.D., Boston, MA. Normal human T cells were primed in vitro for 7 days in the presence of TNP modified monocytes, washed then cultured for 48 hours in the presence of fresh TNP modified autologous monocytes (Mo.) and their proliferation was measured. Rabbit antiserum to TNP inhibited T cell proliferation to fresh TNP modified Mo. as well as to TNP modified Mo. which have been aged for 18 hours. This inhibition resulted from masking of TNP determinants and not from clearance of Mo. processed TNP in a bystander fashion nor from the masking of TNP derivatized DR determinants because the proliferative response of fresh T cells to TNP modified tetanus toxoid antigen pulsed Mo. was not affected by the addition of anti TNP. Turkey antiserum to human p 29, 34 but not to human B2 microglobulin inhibited the proliferative response of primed T cells to TNP modified Mo. Furthermore, a DR related gene dose effect was observed when TNP primed T cells were stimulated in the presence of TNP modified Mo. which shared none, one, or two of their DR antigens with T cells. The present data can be interpreted to indicate that the human T cells which proliferate in response to TNP modified self antigens recognize native TNP in association with products of the DR region. Unlike protein antigens such as TT the TNP hapten may not require processing by Mo. to elicit T cell proliferation.
129