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OKT48 ratio in the blood and in the graft during episodes of human renal allograft rejection
CELLULAR
IMMUNOLOGY
77, 196-201 (1983)
OKT4/8 Ratio in the Blood and in the Graft during Episodes of Human Renal Allograft Rejection EEVA VON WILLEBBAND’ Transplantation Laboratory and Fourth Department of Surgery, University of Helsinki, Helsinki, Finland Received December 20, 1982; accepted January 7, I983
We have analyzed the frequency of T helper (Th) and T suppressor/killer (Ts/k) lymphocytes in the blood and in the renal allograft during episodes of rejection and during quiescence. Monoclonal OKT4 and OKT8 antibodies were used to mark the Th and Ts/k cells, respectively. Density centrifugation-separated mononuclear leukocytes and FACS IV cell sorter or the Staphylococcus aureus rosette assay were used to determine the ratio in the blood, with concordant results. Fine needle aspiration biopsy (FNAB) and the Staph. assay were used to demonstrate the lymphocyte subtypes in the graft. The mean OKT4/8 ratio in the blood was significantly lower in the transplant recipients than in healthy controls (1.1 f 0.7 vs 1.8 + 0.2, respectively, P = 0.000). The individual variation was, however, high and no correlation between the OKT4/ 8 ratio in the blood and the inflammatory episodes in situ was observed. During 19 of the 25 episodes of inflammation, the dominant lymphocyte subtype in the graft was the Ts/k cell. In the remaining six casesit was the Th cell. All rejection episodesof the former type were reversible, in the latter type, four out of six were irreversible.
INTRODUCTION Several different inflammatory pathways may apparently lead to allograft rejection. In renal transplantation in man, the most common is acute cellular rejection characterized by the presence of lymphocytes and blast cells in the graft inflammatory cell cytology (1). During advanced and irreversible rejection mononuclear phagocytes are superimposed on the inflammatory picture (1). A lesscommon cytological pattern of inflammation is dominated (with a minimal or no blastogenic response) by lymphocytes and mononuclear phagocytes (2). This type of cytological picture is encountered particularly during “vascular” rejection. Finally there are patterns of inflammation where mononuclear phagocytes, including monoblasts, dominate the inflammatory cell cytology (2). Several methods have been proposed to correlate changes in the peripheral blood to episodes of rejection (3-8). It has recently been reported that elevated T helper (Th)-T suppressor/killer (Ts/k) cell ratios in the blood are indicative for acute rejection and may be used as a diagnostic aid (9, 10). Becausethis type of finding, if substantiated, would lead to a new important monitoring method in renal trans’ Address all correspondence to Dr. Eeva von Willebrand, Transplantation Laboratory, University of Helsinki, Haartmaninkatu 3, SF 00290 Helsinki 29, Finland. 196 0008-8749183$3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
OKT4/8 RATIO DURING REJECTION
197
plantation in man, we found it important to reexplore the changes in the peripheral blood and correlate them to the inflammatory episodes in situ. Fine needle aspiration biopsy (FNAB) is a new, atraumatic method for continuous monitoring of intragraft events in parenchymal organ transplantation in man. In this communication we have investigated the inflammatory profiles of (first) renal allograft rejection in three types of patients: patients immunosuppressed with azathioprine (AZA) plus methylprednisolone (MP), patients immunosuppressed with cyclosporin A (CyA), and patients immunosuppressed with CyA plus MP. We have analyzed the frequency of Th and Ts/k cells in the blood and in the graft during episodes of inflammation and during quiescence. The results demonstrate that on most occasions the dominant inflammatory lymphocyte in the graft is the Ts/k cell and that no correlation exists between inflammatory episodesof rejection on one hand and changes in the blood Th-Ts/k ratio on the other. MATERIALS
AND METHODS
Patients. Forty recipients of cadaveric renal transplants were included in the study. For immunosuppression, 14 patients received AZA + MP, 12 CyA, and 14 CyA + MP. The patients were on the average 1.5 HLA-AB and 0.5 HLA-DR antigen mismatched with no significant differences between the three groups. All patients were transfused at least three times before transplantation and none of them had cytotoxic antibodies to a panel of 24, or to the donor lymphoid cells before transplantation, Immunosuppression. The first group of patients received AZA 2 mg/kg/day or in the maximal tolerated dose, and 3.6 mg/kg/day of MP tapered to 0.5 mg/kg in 20 days. The second group of patients received 20 mg/kg/day of CyA iv for the first 3 days, tapered to 10 mg/kg/day po on the third day; thereafter the maintenance dose was adjusted according to the level of CyA in plasma, by keeping it as close to 200 rig/ml as possible. The third group of patients received CyA as above, and 3.6 mg/ kg/day of MP tapered to 0 mg/kg/day in 12 days. Diagnosis of rejection. Clinical diagnosis of rejection was based on the generally accepted clinical signs: fever, swelling, and tenderness of the graft, decreased urine output, and increasing serum creatinine, in the absence of pre- and postrenal complications and signs of viral infection. Fine needle aspiration biopsies and blood specimens. To obtain the inflammatory cell profile (see Fig. l), all 40 patients were aspiration biopsied on the average every second day post-transplantation, and concomitantly a blood specimen of approximately 20 ~1 in size was drawn from a fingertip. Blood Th-Ts/k ratio (Fig. 2) was monitored from these patients on 53 occasions. This was performed either from a heparinized blood specimen of 10 ml using FACS IV flow cytometer (Becton Dickinson Co., Sunny Valley, Calif.) or (occasionally) from a fingertip specimen by the Staphylococcusaureus rosette assay,with concordant results (see below). The Th-Ts/k ratio in the graft was determined in 20 patients on 37 occasions (Fig. 2). On 25 occasions the biopsy was obtained during established episodesof rejection; on 7 occasions prior to the onset of rejection and on the remaining 5 occasions after the rejection was overcome. Thus the graft Th-Ts/k ratio was determined in several patients at least twice, either during rejection or during quiescence and rejection. Processing of the FNAB specimen for quantitation of inflammation. The FNAB
198
EEVA VON WILLEBBAND
I
6r
4
2
I
I
I
0
5
10
LI -5
DAYS IN RELATION
A 15
TO ONSET OF REJECTION
FIG. 1. FNAB analysis of the inflammatory profiles of (first) episodes of rejection in patients receiving for immunosuppression azathioprine (AZA) plus methylprednisolone (MP) (top), cyclosporin A (CyA) (middle), or CyA plus MP.
specimen was processedwith a cytocentrifuge, stained with May-Gtinwald-Giemsa (MGG), and the inflammatory changes were quantitated against blood background as described (11). The in situ inflammation is expressed in “corrected increment” units ( 12). 0 AZA+MP
OD a
J I I
3
y’ 0 2-A
I I
'A
15 0 DAYS IN RELATION
A 8
Graft
10 5 215 TO ONSET OF REJECTl0i.j
FIG. 2. OKT4/8 ratio in the blood and in the graft as adjusted to the onset of clinical rejection (vertical dashed line). Circles: patients receiving azathioprine plus methylprednisolone. Squares: patients receiving cyclosporin A. Triangles: patients receiving cyclosporin A plus methylprednisolone. Open figures: reversible rejection; closed figures: irreversible rejection.
OKT4/8 RATIO DURING REJECTION
199
Processing of the heparinized blood specimen. Blood mononuclear leukocytes were isolated by Ficoll-Isopaque (Pharmacia Fine Chemicals, Uppsala, Sweden) density centrifugation and washed in phosphate-buffered saline (PBS). Monitoring of the Th-Ts/k ratio in the blood and in the graft. OKT4 monoclonal antibody was used to recognize the Th and OKT8 the Ts/k subset. The antibodies were purchased from Ortho Pharmaceuticals, Raritan, N.J. Blood mononuclear leukocytes were treated with 1:10 diluted monoclonal antibody, washed,treated with 1:20 diluted FITC-labeled sheepanti-mouse Ig, and washed. The frequency of fluorescent cells was quantitated with FACS IV flow cytometer. With this method of analysis the OKT4/8 ratio in healthy controls is in our hands 1.8 f 0.2 (SD). The OKT4/OKT8 ratio in the graft was determined at least once in each patient and, if heparinized blood was not available, the simultaneously obtained fingertip specimen was processedin identical fashion. Approximately 30 ~1of FNAB material (600,000-3 X lo6 nucleated cells) was divided into two batches, washed, treated with 1:20 diluted monoclonal antibody, washed, and the cell-bound antibody was chased with Staphylococcus aureus Cowan I bacteria (a gift from Prof. H. Wigzell, University of Uppsala, Uppsala, Sweden). As described in detail ( 11, 13), the staphylococci with a high content of protein A bind to the cell-bound antibody, forming rosettes with the antibody-coated cells. When the cells are processedonto microscope slides and stained with MGG, it is possible to identify morphologically the rosette-forming cell OwW ( 11).
RESULTS Projles of the First Episode of Inflammation
The net inflammatory responsesof the first episode of rejection in the three groups of patients are demonstrated in Fig. 1. Most of these episodes commenced during the first 15 days post-transplantation. As is seen in Fig. 1, the background inflammation in patients receiving AZA + MP was lowest and duration of the first inflammatory episode shortest. The background inflammation in patients receiving CyA was higher than in patients receiving CyA + MP. OKT4/8 Ratio in the Graft and in the Blood
Table 1 summarizes the findings in the graft and in the blood during quiescence and during episodes of inflammation. During quiescence the OKT4/8 ratio in the graft was below 1.O on 5 of 12 occasions; in the blood it was below 1.O in 13 of 2 1 occasions. During episodesof inflammation the OKT4/8 ratio in the graft was below 1.0 and 19 of 25 occasions; in the blood it was below 1.0 in 17 of 32 occasions. Ratios below one were thus recorded in the graft significantly more often during episodes of inflammation than during episodes of quiescence (P = 0.040 in x2 test). On the contrary, ratios below one were equally frequent in the blood during episodes of inflammation compared to episodes of quiescence (P = 0.528 in x2 test). Sequential follow-up of the OKT4/8 ratio through the episodes of inflammation is demonstrated in Fig. 2. Although the mean OKT4/OKT8 ratio in the blood of renal allograft recipients was well below that of normal controls (1.1 + 0.7 vs 1.8 f. 0.3, P = O.OOO), there was a wide variation throughout the episode and at no time during rejection could any sensible correlations between the OKT4/8 ratio in the blood and the inflammatory episode in the graft be recorded.
200
EEVA VON WILLEBRAND TABLE 1 OKT4/8 Ratio in the Graft and in the Blood during Inflammatory Episodes of Rejection and during Quiescence OKT4/8 ratio Graft
During inflammation During quiescence P in x2 test
Blood
<1
3-I
19 5
6 7 0.040
>I
17 13
15 8 0.528
In clear contrast there was a distinct depression of the OKT4/8 ratio in the graft which reached its nadir at the onset of clinical rejection and gradually disappeared when the inflammatory episode was overcome. However, in six biopsies taken during episode of inflammation the intragraft OKT4/8 ratio remained high, even elevated. Four of these six grafts were lost. DISCUSSION Although several methods have been proposed for monitoring of the in situ episodes of inflammation (i.e., rejection) from changes taking place in the peripheral blood, many of them have been shown to be inconsistent in later critical analysis (14, 15) and none of them is universally used or has replaced the biopsy. This seemsto be the case also with the Th-Ts/k ratio. In our analysis the OKT4/ 8 ratio was definitely lower in the blood of transplant recipients than in healthy subjects, but large individual variations were apparent, without any correlation to the inflammatory episodesin the graft. This result coincides with several recent results. For example, in a recent international meeting (IX International Meeting of the Transplantation Society, Brighton, England 1982) as many papers were presented suggesting a correlation as a lack of correlation, and in papers indicating that a correlation exists, two entirely divergent opinions emerged: the Th-Ts/k ratio was reported to be either elevated or depressedduring episodes of rejection. Needless to say, in most of these studies, no biopsies were made of the graft to prove the presence or lack of inflammation in situ. The observation that the Ts/k lymphoid cells dominate in the graft during episodes of rejection carries a good correlation to recent findings in rat and man. Platt et al. (16) and Hancock and Atkins (17) have reported an increased frequency of Ts/k lymphoid cells in biopsies of acutely rejecting human renal allografts. Dallman et al. ( 18) and Bhan et al. ( 19) have found the Ts/k subset to dominate in the inflammatory infiltrate of epidermis in skin allografts in rat and man. Renkonen et al. (20) have shown that lymphoid cells with a Ts/k phenotype dominate in rat renal allografts during rejection. This dominance is even stronger if one considers only the “activated” lymphoid cells, i.e., lymphoid celk with a blast cell phenotype. In striking contrast in the recipient spleen, Th blasts dominated throughout the course of rejection. In the FNAB analysis, the pre- and postrejection biopsies represent mainly blood background. Thus most emphasis should be placed on biopsies taken during inflammation. Here, biopsies fell in two distinct categories: in biopsies taken - 1 to + 10
OKT4/8 RATIO DURING REJECTION
201
days after the onset of inflammation, the Ts/k cells dominated on 19 occasions but there was a distinct Th cell predominance on 6 occasions. It is not known whether these two patterns of inflammation represent independent inflammatory pathways of allograft rejection. This is, however, possible as in the latter group five of six patients were on CyA (which drug may deviate the pattern of inflammation; Ref. 2) and four of six patients (including those on CyA and one on AZA) had an irreversible rejection whereas in the former group all rejections were reversible. Finally it should be emphasized that other intragraft events in addition to rejection may modify the inflammation. Hammer et al. (personal communication) have demonstrated with the FNAB that during cytomegalovirus infection of the graft, there is an even stronger Ts/k predominance than during ordinary episodes of rejection; the two conditions may, however, be distinguished by the presence of viral antigen in the graft tubular cells. In bronchoalveolar lavage specimens taken from the lung of human bone marrow transplant recipients, viral infections-verified by the demonstration of the virus-are always associated with an extremely strong Ts/k cell predominance at the site of inflammation (Taskinen et al., unpublished results). ACKNOWLEDGMENTS I thank Dr. P. Hayry and the staff of the Transplantation Laboratory and the Division of Transplantation Surgery for co-operation and Ms. Leena Sarastefor secretarial and editorial help. This study was financed by grant 1 ROl AM 26882-03 from the National Institutes of Health, Bethesda, Maryland, and by grants from the Association of Finnish Life Insurance Companies, and Finska LZkaresallskapet, Helsinki, Finland.
REFERENCES 1. Hayry, P., and von Willebrand, E., &and. J. Immunol. 13, 87, 1981. 2. von Willebrand, E., Taskinen, E., Ahonen, J., and Hiiyry, P., Transplant. Proc. 1982 (in press). 3. Hersh, E. M., Butler, W. T., Rossen, R. D., and Morgan, R. O., Nature (London) 226, 757, 1970. 4. Parker, J. R., and Mowbray, J. F., Transplantation 11, 201, 1971. 5. Kelly, G. E., and Sheil, G. R., Clin. Exp. Immunol. 27, 454, 1977. 6. Stille, C. R., and Sinclair, N. R. StC., Transplant. Proc. XI, 343, 1979. 7. Clancy, J., Jr., Thomas, J. H., Pierce, G. E., Dennison, D. K., and Barth, R. F., Transplant. Proc. XI, 418, 1979. 8. Goulmy, E., Persijn, G., Blokland, E., D’Amaro, J., and van Rood, J. J., Transplantation 31, 210, 1981. 9. Cosimi, A. B., Calvin, R. B., Burton, R. C., Rubin, R. H., Goldstein, G., Kung, P. C., Hansen, W. P., Delmonico, F. L., and Russell, P. S., N. Engl. J. Med. 305, 308, 1981. 10. Ellis, T. M., Berry, C. R., Mendez-Picon, G., Goldman, M. H., Lower, R. R., Lee, H. M., and Mohanakumar, T., Transplantation 33, 317, 1982. 11. HLyry, P., and von Willebrand, E., Ann. Clin. Res. 13, 288, 1981. 12. Hayry, P., von Willebrand, E., Ahonen, J., Eklund, B., and Lautenschlager, I., Ann. Clin. Res. 13, 265, 1981. 13. Ghetie, V., Nilsson, K., and Sjijquist, J., Stand. J. Immunol. 3, 397, 1974. 14. Dimitriu, A., Debray-Sachs, M., Descamps, B., Sultan, C., and Hamburger, J., Transplant. Proc. 3, 1577, 1971. 15. Hayry, P., Ialla, M., Pastemack, A., and Virolainen, M., Ann. Clin. Res. 4, 100, 1972. 16. Platt, J. L., LeBien, T. W., and Michael, A. F., J. Exp: Med. 155, 17, 1982. 17. Hancock, W., and Atkins, C., Transplantation (in press). 18. Datiman, M. J., Mason, D. W., and Webb, M., Eur. J. Iinmunol. 12, 511, 1982. 19. Bhan, A. K., Mihm, M.-C, Jr., and Dvorak, H. F., J. Immunol. 129, 1578, 1982. 20. Renkonen, R., Nemlander, A., Soots,A., van Willebrand, E., and Hayry, P., submitted for publication.
IMMUNOLOGY
77, 196-201 (1983)
OKT4/8 Ratio in the Blood and in the Graft during Episodes of Human Renal Allograft Rejection EEVA VON WILLEBBAND’ Transplantation Laboratory and Fourth Department of Surgery, University of Helsinki, Helsinki, Finland Received December 20, 1982; accepted January 7, I983
We have analyzed the frequency of T helper (Th) and T suppressor/killer (Ts/k) lymphocytes in the blood and in the renal allograft during episodes of rejection and during quiescence. Monoclonal OKT4 and OKT8 antibodies were used to mark the Th and Ts/k cells, respectively. Density centrifugation-separated mononuclear leukocytes and FACS IV cell sorter or the Staphylococcus aureus rosette assay were used to determine the ratio in the blood, with concordant results. Fine needle aspiration biopsy (FNAB) and the Staph. assay were used to demonstrate the lymphocyte subtypes in the graft. The mean OKT4/8 ratio in the blood was significantly lower in the transplant recipients than in healthy controls (1.1 f 0.7 vs 1.8 + 0.2, respectively, P = 0.000). The individual variation was, however, high and no correlation between the OKT4/ 8 ratio in the blood and the inflammatory episodes in situ was observed. During 19 of the 25 episodes of inflammation, the dominant lymphocyte subtype in the graft was the Ts/k cell. In the remaining six casesit was the Th cell. All rejection episodesof the former type were reversible, in the latter type, four out of six were irreversible.
INTRODUCTION Several different inflammatory pathways may apparently lead to allograft rejection. In renal transplantation in man, the most common is acute cellular rejection characterized by the presence of lymphocytes and blast cells in the graft inflammatory cell cytology (1). During advanced and irreversible rejection mononuclear phagocytes are superimposed on the inflammatory picture (1). A lesscommon cytological pattern of inflammation is dominated (with a minimal or no blastogenic response) by lymphocytes and mononuclear phagocytes (2). This type of cytological picture is encountered particularly during “vascular” rejection. Finally there are patterns of inflammation where mononuclear phagocytes, including monoblasts, dominate the inflammatory cell cytology (2). Several methods have been proposed to correlate changes in the peripheral blood to episodes of rejection (3-8). It has recently been reported that elevated T helper (Th)-T suppressor/killer (Ts/k) cell ratios in the blood are indicative for acute rejection and may be used as a diagnostic aid (9, 10). Becausethis type of finding, if substantiated, would lead to a new important monitoring method in renal trans’ Address all correspondence to Dr. Eeva von Willebrand, Transplantation Laboratory, University of Helsinki, Haartmaninkatu 3, SF 00290 Helsinki 29, Finland. 196 0008-8749183$3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
OKT4/8 RATIO DURING REJECTION
197
plantation in man, we found it important to reexplore the changes in the peripheral blood and correlate them to the inflammatory episodes in situ. Fine needle aspiration biopsy (FNAB) is a new, atraumatic method for continuous monitoring of intragraft events in parenchymal organ transplantation in man. In this communication we have investigated the inflammatory profiles of (first) renal allograft rejection in three types of patients: patients immunosuppressed with azathioprine (AZA) plus methylprednisolone (MP), patients immunosuppressed with cyclosporin A (CyA), and patients immunosuppressed with CyA plus MP. We have analyzed the frequency of Th and Ts/k cells in the blood and in the graft during episodes of inflammation and during quiescence. The results demonstrate that on most occasions the dominant inflammatory lymphocyte in the graft is the Ts/k cell and that no correlation exists between inflammatory episodesof rejection on one hand and changes in the blood Th-Ts/k ratio on the other. MATERIALS
AND METHODS
Patients. Forty recipients of cadaveric renal transplants were included in the study. For immunosuppression, 14 patients received AZA + MP, 12 CyA, and 14 CyA + MP. The patients were on the average 1.5 HLA-AB and 0.5 HLA-DR antigen mismatched with no significant differences between the three groups. All patients were transfused at least three times before transplantation and none of them had cytotoxic antibodies to a panel of 24, or to the donor lymphoid cells before transplantation, Immunosuppression. The first group of patients received AZA 2 mg/kg/day or in the maximal tolerated dose, and 3.6 mg/kg/day of MP tapered to 0.5 mg/kg in 20 days. The second group of patients received 20 mg/kg/day of CyA iv for the first 3 days, tapered to 10 mg/kg/day po on the third day; thereafter the maintenance dose was adjusted according to the level of CyA in plasma, by keeping it as close to 200 rig/ml as possible. The third group of patients received CyA as above, and 3.6 mg/ kg/day of MP tapered to 0 mg/kg/day in 12 days. Diagnosis of rejection. Clinical diagnosis of rejection was based on the generally accepted clinical signs: fever, swelling, and tenderness of the graft, decreased urine output, and increasing serum creatinine, in the absence of pre- and postrenal complications and signs of viral infection. Fine needle aspiration biopsies and blood specimens. To obtain the inflammatory cell profile (see Fig. l), all 40 patients were aspiration biopsied on the average every second day post-transplantation, and concomitantly a blood specimen of approximately 20 ~1 in size was drawn from a fingertip. Blood Th-Ts/k ratio (Fig. 2) was monitored from these patients on 53 occasions. This was performed either from a heparinized blood specimen of 10 ml using FACS IV flow cytometer (Becton Dickinson Co., Sunny Valley, Calif.) or (occasionally) from a fingertip specimen by the Staphylococcusaureus rosette assay,with concordant results (see below). The Th-Ts/k ratio in the graft was determined in 20 patients on 37 occasions (Fig. 2). On 25 occasions the biopsy was obtained during established episodesof rejection; on 7 occasions prior to the onset of rejection and on the remaining 5 occasions after the rejection was overcome. Thus the graft Th-Ts/k ratio was determined in several patients at least twice, either during rejection or during quiescence and rejection. Processing of the FNAB specimen for quantitation of inflammation. The FNAB
198
EEVA VON WILLEBBAND
I
6r
4
2
I
I
I
0
5
10
LI -5
DAYS IN RELATION
A 15
TO ONSET OF REJECTION
FIG. 1. FNAB analysis of the inflammatory profiles of (first) episodes of rejection in patients receiving for immunosuppression azathioprine (AZA) plus methylprednisolone (MP) (top), cyclosporin A (CyA) (middle), or CyA plus MP.
specimen was processedwith a cytocentrifuge, stained with May-Gtinwald-Giemsa (MGG), and the inflammatory changes were quantitated against blood background as described (11). The in situ inflammation is expressed in “corrected increment” units ( 12). 0 AZA+MP
OD a
J I I
3
y’ 0 2-A
I I
'A
15 0 DAYS IN RELATION
A 8
Graft
10 5 215 TO ONSET OF REJECTl0i.j
FIG. 2. OKT4/8 ratio in the blood and in the graft as adjusted to the onset of clinical rejection (vertical dashed line). Circles: patients receiving azathioprine plus methylprednisolone. Squares: patients receiving cyclosporin A. Triangles: patients receiving cyclosporin A plus methylprednisolone. Open figures: reversible rejection; closed figures: irreversible rejection.
OKT4/8 RATIO DURING REJECTION
199
Processing of the heparinized blood specimen. Blood mononuclear leukocytes were isolated by Ficoll-Isopaque (Pharmacia Fine Chemicals, Uppsala, Sweden) density centrifugation and washed in phosphate-buffered saline (PBS). Monitoring of the Th-Ts/k ratio in the blood and in the graft. OKT4 monoclonal antibody was used to recognize the Th and OKT8 the Ts/k subset. The antibodies were purchased from Ortho Pharmaceuticals, Raritan, N.J. Blood mononuclear leukocytes were treated with 1:10 diluted monoclonal antibody, washed,treated with 1:20 diluted FITC-labeled sheepanti-mouse Ig, and washed. The frequency of fluorescent cells was quantitated with FACS IV flow cytometer. With this method of analysis the OKT4/8 ratio in healthy controls is in our hands 1.8 f 0.2 (SD). The OKT4/OKT8 ratio in the graft was determined at least once in each patient and, if heparinized blood was not available, the simultaneously obtained fingertip specimen was processedin identical fashion. Approximately 30 ~1of FNAB material (600,000-3 X lo6 nucleated cells) was divided into two batches, washed, treated with 1:20 diluted monoclonal antibody, washed, and the cell-bound antibody was chased with Staphylococcus aureus Cowan I bacteria (a gift from Prof. H. Wigzell, University of Uppsala, Uppsala, Sweden). As described in detail ( 11, 13), the staphylococci with a high content of protein A bind to the cell-bound antibody, forming rosettes with the antibody-coated cells. When the cells are processedonto microscope slides and stained with MGG, it is possible to identify morphologically the rosette-forming cell OwW ( 11).
RESULTS Projles of the First Episode of Inflammation
The net inflammatory responsesof the first episode of rejection in the three groups of patients are demonstrated in Fig. 1. Most of these episodes commenced during the first 15 days post-transplantation. As is seen in Fig. 1, the background inflammation in patients receiving AZA + MP was lowest and duration of the first inflammatory episode shortest. The background inflammation in patients receiving CyA was higher than in patients receiving CyA + MP. OKT4/8 Ratio in the Graft and in the Blood
Table 1 summarizes the findings in the graft and in the blood during quiescence and during episodes of inflammation. During quiescence the OKT4/8 ratio in the graft was below 1.O on 5 of 12 occasions; in the blood it was below 1.O in 13 of 2 1 occasions. During episodesof inflammation the OKT4/8 ratio in the graft was below 1.0 and 19 of 25 occasions; in the blood it was below 1.0 in 17 of 32 occasions. Ratios below one were thus recorded in the graft significantly more often during episodes of inflammation than during episodes of quiescence (P = 0.040 in x2 test). On the contrary, ratios below one were equally frequent in the blood during episodes of inflammation compared to episodes of quiescence (P = 0.528 in x2 test). Sequential follow-up of the OKT4/8 ratio through the episodes of inflammation is demonstrated in Fig. 2. Although the mean OKT4/OKT8 ratio in the blood of renal allograft recipients was well below that of normal controls (1.1 + 0.7 vs 1.8 f. 0.3, P = O.OOO), there was a wide variation throughout the episode and at no time during rejection could any sensible correlations between the OKT4/8 ratio in the blood and the inflammatory episode in the graft be recorded.
200
EEVA VON WILLEBRAND TABLE 1 OKT4/8 Ratio in the Graft and in the Blood during Inflammatory Episodes of Rejection and during Quiescence OKT4/8 ratio Graft
During inflammation During quiescence P in x2 test
Blood
<1
3-I
19 5
6 7 0.040
>I
17 13
15 8 0.528
In clear contrast there was a distinct depression of the OKT4/8 ratio in the graft which reached its nadir at the onset of clinical rejection and gradually disappeared when the inflammatory episode was overcome. However, in six biopsies taken during episode of inflammation the intragraft OKT4/8 ratio remained high, even elevated. Four of these six grafts were lost. DISCUSSION Although several methods have been proposed for monitoring of the in situ episodes of inflammation (i.e., rejection) from changes taking place in the peripheral blood, many of them have been shown to be inconsistent in later critical analysis (14, 15) and none of them is universally used or has replaced the biopsy. This seemsto be the case also with the Th-Ts/k ratio. In our analysis the OKT4/ 8 ratio was definitely lower in the blood of transplant recipients than in healthy subjects, but large individual variations were apparent, without any correlation to the inflammatory episodesin the graft. This result coincides with several recent results. For example, in a recent international meeting (IX International Meeting of the Transplantation Society, Brighton, England 1982) as many papers were presented suggesting a correlation as a lack of correlation, and in papers indicating that a correlation exists, two entirely divergent opinions emerged: the Th-Ts/k ratio was reported to be either elevated or depressedduring episodes of rejection. Needless to say, in most of these studies, no biopsies were made of the graft to prove the presence or lack of inflammation in situ. The observation that the Ts/k lymphoid cells dominate in the graft during episodes of rejection carries a good correlation to recent findings in rat and man. Platt et al. (16) and Hancock and Atkins (17) have reported an increased frequency of Ts/k lymphoid cells in biopsies of acutely rejecting human renal allografts. Dallman et al. ( 18) and Bhan et al. ( 19) have found the Ts/k subset to dominate in the inflammatory infiltrate of epidermis in skin allografts in rat and man. Renkonen et al. (20) have shown that lymphoid cells with a Ts/k phenotype dominate in rat renal allografts during rejection. This dominance is even stronger if one considers only the “activated” lymphoid cells, i.e., lymphoid celk with a blast cell phenotype. In striking contrast in the recipient spleen, Th blasts dominated throughout the course of rejection. In the FNAB analysis, the pre- and postrejection biopsies represent mainly blood background. Thus most emphasis should be placed on biopsies taken during inflammation. Here, biopsies fell in two distinct categories: in biopsies taken - 1 to + 10
OKT4/8 RATIO DURING REJECTION
201
days after the onset of inflammation, the Ts/k cells dominated on 19 occasions but there was a distinct Th cell predominance on 6 occasions. It is not known whether these two patterns of inflammation represent independent inflammatory pathways of allograft rejection. This is, however, possible as in the latter group five of six patients were on CyA (which drug may deviate the pattern of inflammation; Ref. 2) and four of six patients (including those on CyA and one on AZA) had an irreversible rejection whereas in the former group all rejections were reversible. Finally it should be emphasized that other intragraft events in addition to rejection may modify the inflammation. Hammer et al. (personal communication) have demonstrated with the FNAB that during cytomegalovirus infection of the graft, there is an even stronger Ts/k predominance than during ordinary episodes of rejection; the two conditions may, however, be distinguished by the presence of viral antigen in the graft tubular cells. In bronchoalveolar lavage specimens taken from the lung of human bone marrow transplant recipients, viral infections-verified by the demonstration of the virus-are always associated with an extremely strong Ts/k cell predominance at the site of inflammation (Taskinen et al., unpublished results). ACKNOWLEDGMENTS I thank Dr. P. Hayry and the staff of the Transplantation Laboratory and the Division of Transplantation Surgery for co-operation and Ms. Leena Sarastefor secretarial and editorial help. This study was financed by grant 1 ROl AM 26882-03 from the National Institutes of Health, Bethesda, Maryland, and by grants from the Association of Finnish Life Insurance Companies, and Finska LZkaresallskapet, Helsinki, Finland.
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