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Two-color Immunofluorescence Flow Cytometric Analysis of Lymphocytes in Long-term Renal Allograft Recipients
0022-534 7/89/1413-0492$2.00/0 Vol. 141, March
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright© 1989 by The Williams & Wilkins Co.
TWO-COLOR IMMUNOFLUORESCENCE FLOW CYTOMETRIC ANALYSIS OF LYMPHOCYTES IN LONG-TERM RENAL ALLOGRAFT RECIPIENTS MICHAEL ZIEGELBAUM, RAFAEL VALENZUELA, JOSEPH P. HAYES, DONALD STEINMULLER, STEVAN B. STREEM AND ANDREW C. NOVICK* From the Departments of Uro/,ogy, Immunopathology, and Hypertension and Nephro/,ogy, The Cleveland Clinic Foundation, Cleveland, Ohio
ABSTRACT
We used 2-color immunofluorescence flow cytometry (FACS 440) and monoclonal antibodies (Becton-Dickinson) to study peripheral blood lymphocyte subsets from 26 patients with well functioning renal allografts for longer than 5 years. Comparisons werEt made to a group of 25 healthy volunteers and 25 dialysis patients awaiting renal transplantation. As anticipated,. there was no significant difference with respect to the absolute number or percentage of activated (Tll + and HLA-DR+) and cytotoxic (Leu-2+ and Leu-15-) T lymphocytes among the 3 groups. However, there was a significantly decreased absolute number (p equals 0.0001) and percentage (p equals 0.0001) of suppressor cells (Leu-2+ and Leu-15+) in the transplant patients compared to the healthy control group. No significant difference existed between the transplant and dialysis groups. There also was an increase in the percentage (p equals 0.002) but not in the absolute number of T helper lymphocytes (Leu-3+ and Leu-8-) in the transplant population compared to healthy controls. No significant difference existed between the transplant and dialysis groups. These findings suggest that a normal value of cytotoxic (Leu-2+ and Leu-15-) and activated (Tll + and HLA-Dr+) T lymphocytes may be a good prognostic indicator of long-term survival of renal allografts. Also, the highly significant decrease in the number of Leu-2+ and Leu-15+ cells in long-term, well functioning allografts indicates that additional functional characterization of this subset may be necessary. (J. Ural., 141: 492-494, 1989) The ultimate success of renal transplantation is dependent MATERIALS AND METHODS on many factors. Perhaps most important in the long-term survival of the renal allograft is the immunological tolerance of Patients. We studied 26 long-term (more than 5 years) renal the host immune system toward the transplanted organ. transplant recipients. The 20 men and 6 women ranged from Knowledge of the immune status in the long-term recipient 24 to 68 years old (mean age 43.5 years). All patients have been and how that differs, if at all, from normal is required to followed for 6 to 20 years (mean 11 years). Of the patients 14 understand better the basic difference between long-term graft had undergone live donor renal transplants and 12 received acceptance and rejection. cadaveric allografts. At evaluation all patients had serum creRecent advances in monoclonal antibody technology and atinine levels of 0.8 to 2.1 mg.dl., with a mean serum creatinine immunofluorescence flow cytometric techniques have enabled of 1.25 mg.dl. (normal 0.7 to 1.4 mg./dl.). All but 1 patient were investigators to identify a variety of lymphocyte subsets based taking standard immunosuppressive agents, including azathion surface antigens. 1 We have used 2-color immunofluorescence oprine (1 to 1.5 mg./kg. dosage) and prednisone (10 to 15 mg. flow cytometry (FACS 440) and monoclonal antibodies to eval- per day). One patient had discontinued medications against uate phenotypically the peripheral blood lymphocytes of a . medical advice although he apparently suffered no ill-effects. group of long-term renal graft recipients. The control population consisted of 25 healthy volunteers (6 Previously, investigators have used multicolor flow cytometry men and 19 women between 22 and 40 years old, with a mean to evaluate populations of natural killer cells. 2 - 6 However, other age of 31.7 years). Also, 25 patients on dialysis (19 men and 6 populations (that is helper, suppressor, cytotoxic and activated women between 17 and 69 years old, with a mean age of 41 T lymphocytes) have not been investigated extensively. Thus, years) who were awaiting renal transplantation were chosen to we studied 26 patients with long-term renal allograft survival serve as pre-transplantation controls. to define a variety of lymphocyte phenotypes, including total Cell preparation flow cytometry analysis. Heparinized periphT cells (Tll +), T helper cells (Leu-3+/Leu-8-), T suppressor eral blood was obtained from all subjects for immune monitorcells (Leu-2+/Leu-15+), T cytotoxic cells (Leu-2+/Leu-15-), ing. An additional sample was examined for a total white blood activated T lymphocytes (Tll+ HLA-DR+) and various types count and differential, thus, defining the total number of lymof natural killer cells (Leu-7+/Leu-n-, Leu-7-/Leu-n+ and phocytes in each patient. Leu-7+/Leu-11+). We hope that this information will help to Lymphocytes were isolated from the heparinized samples by delineate the mechanism of long-term renal allograft accept- centrifugation on Ficoll-Hypaque gradients that included inance and rejection, and clinically detectable markers thereof. cubation with carbonyl iron to remove monocytes. Lymphocyte In this way we ultimately may be capable of identifying and staining was accomplished using fluorescein isothiocyanateperhaps treating rejection at an earlier point and potentially conjugated Tll,t and Leu-3, Leu-2, Leu-7, and phycoerythrineffect a greater number of reversals. conjugated HLA-DR, Leu-8, Leu-15 and Leu-11. Each lymphocyte aliquot was stained with 1 of the following combinations Accepted for publication August 30, 1988.
* Requests for reprints: Department of Urology, The Cleveland Clinic Foundation, 1 Clinic Center, 9500 Euclid Ave., Cleveland, Ohio 44195-5041.
492
t Coulter Corp., Hialeah, Florida.
FLOW CYTOME'I'RIC ANALYSIS OF LYMPHOCYTES IN RENAL ALLOGR.AFT RECIPIENTS
of monoclonal antibodies: Tll/HLA-DR, Leu-2/Leu-15, Leu3/Leu-8 and Leu-7/Leu-11. At least 10,000 cells from each preparation were analyzed on a Becton-Dickinson Fluorescence Activated Cell Sorter (FACS 440)* using a computer system (Consort 40) and appropriate software. Lymphocytes were gated using forward and 90-degree scatter signals, and then analyzed for red and green fluorescence. As controls for nonspecific fluorescence lymphocyte aliquots were stained with irrelevant, isotype matched, fluorescein-isothiocyanate and phycoerythrin-labeled mouse immunoglobulin. Analysis of variance was used to detect differences among test groups. A p value of ;;i;0.01 was considered significant. When the p value was significant specific F tests were done to detect pairwise difference. RESULTS
Comparisons were made between control and patient groups with respect to each lymphocyte subset. Tables 1 and 2 refer to absolute numbers of cells as well as the relative percentage of each cell type. Table 1 compares the transplant and healthy groups, while table 2 compares transplant and dialysis groups. The absolute number as well as percentage of cells co-expressing the Tll and HLA-DR antigens (activated T lymphocytes) were not significantly different among the 3 test groups. The absolute number but not relative percentage of the cells expressing Leu-11 but not Leu-7 (Leu-r/Leu-11+) was depressed significantly in the transplant group compared to the healthy controls (68.25 ± 99.8 versus 128.88 ± 65.6 standard deviation, p = 0.003). Similarly, Leu-T /Leu-11 + was depressed in the dialysis group compared to healthy controls (50.39 ± 26.6 versus 128.88 ± 65.6, p = 0.0002). No statistical difference existed between the dialysis and transplant groups. Cells co-expressing Leu-7 and Leu-11 (Leu-7+ /Leu-11 +) were significantly elevated in the transplant patients with respect to * Becton-Dickinson, Mountain View, California.
TABLE
493
dialysis patients ± 148. 7 versus 69.62 ± 48, p = No difference was noted between the transplant patients and healthy controls with respect to Leu-7+/Leu-11+. Also, no significant differences were noted among those 3 groups with respect to Leu -7+ /Leu -11 - . There was a significant elevation in the percentage (10.78 ± 8.1 versus 5.56 ± 2.3, p = 0.002) but not the absolute number (175.82 ± 187.3 versus 107.08 ± 48.9, not significant) of cells with the Leu-3+ /Leu-8- phenotype (T helpers) between the transplantation group and healthy controls. A trend towards a higher percentage but not absolute number of Leu-3+ /Leu-8was noted in the dialysis patients compared to the control group (9.07 ± 5.7 versus 5.56 ± 2.3, p = 0.04). No difference was noted between transplant and dialysis patients with respect to Leu-3+ /Leu-8-. There was no significant difference among the 3 groups with respect to the Leu-3+ /8+ cells. Lymphocytes co-expressing the Leu-15/Leu-2 phenotypes (T suppressors) were significantly depressed in the transplant patients compared to the healthy controls. This was true for absolute (68.14 ± 56.8 versus 170.33 ± 72.9, p = 0.0001) and percentage (4.23 ± 2.8 versus 8.91 ± 3.8, p = 0.0001) values. The absolute numbers of Leu-2+ /Leu-15+ in the dialysis patients also were lower than normal (87.42 ± 66.9, p = 0.0001) but they were of borderline significance with respect to percentage decrease (6.45 ± 3.8, p = 0.016). While the absolute levels of Leu-2+/Leu-15+ cells in the transplant and dialysis groups were not statistically different, there was a trend for the percentage of cells to be lower in the transplant population than in the dialysis group (p = 0.03). There was no significant difference in the absolute or relative values of Leu-2+ /Leu-15cells (T cytotoxic) among the 3 groups. DISCUSSION AND CONCLUSION
Immune monitoring may become a useful tool to identify patients undergoing allograft rejection, thus, affording earlier and perhaps more effective treatment. 7 For this to happen a clear understanding is needed of the immunological status of
l. Absolute value in cells per µ.l. (relative value in per cent) in transplant and healthy groups.
Cell Type*
Healthy (25 pts.)
White blood cells Lymphocytes Tll+ Tll+/HLA-DW Leu-7* /Leu-11 + Leu-T/Leu-11+ Leu-7+ /Leu-11Leu-3* /Leu-sLeu-3* /Leu-8* Leu-2+ /Leu-15+ Leu-2+/Leu-15-
6,220.00 ± 1,941.60 ± 1,674.20 ± 77.27 ± 160.80 ± 128.88 ± 155.80 ± 107.00 ± 848.19 ± 170.33 ± 422.83 ±
1,322.2 350.3 (32.06 311.4 (86.32 51.5 (5.44 92.8 (8.25 65.6 (6.54 101.8 (8.49 48.9 (5.56 250.9 (43.44 72.9 (8.91 128.1 (21.74
Transplant (26 pts.) ± ± ± ±
± ± ± ±
± ±
6.9) 4.8) 2.5) 4.7) 2.8) 6.3) 2.3) 9.3) 3.8) 5.1)
8.253.90 ± 1,544.83 ± 1,416.30 ± 86.99 ± 159.40 ± 68.25 ± 204.48 ± 175.82 ± 600.59 ± 68.14 ± 370.06 ±
2,051.0 718.7 (19.67 680.8 (91.10 31.7 (5.88 148.7 (10.34 99.8 (4.58 268.1 (13.03 187.3 (10.78 407.8 (38.70 56.8 (4.23 268.1 (23.18
± 8.8) ± 6.2) ± 2.8) ± 9.8) ± 6.1) ± 15.5) ± 8.1) ± 16.8) ± 2.8) ± 10.8)
P Value 0.0009 Not significant (0.0001) Not significant (0.001) Not significant (not significant) Not significant (not significant) 0.003 (not significant) Not significant (not significant) Not significant (0.002) Not significant (not significant) 0.0001 (0.0001) Not significant (not significant)
Values represent means ± 1 standard deviation.
* Tll +-total T. Tll * HLA-activated T. Leu-7* /Leu-11 +-natural killer (variable functional activity). Leu-7-/Leu-ll + -natural killer (strong functional activity). Leu-7* /Leu-ll - -natural killer (weak functional activity). Leu-3*/Leu-8--T helper. Leu-2* /Leu-15* -T suppressor. Leu-3* /Leu-8* -T inducer. Leu-2* / Leu-15--T cytotoxic.
TABLE 2.
Absolute value in cells per µ,l. (relative value in per cent) in transplant and dialysis groups
Cell Type*
Transplant (26 pts.)
White blood cells Lymphocytes Tll* Tll*/HLA-DR* Leu-7+/Leu-11 * Leu-T /Leu-11 * Leu-7*/Leu-11Leu-3* /Leu-8* Leu-3* /Leu-8Leu-2+/Leu-15* Leu-2*/Leu-15-
8,253.90 ± 2,051 1,546.83 ± 718.7 (19.67 ± 8.8) 1,416.3 ± 680.9 (91.10 ± 6.2) 86.99 ± 31.7 (5.88 ± 2.8) 159.4 ± 148.7 (10.34 ± 9.8) 68.25 ± 99.8 (4.58 ± 6.1) 204.48 ± 268.1 (13.03 ± 15.5) 600.59 ± 407.8 (38. 70 ± 16.8) 175.82 ± 187.3 (10. 78 ± 8.1) 68.14 ± 56.8 (4.23 ± 2.8) 370.06 ± 268.1 (23.18 ± 10.3)
Dialysis (25 pts.) 6,676.0 ± 2,671 1,480.6 ± 719 1,327.34 ± 645.7 68.55 ± 53.7 69.62 ± 48.0 50.39 ± 26.6 163.61 ± 203.7 671.38 ± 369.2 144.32 ± 131.2 87.42 ± 66.9 333.82 ± 202. 7
(23.80 ± 10.6) (89.360 ± 3.6) (5.09 ± 2.5) (5.72 ± 5.4) (3.98 ± 2.5) (9.28 ± 7.5) (44.96 ± 13.2) (9.07 ± 5.7) (6.45 ± 3.8) (22.62 ± 7. 7)
P Value 0.009 Not significant (not significant) Not significant (not significant) Not significant (not significant) 0.003 (not significant) Not significant (not significant) Not significant (not significant) Not significant (not significant) Not significant (not significant) Not significant (not significant) Not significant (not significant)
Values represent means ± 1 standard deviation.
* Tll *-total T. Tll + HLA-activated T. Leu-7* /Leu-11 *-natural killer (variable functional activity). Leu-T/Leu-11 *-natural killer (strong functional activity). Leu-7* /Leu-Ii--natural killer (weak functional activity). Leu-3* /Leu-8--T helper. Leu-2* /Leu-15* -T suppressor. Leu-3* /Leu-8+ -T inducer. Leu-2* / Leu-15--T cytotoxic.
494
ZIEGELBAUM AND ASSOCIATES
the patient with adequate immunosuppression and good renal function. Our data are consistent with earlier reports regarding subpopulations of natural killer cells.s-,;,s-w In contrast to patients with acute rejection, long-term survivors have normal numbers of activated T lymphocytes. Questions must be raised regarding the role of the suppressor cell (Leu-2+ /Leu-15+) population in healthy renal transplant patients. A positive relationship between the number of peripheral blood activated T cells (Tll/HLA-DR) and allograft rejection in renal transplant patients has been found previously. 11 In our study no significant difference was noted among the healthy controls and the 2 groups of patients with respect to the amount of activated T lymphocytes. Thus, a low level of Tll/HLA-DR may be an indication of adequate immunosuppression and good prognosis. Other investigators have demonstrated a decrease in the most cytotoxic subsets of natural killer cells in the transplant patient.2-G,9, 10 This is seen by the diminished activity displayed in natural killer functional assays/ 0 • 8 - 10 as well as a decrease in the number of cells with a Leu-T/Leu-11+ phenotype.>-G Our results corroborate that finding with respect to absolute values of natural killer cells. Thus, a diminution in natural killer cell activity also appears to be a component of a long-term well functioning allograft in an adequately immunosuppressed recipient. In 1985 Fregona and associates noted a decrease in helperinducer cells with a relative increase in suppressor-cytotoxic cells as defined by the single monoclonal antibody Leu-3 and Leu-2, respectively.'" Redlich and associates demonstrated no change in either suppressor/cytotoxic or helper/inducer population using only single color immunofluorescence techniques. 1 " With a functional assay for suppressor cells Redlich and associates demonstrated decreased suppressor cell activity in renal transplant patients despite no phenotypic differences with single color techniques. Our data, derived from 2-color immunofluorescence methods, support the functional observations of Redlich and associates of diminished suppressor cell activity. The absolute number and proportion of Leu-2+ /Leu-15+ cells are significantly depressed in the transplant patient group. Also, the increase in the relative value of Leu-3+ /Leu-8(helper) cells supports their observation that the fraction of Leu-3+ cells increases as graft longevity increases. 11 The explanation for a decrease of suppressor cells in the well functioning renal allograft patient is not apparent. The finding of depressed Leu-2+ /Leu-15+ in dialysis patients raises the question of whether the finding in transplant patients is due to the immunosuppression or if that population has been affected permanently by the pre-transplant azotemia or dialysis. Perhaps Leu-2+ /Leu-15+ identifies cells other than those with suppressor function and that population is depressed. It has been suggested that Leu-2+ /Leu-15+ may be a heterogeneous population 14 of cells not all of which have suppressor function. Alternatively, the decrease seen in the peripheral blood may be related partly to the sequestration of specific suppressor cells in the region of the allograft. In summary, with 2-color immunofluorescence flow cytometric techniques, we have confirmed previous reports of diminished natural killer cell activity in the adequately immunosup-
pressed long-term renal allograft recipient. In addition, our findings regarding levels of suppressor, helper and activated T cells may help to define the immunological norm for these patients. This information is not applicable clinically to date and additional work is needed to delineate better the mechanism of long-term graft acceptance. Rebecca Turinic and Gordon Burgess provided technical assistance, Theresa Babiak provided statistical assistance and patient coordination was performed by Donna Granetto and Claudia Swift. REFERENCES 1. Guttmann, R. D. and Poulsen, R.: Fluorescence activated cell sorter analysis of lymphocytes following renal allotransplantation. Transplant. Proc., 13: 1579, 1981. 2, Legendre, C. M., Guttmann, R. D. and Yip, G. H.: Natural killer cell subsets in long-term renal allograft recipients. A phenotypic and functional study, Transplantation, 42: 347, 1986. 3. Legendre, C. M., Guttmann, R. D. and Peters, L.: Two-color flow cytometry analysis of NK cells in long-term renal allograft recipients. TTansplant. Proc., 18: 156, 1986. 4. Yip, G. H., Legendre, C. M., Rodrigues, G. A. and Guttmann, R. D.: Large granular lymphocyte subsets in long-term renal allograft recipients. Transplant. Proc., 19: 3396, 1987. 5. Legendre, C. M., Yip, G. H., Rodrigues, G. A., Forbes, C. and Guttmann, R. D.: Characterization of an expanded large granular lymphocyte subset lacking natural killer activity present in renal allograft recipients. Transplantation, 43: 229, 1987. 6. Legendre, C. M., Yip, G. H., Rodrigues, G. A., Forbes, R. D. C. and Guttmann, R. D.: Abnormal expansion of a large granular lymphocyte subset that lacks natural killer activity in long-term renal allograft recipients. Transplant. Proc., 19: 1575, 1987. 7. Binkley, W. F., Valenzuela, R., Braun, W. E., Deodhar, S. D., Novick, A. C., Steinmuller, D. R. and Williams, G.: Flow cytometry quantitation of peripheral blood T-cell subsets as a monitor of renal allograft rejection. Cleveland Clin. Quart., 53: 249, 1986. 8. Waltzer, W. C., Bachvaroff, R. J., Anaise, D. and Rapaport, F. T.: Natural killer activity after renal transplantation. Transplant. Proc., 16: 1527, 1984. 9. Ramsey, K. M., Djeu, J. Y. and Rook, A. H.: Decreased circulating large granular lymphocytes associated with depressed natural killer cell activity in renal transplant recipients. Transplantation, 38: 351, 1984. 10. Guillou, P. J., Hegarty, J., Ramsden, C., Davison, A. M., Will, E. J. and Giles, G. R.: Changes in human natural killer activity early and late after renal transplantation using conventional immunosuppression. Transplantation, 33: 414, 1982. 11. Hayes, J.M., Valenzuela, R., Novick, A. C., Steinmuller, D.R. and Williams, G.: Correlation between two-color flow cytometry quantitation of activated T cells and acute allograft rejection. Transplant. Proc., 19: 1605, 1987. 12. Fregona, I., Guttmann, R. D. and Jean, R.: HNK-1+ (Leu-7) and other lymphocyte subsets in long-term survivors with renal allotransplants. Transplantation, 39: 25, 1985. 13. Redlich, P. N., Leapman, S. B., Marder, P., Schmidtke, J., Smith, E. J., Fineberg, N. and Filo, R. S.: Evaluation of lymphocyte subsets and suppressor cell function in long-term renal transplant recipients. Transplant. Proc., 16: 1555, 1984. 14. Rich, R.R., el Masry, M. N. and Fox, E. J.: Human suppressor T cells: induction, differentiation, and regulatory functions. Hum. Immunol., 17: 369, 1986.
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright© 1989 by The Williams & Wilkins Co.
TWO-COLOR IMMUNOFLUORESCENCE FLOW CYTOMETRIC ANALYSIS OF LYMPHOCYTES IN LONG-TERM RENAL ALLOGRAFT RECIPIENTS MICHAEL ZIEGELBAUM, RAFAEL VALENZUELA, JOSEPH P. HAYES, DONALD STEINMULLER, STEVAN B. STREEM AND ANDREW C. NOVICK* From the Departments of Uro/,ogy, Immunopathology, and Hypertension and Nephro/,ogy, The Cleveland Clinic Foundation, Cleveland, Ohio
ABSTRACT
We used 2-color immunofluorescence flow cytometry (FACS 440) and monoclonal antibodies (Becton-Dickinson) to study peripheral blood lymphocyte subsets from 26 patients with well functioning renal allografts for longer than 5 years. Comparisons werEt made to a group of 25 healthy volunteers and 25 dialysis patients awaiting renal transplantation. As anticipated,. there was no significant difference with respect to the absolute number or percentage of activated (Tll + and HLA-DR+) and cytotoxic (Leu-2+ and Leu-15-) T lymphocytes among the 3 groups. However, there was a significantly decreased absolute number (p equals 0.0001) and percentage (p equals 0.0001) of suppressor cells (Leu-2+ and Leu-15+) in the transplant patients compared to the healthy control group. No significant difference existed between the transplant and dialysis groups. There also was an increase in the percentage (p equals 0.002) but not in the absolute number of T helper lymphocytes (Leu-3+ and Leu-8-) in the transplant population compared to healthy controls. No significant difference existed between the transplant and dialysis groups. These findings suggest that a normal value of cytotoxic (Leu-2+ and Leu-15-) and activated (Tll + and HLA-Dr+) T lymphocytes may be a good prognostic indicator of long-term survival of renal allografts. Also, the highly significant decrease in the number of Leu-2+ and Leu-15+ cells in long-term, well functioning allografts indicates that additional functional characterization of this subset may be necessary. (J. Ural., 141: 492-494, 1989) The ultimate success of renal transplantation is dependent MATERIALS AND METHODS on many factors. Perhaps most important in the long-term survival of the renal allograft is the immunological tolerance of Patients. We studied 26 long-term (more than 5 years) renal the host immune system toward the transplanted organ. transplant recipients. The 20 men and 6 women ranged from Knowledge of the immune status in the long-term recipient 24 to 68 years old (mean age 43.5 years). All patients have been and how that differs, if at all, from normal is required to followed for 6 to 20 years (mean 11 years). Of the patients 14 understand better the basic difference between long-term graft had undergone live donor renal transplants and 12 received acceptance and rejection. cadaveric allografts. At evaluation all patients had serum creRecent advances in monoclonal antibody technology and atinine levels of 0.8 to 2.1 mg.dl., with a mean serum creatinine immunofluorescence flow cytometric techniques have enabled of 1.25 mg.dl. (normal 0.7 to 1.4 mg./dl.). All but 1 patient were investigators to identify a variety of lymphocyte subsets based taking standard immunosuppressive agents, including azathion surface antigens. 1 We have used 2-color immunofluorescence oprine (1 to 1.5 mg./kg. dosage) and prednisone (10 to 15 mg. flow cytometry (FACS 440) and monoclonal antibodies to eval- per day). One patient had discontinued medications against uate phenotypically the peripheral blood lymphocytes of a . medical advice although he apparently suffered no ill-effects. group of long-term renal graft recipients. The control population consisted of 25 healthy volunteers (6 Previously, investigators have used multicolor flow cytometry men and 19 women between 22 and 40 years old, with a mean to evaluate populations of natural killer cells. 2 - 6 However, other age of 31.7 years). Also, 25 patients on dialysis (19 men and 6 populations (that is helper, suppressor, cytotoxic and activated women between 17 and 69 years old, with a mean age of 41 T lymphocytes) have not been investigated extensively. Thus, years) who were awaiting renal transplantation were chosen to we studied 26 patients with long-term renal allograft survival serve as pre-transplantation controls. to define a variety of lymphocyte phenotypes, including total Cell preparation flow cytometry analysis. Heparinized periphT cells (Tll +), T helper cells (Leu-3+/Leu-8-), T suppressor eral blood was obtained from all subjects for immune monitorcells (Leu-2+/Leu-15+), T cytotoxic cells (Leu-2+/Leu-15-), ing. An additional sample was examined for a total white blood activated T lymphocytes (Tll+ HLA-DR+) and various types count and differential, thus, defining the total number of lymof natural killer cells (Leu-7+/Leu-n-, Leu-7-/Leu-n+ and phocytes in each patient. Leu-7+/Leu-11+). We hope that this information will help to Lymphocytes were isolated from the heparinized samples by delineate the mechanism of long-term renal allograft accept- centrifugation on Ficoll-Hypaque gradients that included inance and rejection, and clinically detectable markers thereof. cubation with carbonyl iron to remove monocytes. Lymphocyte In this way we ultimately may be capable of identifying and staining was accomplished using fluorescein isothiocyanateperhaps treating rejection at an earlier point and potentially conjugated Tll,t and Leu-3, Leu-2, Leu-7, and phycoerythrineffect a greater number of reversals. conjugated HLA-DR, Leu-8, Leu-15 and Leu-11. Each lymphocyte aliquot was stained with 1 of the following combinations Accepted for publication August 30, 1988.
* Requests for reprints: Department of Urology, The Cleveland Clinic Foundation, 1 Clinic Center, 9500 Euclid Ave., Cleveland, Ohio 44195-5041.
492
t Coulter Corp., Hialeah, Florida.
FLOW CYTOME'I'RIC ANALYSIS OF LYMPHOCYTES IN RENAL ALLOGR.AFT RECIPIENTS
of monoclonal antibodies: Tll/HLA-DR, Leu-2/Leu-15, Leu3/Leu-8 and Leu-7/Leu-11. At least 10,000 cells from each preparation were analyzed on a Becton-Dickinson Fluorescence Activated Cell Sorter (FACS 440)* using a computer system (Consort 40) and appropriate software. Lymphocytes were gated using forward and 90-degree scatter signals, and then analyzed for red and green fluorescence. As controls for nonspecific fluorescence lymphocyte aliquots were stained with irrelevant, isotype matched, fluorescein-isothiocyanate and phycoerythrin-labeled mouse immunoglobulin. Analysis of variance was used to detect differences among test groups. A p value of ;;i;0.01 was considered significant. When the p value was significant specific F tests were done to detect pairwise difference. RESULTS
Comparisons were made between control and patient groups with respect to each lymphocyte subset. Tables 1 and 2 refer to absolute numbers of cells as well as the relative percentage of each cell type. Table 1 compares the transplant and healthy groups, while table 2 compares transplant and dialysis groups. The absolute number as well as percentage of cells co-expressing the Tll and HLA-DR antigens (activated T lymphocytes) were not significantly different among the 3 test groups. The absolute number but not relative percentage of the cells expressing Leu-11 but not Leu-7 (Leu-r/Leu-11+) was depressed significantly in the transplant group compared to the healthy controls (68.25 ± 99.8 versus 128.88 ± 65.6 standard deviation, p = 0.003). Similarly, Leu-T /Leu-11 + was depressed in the dialysis group compared to healthy controls (50.39 ± 26.6 versus 128.88 ± 65.6, p = 0.0002). No statistical difference existed between the dialysis and transplant groups. Cells co-expressing Leu-7 and Leu-11 (Leu-7+ /Leu-11 +) were significantly elevated in the transplant patients with respect to * Becton-Dickinson, Mountain View, California.
TABLE
493
dialysis patients ± 148. 7 versus 69.62 ± 48, p = No difference was noted between the transplant patients and healthy controls with respect to Leu-7+/Leu-11+. Also, no significant differences were noted among those 3 groups with respect to Leu -7+ /Leu -11 - . There was a significant elevation in the percentage (10.78 ± 8.1 versus 5.56 ± 2.3, p = 0.002) but not the absolute number (175.82 ± 187.3 versus 107.08 ± 48.9, not significant) of cells with the Leu-3+ /Leu-8- phenotype (T helpers) between the transplantation group and healthy controls. A trend towards a higher percentage but not absolute number of Leu-3+ /Leu-8was noted in the dialysis patients compared to the control group (9.07 ± 5.7 versus 5.56 ± 2.3, p = 0.04). No difference was noted between transplant and dialysis patients with respect to Leu-3+ /Leu-8-. There was no significant difference among the 3 groups with respect to the Leu-3+ /8+ cells. Lymphocytes co-expressing the Leu-15/Leu-2 phenotypes (T suppressors) were significantly depressed in the transplant patients compared to the healthy controls. This was true for absolute (68.14 ± 56.8 versus 170.33 ± 72.9, p = 0.0001) and percentage (4.23 ± 2.8 versus 8.91 ± 3.8, p = 0.0001) values. The absolute numbers of Leu-2+ /Leu-15+ in the dialysis patients also were lower than normal (87.42 ± 66.9, p = 0.0001) but they were of borderline significance with respect to percentage decrease (6.45 ± 3.8, p = 0.016). While the absolute levels of Leu-2+/Leu-15+ cells in the transplant and dialysis groups were not statistically different, there was a trend for the percentage of cells to be lower in the transplant population than in the dialysis group (p = 0.03). There was no significant difference in the absolute or relative values of Leu-2+ /Leu-15cells (T cytotoxic) among the 3 groups. DISCUSSION AND CONCLUSION
Immune monitoring may become a useful tool to identify patients undergoing allograft rejection, thus, affording earlier and perhaps more effective treatment. 7 For this to happen a clear understanding is needed of the immunological status of
l. Absolute value in cells per µ.l. (relative value in per cent) in transplant and healthy groups.
Cell Type*
Healthy (25 pts.)
White blood cells Lymphocytes Tll+ Tll+/HLA-DW Leu-7* /Leu-11 + Leu-T/Leu-11+ Leu-7+ /Leu-11Leu-3* /Leu-sLeu-3* /Leu-8* Leu-2+ /Leu-15+ Leu-2+/Leu-15-
6,220.00 ± 1,941.60 ± 1,674.20 ± 77.27 ± 160.80 ± 128.88 ± 155.80 ± 107.00 ± 848.19 ± 170.33 ± 422.83 ±
1,322.2 350.3 (32.06 311.4 (86.32 51.5 (5.44 92.8 (8.25 65.6 (6.54 101.8 (8.49 48.9 (5.56 250.9 (43.44 72.9 (8.91 128.1 (21.74
Transplant (26 pts.) ± ± ± ±
± ± ± ±
± ±
6.9) 4.8) 2.5) 4.7) 2.8) 6.3) 2.3) 9.3) 3.8) 5.1)
8.253.90 ± 1,544.83 ± 1,416.30 ± 86.99 ± 159.40 ± 68.25 ± 204.48 ± 175.82 ± 600.59 ± 68.14 ± 370.06 ±
2,051.0 718.7 (19.67 680.8 (91.10 31.7 (5.88 148.7 (10.34 99.8 (4.58 268.1 (13.03 187.3 (10.78 407.8 (38.70 56.8 (4.23 268.1 (23.18
± 8.8) ± 6.2) ± 2.8) ± 9.8) ± 6.1) ± 15.5) ± 8.1) ± 16.8) ± 2.8) ± 10.8)
P Value 0.0009 Not significant (0.0001) Not significant (0.001) Not significant (not significant) Not significant (not significant) 0.003 (not significant) Not significant (not significant) Not significant (0.002) Not significant (not significant) 0.0001 (0.0001) Not significant (not significant)
Values represent means ± 1 standard deviation.
* Tll +-total T. Tll * HLA-activated T. Leu-7* /Leu-11 +-natural killer (variable functional activity). Leu-7-/Leu-ll + -natural killer (strong functional activity). Leu-7* /Leu-ll - -natural killer (weak functional activity). Leu-3*/Leu-8--T helper. Leu-2* /Leu-15* -T suppressor. Leu-3* /Leu-8* -T inducer. Leu-2* / Leu-15--T cytotoxic.
TABLE 2.
Absolute value in cells per µ,l. (relative value in per cent) in transplant and dialysis groups
Cell Type*
Transplant (26 pts.)
White blood cells Lymphocytes Tll* Tll*/HLA-DR* Leu-7+/Leu-11 * Leu-T /Leu-11 * Leu-7*/Leu-11Leu-3* /Leu-8* Leu-3* /Leu-8Leu-2+/Leu-15* Leu-2*/Leu-15-
8,253.90 ± 2,051 1,546.83 ± 718.7 (19.67 ± 8.8) 1,416.3 ± 680.9 (91.10 ± 6.2) 86.99 ± 31.7 (5.88 ± 2.8) 159.4 ± 148.7 (10.34 ± 9.8) 68.25 ± 99.8 (4.58 ± 6.1) 204.48 ± 268.1 (13.03 ± 15.5) 600.59 ± 407.8 (38. 70 ± 16.8) 175.82 ± 187.3 (10. 78 ± 8.1) 68.14 ± 56.8 (4.23 ± 2.8) 370.06 ± 268.1 (23.18 ± 10.3)
Dialysis (25 pts.) 6,676.0 ± 2,671 1,480.6 ± 719 1,327.34 ± 645.7 68.55 ± 53.7 69.62 ± 48.0 50.39 ± 26.6 163.61 ± 203.7 671.38 ± 369.2 144.32 ± 131.2 87.42 ± 66.9 333.82 ± 202. 7
(23.80 ± 10.6) (89.360 ± 3.6) (5.09 ± 2.5) (5.72 ± 5.4) (3.98 ± 2.5) (9.28 ± 7.5) (44.96 ± 13.2) (9.07 ± 5.7) (6.45 ± 3.8) (22.62 ± 7. 7)
P Value 0.009 Not significant (not significant) Not significant (not significant) Not significant (not significant) 0.003 (not significant) Not significant (not significant) Not significant (not significant) Not significant (not significant) Not significant (not significant) Not significant (not significant) Not significant (not significant)
Values represent means ± 1 standard deviation.
* Tll *-total T. Tll + HLA-activated T. Leu-7* /Leu-11 *-natural killer (variable functional activity). Leu-T/Leu-11 *-natural killer (strong functional activity). Leu-7* /Leu-Ii--natural killer (weak functional activity). Leu-3* /Leu-8--T helper. Leu-2* /Leu-15* -T suppressor. Leu-3* /Leu-8+ -T inducer. Leu-2* / Leu-15--T cytotoxic.
494
ZIEGELBAUM AND ASSOCIATES
the patient with adequate immunosuppression and good renal function. Our data are consistent with earlier reports regarding subpopulations of natural killer cells.s-,;,s-w In contrast to patients with acute rejection, long-term survivors have normal numbers of activated T lymphocytes. Questions must be raised regarding the role of the suppressor cell (Leu-2+ /Leu-15+) population in healthy renal transplant patients. A positive relationship between the number of peripheral blood activated T cells (Tll/HLA-DR) and allograft rejection in renal transplant patients has been found previously. 11 In our study no significant difference was noted among the healthy controls and the 2 groups of patients with respect to the amount of activated T lymphocytes. Thus, a low level of Tll/HLA-DR may be an indication of adequate immunosuppression and good prognosis. Other investigators have demonstrated a decrease in the most cytotoxic subsets of natural killer cells in the transplant patient.2-G,9, 10 This is seen by the diminished activity displayed in natural killer functional assays/ 0 • 8 - 10 as well as a decrease in the number of cells with a Leu-T/Leu-11+ phenotype.>-G Our results corroborate that finding with respect to absolute values of natural killer cells. Thus, a diminution in natural killer cell activity also appears to be a component of a long-term well functioning allograft in an adequately immunosuppressed recipient. In 1985 Fregona and associates noted a decrease in helperinducer cells with a relative increase in suppressor-cytotoxic cells as defined by the single monoclonal antibody Leu-3 and Leu-2, respectively.'" Redlich and associates demonstrated no change in either suppressor/cytotoxic or helper/inducer population using only single color immunofluorescence techniques. 1 " With a functional assay for suppressor cells Redlich and associates demonstrated decreased suppressor cell activity in renal transplant patients despite no phenotypic differences with single color techniques. Our data, derived from 2-color immunofluorescence methods, support the functional observations of Redlich and associates of diminished suppressor cell activity. The absolute number and proportion of Leu-2+ /Leu-15+ cells are significantly depressed in the transplant patient group. Also, the increase in the relative value of Leu-3+ /Leu-8(helper) cells supports their observation that the fraction of Leu-3+ cells increases as graft longevity increases. 11 The explanation for a decrease of suppressor cells in the well functioning renal allograft patient is not apparent. The finding of depressed Leu-2+ /Leu-15+ in dialysis patients raises the question of whether the finding in transplant patients is due to the immunosuppression or if that population has been affected permanently by the pre-transplant azotemia or dialysis. Perhaps Leu-2+ /Leu-15+ identifies cells other than those with suppressor function and that population is depressed. It has been suggested that Leu-2+ /Leu-15+ may be a heterogeneous population 14 of cells not all of which have suppressor function. Alternatively, the decrease seen in the peripheral blood may be related partly to the sequestration of specific suppressor cells in the region of the allograft. In summary, with 2-color immunofluorescence flow cytometric techniques, we have confirmed previous reports of diminished natural killer cell activity in the adequately immunosup-
pressed long-term renal allograft recipient. In addition, our findings regarding levels of suppressor, helper and activated T cells may help to define the immunological norm for these patients. This information is not applicable clinically to date and additional work is needed to delineate better the mechanism of long-term graft acceptance. Rebecca Turinic and Gordon Burgess provided technical assistance, Theresa Babiak provided statistical assistance and patient coordination was performed by Donna Granetto and Claudia Swift. REFERENCES 1. Guttmann, R. D. and Poulsen, R.: Fluorescence activated cell sorter analysis of lymphocytes following renal allotransplantation. Transplant. Proc., 13: 1579, 1981. 2, Legendre, C. M., Guttmann, R. D. and Yip, G. H.: Natural killer cell subsets in long-term renal allograft recipients. A phenotypic and functional study, Transplantation, 42: 347, 1986. 3. Legendre, C. M., Guttmann, R. D. and Peters, L.: Two-color flow cytometry analysis of NK cells in long-term renal allograft recipients. TTansplant. Proc., 18: 156, 1986. 4. Yip, G. H., Legendre, C. M., Rodrigues, G. A. and Guttmann, R. D.: Large granular lymphocyte subsets in long-term renal allograft recipients. Transplant. Proc., 19: 3396, 1987. 5. Legendre, C. M., Yip, G. H., Rodrigues, G. A., Forbes, C. and Guttmann, R. D.: Characterization of an expanded large granular lymphocyte subset lacking natural killer activity present in renal allograft recipients. Transplantation, 43: 229, 1987. 6. Legendre, C. M., Yip, G. H., Rodrigues, G. A., Forbes, R. D. C. and Guttmann, R. D.: Abnormal expansion of a large granular lymphocyte subset that lacks natural killer activity in long-term renal allograft recipients. Transplant. Proc., 19: 1575, 1987. 7. Binkley, W. F., Valenzuela, R., Braun, W. E., Deodhar, S. D., Novick, A. C., Steinmuller, D. R. and Williams, G.: Flow cytometry quantitation of peripheral blood T-cell subsets as a monitor of renal allograft rejection. Cleveland Clin. Quart., 53: 249, 1986. 8. Waltzer, W. C., Bachvaroff, R. J., Anaise, D. and Rapaport, F. T.: Natural killer activity after renal transplantation. Transplant. Proc., 16: 1527, 1984. 9. Ramsey, K. M., Djeu, J. Y. and Rook, A. H.: Decreased circulating large granular lymphocytes associated with depressed natural killer cell activity in renal transplant recipients. Transplantation, 38: 351, 1984. 10. Guillou, P. J., Hegarty, J., Ramsden, C., Davison, A. M., Will, E. J. and Giles, G. R.: Changes in human natural killer activity early and late after renal transplantation using conventional immunosuppression. Transplantation, 33: 414, 1982. 11. Hayes, J.M., Valenzuela, R., Novick, A. C., Steinmuller, D.R. and Williams, G.: Correlation between two-color flow cytometry quantitation of activated T cells and acute allograft rejection. Transplant. Proc., 19: 1605, 1987. 12. Fregona, I., Guttmann, R. D. and Jean, R.: HNK-1+ (Leu-7) and other lymphocyte subsets in long-term survivors with renal allotransplants. Transplantation, 39: 25, 1985. 13. Redlich, P. N., Leapman, S. B., Marder, P., Schmidtke, J., Smith, E. J., Fineberg, N. and Filo, R. S.: Evaluation of lymphocyte subsets and suppressor cell function in long-term renal transplant recipients. Transplant. Proc., 16: 1555, 1984. 14. Rich, R.R., el Masry, M. N. and Fox, E. J.: Human suppressor T cells: induction, differentiation, and regulatory functions. Hum. Immunol., 17: 369, 1986.