Tissue Antigens (1974), 4, 106--114 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s) ‘The Use of in vitro Lymphocyte Responsiveness to Specific Mitogenic Agents in the Immunological Monitoring of Human Renal Allograft Recipients DAVID COPELAND, ABDUR RAsnIv, THOMAS STEWART ANL) JULES HARRIS Laboratory of Irrirriunology and the Renal Metabolic Unit, Department of Medicine, University of Ottawa and Ottawa General Hospital, Ottawa, Canada In the course of immunologically monitoring the clinical progress of 16 renal allograft re- cipients, blood was drawn at serial intervals and patient in uitro lymphocyte reactivity to streptolysin-0 and streptokinase-streptodornase studied. In uitro lymphocyte responsiveness to these specific mitogens provided no consistent guide to the adequacy of immunosuppression for maintenance of stable renal function. Variations in responsiveness gave no indication of either impending or actual rejection crises. Short-term culture (4 h ) assessment of phyto- hemagglutinin responsiveness using the ZH-uridine method also failed to provide clinically useful information for these purposes. We conclude that conventional specific and non-specific mitogenic assays of lymphocyte functional integrity cannot be used to evaluate either the effectiveness of immunosuppressive medication or the development of rejection episodes. Received for publication 26 June, accepted 13 Novembtv 1973 A variety of tests have been proposed to regulate the dosage of those immunosup- pressive drugs used to prevent rejection crises in renal allograft recipients (Ham- burger 1972, Pag6 et al. 1971, Posen et al. 1971). The in uitro response of lympho- cytes to the non-specific mitogen phyto- heniagglutinin (PHA) has come to be re- garded as one index of the normal immu- nological functioning of those cells (Bach & Hirschhorn 1965). The serial monitor- ing of PHA-reactivity in allografts reci- pients might be expected to provide some guidance as to the adequacy of immuno- Presented in part at the 42nd Annual Meeting of the Royal College of Physicians and Sur- geons of Canada, Edmonton, Alberta, Canada, January 24-27, 1973. This work was supported by the Medical Research Council of Canada Operating Grant MA-3902.
Transcript
Tissue Antigens (1974), 4, 106--114
Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)
‘The Use of in vitro Lymphocyte Responsiveness to Specific Mitogenic Agents in
the Immunological Monitoring of Human Renal Allograft Recipients
DAVID COPELAND, ABDUR RAsnIv, T H O M A S STEWART ANL) JULES HARRIS
Laboratory of Irrirriunology and the Renal Metabolic Unit, Department of Medicine,
University of Ottawa and Ottawa General Hospital, Ottawa, Canada
In the course of immunologically monitoring the clinical progress of 16 renal allograft re- cipients, blood was drawn at serial intervals and patient in uitro lymphocyte reactivity to streptolysin-0 and streptokinase-streptodornase studied. I n uitro lymphocyte responsiveness to these specific mitogens provided no consistent guide to the adequacy of immunosuppression for maintenance of stable renal function. Variations in responsiveness gave no indication of either impending or actual rejection crises. Short-term culture (4 h ) assessment of phyto- hemagglutinin responsiveness using the ZH-uridine method also failed to provide clinically useful information for these purposes. We conclude that conventional specific and non-specific mitogenic assays of lymphocyte functional integrity cannot be used to evaluate either the effectiveness of immunosuppressive medication or the development of rejection episodes.
Received for publication 26 June, accepted 13 Novembtv 1973
A variety of tests have been proposed to regulate the dosage of those immunosup- pressive drugs used to prevent rejection crises in renal allograft recipients (Ham- burger 1972, Pag6 et al. 1971, Posen et al. 1971). The in uitro response of lympho- cytes to the non-specific mitogen phyto-
heniagglutinin (PHA) has come to be re- garded as one index of the normal immu- nological functioning of those cells (Bach & Hirschhorn 1965). The serial monitor- ing of PHA-reactivity in allografts reci- pients might be expected to provide some guidance as to the adequacy of immuno-
Presented in part a t the 42nd Annual Meeting of the Royal College of Physicians and Sur- geons of Canada, Edmonton, Alberta, Canada, January 24-27, 1973. This work was supported by the Medical Research Council of Canada Operating Grant MA-3902.
1MMUNOI.OGICAL MONITORING WITH SPECIFIC MITOGENIC AGENTS 107
suppressive medication. We have previous- ly reported observations showing that this test is of no practical value in clinical transplantation except in circumstances where the phenomenon of “PHA escape” occurs (PagC et al. 1971, Harris et al. 1972). “PHA escape” refers to a situation in which, when a rejection crisis develops, lymphocytes may recover their normal re- sponse to PHA in the face of a dose of im- munosuppressive drugs which previously suppressed them. In this report we give an account of our experience in attempting to develop a more rapid in vitro assay for PHA responsiveness in the hope of iden- tifying “PHA escape” and associated rejec- tion crises on the actual day on which they occur. Our previous studies have involved 5 day incubation periods with PHA deter- minations becoming available between 5 and 7 days after overt clinical rejection had become manifest. We also evaluated the usefulness of following the in vitro lym- phocyte responses to the specific mitogens streptolysin-0 (SLO) and streptokinase- streptodornase (Varidase) as a means of identifying rejection crises and of modu- lating dosages of immunosuppressive drugs to obtain immunological quiescence. We postulated that successful suppression of this parameter of lymphocyte function might be associated with a more benign and immunologically subdued clinical course.
Materials and Methods Patients and Controls The patient group was comprised of 16 renal allograft recipients - 12 males and four females. Their ages ranged from 17 to 52 years, the median being 25 years. All had a pregraft diagnosis of chronic glome- rulonephritis and all had received cadaver kidneys. All patients investigated were free of infection at the time of study. ’
The control population, for whom our normal laboratory range values are cited, consisted of 42 subjects - 22 male and 20 female - chosen mainly from our hospital staff. Their median age was 29 years (range from 11 to 50 years). Control sub- jects were on no medication when studied immunologically and were selected to ex- clude persons with allergies or bacterial and viral infection.
Lymphocyte Culture Blood for use in the study was drawn into a syringe premoistened with preservative- free heparin (Heparin Sodium, United States Pharmacopeia, Fischer, Ottawa, Ca- nada). The blood was then mixed and se- dimented with 2 ml dextran/lO ml of blood (dextran 6 % w./vol. in saline, Ab- bott, North Chicago, Illinois) a t 37’ C for 1 h. The white cell rich plasma was then drawn off and used to set up cultures com- prised of 106 lymphocytes in 13X 100 mm Pyrex glass round bottom-screw top tubes together with 1 ml of autochthonous plas- ma and 2 ml of Eagle’s spinner modified minimum essential medium (Grand Island Biologicals, Grand Island, New York) , sup- plemented with 20 mM/1 of glutamine and penicillin-streptomycin solution. For PHA studies cultures were stimulated with 0.05 1111 of PHA (PHA-M, Difco Laboratories, Detroit, Michigan). These were either ( i ) incubated for 5 days in 5 % C 0 2 in air at 37” C and harvested for liquid scintillation counting after a terminal 3 h incubation with 2 pc of 3H-thymidine, specific activ- ity 6.7 cJmM (New England Nuclear Corp., Montreal, Quebec) ; or ( i i ) har- vested at 1 to 4 h intervals after being pulsed at the start of culture with 2 pc 3H-uridine, specific activity 26.0 c/mM (New England Nuclear Corp., Montreal, Quebec). The method of harvest and the technique of processing for liquid scintilla- tion counting has been published previous-
108 COPEI.AND ET AL.
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ly (PagC et al. 1971). For studying specific mitogen responses, individual 3 ml cultures containing 106 lymphocytes were stim- ulated with either 0.1 ml SLO (Difco Lab- oratories, Detroit, Michigan) or Varidase (Lederle, Pearl River, New York; prepara- tion reconstituted with 2 ml saline and used following 24 h dialysis). These cul- tures were incubated for 5 days at 37’ C in an atmosphere of 5 ”/. C 0 2 in air and handled in the same way as those cultures with PHA harvested after a 5-day incuba- tion period. For both specific and non- specific mitogen-stimulated cultures, un- stimulated control determinations were subtracted from the results of stimulated cultures to give a final value. Results are expressed as counts per min (cpm) per 106 lymphocytes.
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Results Specific Mitogen Stimulation Fig. 1 shows results obtained with SLO and Varidase studies 1 week before, during and 1 week after 1 1 investigated rejection
crises. A rejection crisis was diagnosed in renal allograft patients when the serum creatinine showed a rise of greater than 0.2 mg/100 ml (PagC et al. 1971). Values are reported as cpm/l06 lymphocytes. The vertical bars in the figure give ranges; the horizontal lines through the bars show me- dian values. The normal values for our laboratory are for SLO: median response 45,835 cpm (range: 4,769 cpm-204,337 cpm) and for Varidase: median response 39,O 12 cpm (range: 4,693 cpm-266,477 cpm). The lower limit for our laboratory is indicated in Fig. 1 by the small broken line at the upper end of the bar. There was no significant rise or fall in count before, during or after rejection. Varidase values were: at 1 week before rejection, median 380 cpni (range: 0-27,509 cpm), at rejec- tion, median 212 cpm (range: 0-52,445 cpm) and 1 week after rejection, median 495 cprn (range: 0-1,878 cpm). SLO val- ues were: a t 1 week before rejection, me- dian 1,135 cpm (range: 0-68,429 cpm), at rejection, median 3,750 cpm (range: 0- 82,044 cpm) and 1 week after rejection,
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-1 W E E K R E J E C T I O N t l W E E K Figure I . In vitro lymphocyte responses to the specific mitogens strepto- lysin-0 and streptokinase-streptodornase before, during and after 11 rejec- tion crises.
[MMUNOLOGICAL MONITORING WITH SPECIFIC MITOGENIC AGENTS I09
1,263 cpm (range: 0-11,937 cpm). These determinations, then, provided no immu- nological evidence of impending rejection crises. There was no significant elevation or depression of responsiveness at the time of rejection or in the interval prior to' re- jection which might indicate unusual lym- phocyte reactivity. I t was noteworthy, too, that the management of rejection crises with actinomycin D and increased doses of steroid did not significantly affect respon- siveness to SLO and Varidase in the 1- week period following rejection.
Fig. 2 depicts a 12-week portion of the clinical course of patient R.P. Renal func- tion remained unimpaired during this pe- riod while the patient was receiving a con- stant 250 mg of azathioprine and 20 mg of prednisone per day. Despite this dosage of
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immunosuppressive medication, Varidase and SLO responsiveness, shown in the top two panels of the Figure, remained sub- stantially within the normal range. Values charted can be seen to lie above our lab- oratory's lower limit of normal for these determinations and are approximately dis- tributed about our normal median values. These in vitro parameters of immune func- tion, therefore, could not have been used to adjust the dosage of immunosuppressive medication,
Fig. 3 graphs a 12-week period during the irnmunologically quiescent course of patient Y.L. The dosage of prednisone va- ried between 20 and 40 mg per day and that of azathioprine varied between 50 and 100 mg per day. O n this dosage of medi- cation there was some fluctuation in SLO
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Figure 2. Clinical course of patient R.P. illustrating fluctuations in lympho- cyte reactivity on constant doses of dzathioprine and prednisone.
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Figure 3. Clinical course of patient Y.L. illustrating fluctuating suppression of lymphocyte reactivity to specific mitogens SLO and Varidase.
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Figu7e 4 . Clinical course of patient G.M. illustrating lack of correlation between in uitro IyIiipliwyte responsiveness to specific and non-specific mitogens.
IMMUNOLOGICAL MONITORING WITH SPECIFIC MITOGENIC AGENTS 111
F.L. 45,000 40,000 VARIDASE RESPONSE
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Figure 5 . Clinical course of patient F.L. illustrating lack of correlation between in uitro responsiveness to specific and non-specific mitogens.
and Varidase responsiveness but all but one of the results obtained during the time of study were below the lower limit of normal. This might, perhaps, have been taken as an indication that a correct and effective dose of drug was being adminis- tered, but we offer it as an example of the extreme variability of responsiveness that we have recorded i.e. counts both high and low on varying doses of azathioprine and prednisone, offering no consistent guide to dose adjustment.
Nor is there any constant or predictable concordance between responsiveness to specific and non-specific rnitogenic agents. Fig. 4 illustrates the in uitro lymphocyte reactivity of patient G.M. who received 150 mg azathioprine and 30 mg prednisone prr day, while being investigated over a
12 week period. Lower normal limits for the rnitogenic responses charted are given by the dotted line in each panel. I n our laboratory normal median PHA respon- siveness for 106 lymphocytes is 44,376 cprn (range: 20,342 cpm-l12,510 cpm). There is no clearly defined pattern of responsive- ness despite a constant dose schedule of immunosupressive drugs and only infre- quently are all three determinations either elevated or depressed. In any case, the values obtained provided no information which could be usefully applied to drug dose adjustment.
A similar situation is depicted in Fig. 5 which traces the clinical course of patient F.L., who was maintained on a fixed dose of 150 mg azathioprine and 20 mg predni- sone per day over a 12-week interval free
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of rejection episodes. Once more there is a fluctuating variability in responsiveness which offers no guidance for manipulation of dose scheduling.
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Response to PHA In the course of these investigations we sought to develop a rapid in vitro assay for PHA responsiveness. We have been able to show that the 4 h uptake of 3H-uridine following PHA stimulation gives a useful measure of lymphocyte responsiveness to this mitogen. This work is based on origi- nal studies done by Forsdyke ( 1967, 1968a, 1968b), Data illustrating the assay are charted in Fig. 6. The ordinate gives cpm/ 106 lymphocytes - the abscissa, the time in hours. The horizontal bars are drawn through median values. These are at 1 h
158 cpm (range : 34 cpm-243 cpm) , at 2 h 250 cpm (range: 69 cpm-515 cpm), at 3 h 413 cpm (range: 176 cpm423 cpm) and at 4 h 555 cpm (range: 389 cpm-1,219 cpm). Each value graphed represents the median of triplicate determinations. Varia- tion is t 10 (r. Background counts are subtracted in each case. We had, then, a reasonably accurate 4 h measure of PHA reactivity, the results of which could be available on the day of study, and we sought next to utilize the test in the hope of obtaining a rapid identification of “PHA escape” and hence of rejection crises.
Unfortunately, the 4 h assay does not appear to be useful for this purpose. Fig. 7 shows 4 h determinations obtained during these rejection crises. The left hand panel graphs 15 observations obtained with three
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F i g w e 6 . Four-hour in z d r o lymphocyte uptake of sH-uridine following PHA stimulation.
IMMUNOLOGICAL MONITORING WITH SPECIFIC MITOGENIC AGENTS
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R E J E C T I O N Figure 7. Four-hour 3H-uridine uptake studies following PHA stimulation before and at the time of rejection episodes.
patients during the 2 to 3 months period prior to reiection. The range of values is
Table 1 PHA determinations
median of these. The right hand panel charts 11 values recorded during the actual time of rejection before increases were made in immunosuppressive drugs. The central panel shows the normal response. There is no significant difference between PHA reactivity before and at the time of rejection episodes; median responsiveness prior to the time of rejection is 303 cpm (range: 156 cpm-888 cpm) and at the actual time of rejection the median respon- se is 475 cpm (range: 145 cpm-1,020 cpm).
A possible explanation for the failure of the SH-uridine method to detect “PHA escape” may lie in the lack of correlation between 4-hour 3H-uridine counts and 5-day 3H-thymidine counts following PHA stimulation. In one column Table 1 lists in
a Control value subtracted from total counts to give final cpms cited.
rank order PHA stimulation results ob- tained in nine normal subjects with the 3H-uridine method. Again these represent the median of triplicate determinations. Opposite each value is the corresponding 3H-thymidine count obtained after 5 days of incubation. Cultures were set up a t the
114 COPEI.AND ET AL.
same time from the same subject. These counts, too, are the medians of triplicate values. The 3H-thymidine results do' not follow the same rank order as for 3H-uri- dine. The fluctuation in individual uridine control values may also affect these results.
DiJcussion These experiments were undertaken to de- termine whether perturbation (elevation or depression) in lymphocyte reactivity might herald impending rejection crises. Some recent work in a different system has sug- gested that mixed lymphocyte reactivity may fall off in the period preceding a re- jection episode (Hattler & Miller 1972). Our results show that patient lymphocyte reactivity to antigens, to which there is established in v i t ro sensitivity, fluctuate under the influence of immunosuppressive medication. There is no clearly defined pattern to these oscillations. The cases used to illustrate this finding were selected among patients who, for the most part, had been on fixed doses of immunosuppressive drugs for periods of up to 3 months. This was done to minimize the possible effect of dose change on lymphocyte activity. The cases still demonstrate considerable and unpredictable alterations in lymphocyte function. These have no discernible pat- tern and are without value for the purpose of modulating the doses of irnmunosup- pressive drugs.
The monitoring of PHA reactivity has only a limited use in clinical transplanta- tion. Our attempts to develop a rapidly rc- portable assay for this index of normal lymphocyte function failed to provide a practical test system for clinical applica- tion.
Our results taken together lead us to the conclusion that there is no benefit in ap- plying conventional in v i t ro methods for the measurement of lymphocyte function to the immunological monitoring of trans-
plant patients. I t may be, furthermore, that these will provide no guide for the applica- tion of immunosuppressive drugs in the management of autoimmune diseases.
References Bach, F. H. & Hirschhorn, K. (1965) The in
vitro immune response of peripheral blood lymphocytes. Sem. Hemat. 2, 68-89.
Forsdyke, D. R. (1967) Quantitative nucleic acid changes during phytohemagglutinin- induced lymphocyte transformation in vitro. Dependence of the response on phytohemag- glutinin-serum rate. Riochem. J . 105, 679-684.
Forsdyke, D. R. (1968a) Studies on the incor- poration of (5-3H) uridine during activation and transformation of lymphocytes induced by phytohemagglutinin. Dependence on the in- corporation rate on uridine concentration a t certain critical concentrations. Biochem. J . 107, 197-205.
Forsdyke, D. R. (1968b) Incorporation of (5- 3 H ) uridine and attachment of cells to glass during activation of lymphocytes induced by phytohemagglutinin. Biochem. J . 108, 297.- 302.
Hamburger, J . ( 1972) The inirnunological follow-up of renal allograft recipients. Proc. roy. Soc. Med. 65, 1051-1056.
Harris, J., Bagai, R., Rashid, A., Hyslop, D. & Stewart, T. (1972) Nucleic acid synthesis in peripheral blood lymphocytes as an indicator of rejection. Transplant. Proc. 4, 659-663.
Hattler, B. G. Jr. & Miller, J. (1972) Changes in human mixed lymphocyte culture reac- tivity as an indicator of kidney rejection. Transplant. Proc. 4, 655-657.
Page, D., Posen, G., Stewart, T. & Harris, J . ( 1971 ) Immunological detection of renal allo- graft rejection in man. Transplantation 12, 341-347.
Posen, G., Harris, J., Page, D. & Stewart, 'r. ( 197 1 ) Reactive lymphocyte blastogenesis (RLB) in the immunological assessment of human renal allograft recipients. Proc. Europ. Dial. Transplant. Assoc. p. 270-278. Williams & Wilkins, Baltimore.
Address: Dr. Jules Harris Department of Medicine Ottawa General Hospital Ottawa Ontario, Canada K I N 5C8
