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Could expression of co-stimulatory molecules on B-PBL condition the acceptance or rejection of human liver grafts?
Could Expression of Co-Stimulatory Molecules on B-PBL Condition the Acceptance or Rejection of Human Liver Grafts? A. Minguela, F. Sa´nchez-Bueno, L. Marı´n, M. Miras, J.A. Pons, M. Muro, A. Torı´o, A.M. Garcı´a-Alonso, R. Robles, P. Parrilla, P. Ramirez, and M.R. A´lvarez-Lo´pez
A
MONG other factors,1 a low expression of MHC and co-stimulatory molecules in potential antigen-presenting cells (APCs) from liver grafts makes these organs show a special tolerogenic behavior,2 probably by inducing specific anergy of recipient Th lymphocytes.3,4 However, other nonprofessional APCs in the periphery can indirectly present allo-antigens and trigger a strong alloresponse depending on their co-stimulatory molecule expression and therefore of their capacity to efficiently stimulate Th lymphocytes.5 It is well known that dendritic cells, macrophages, and activated B lymphocytes with optimal expression of B7 molecules (CD80 and CD86) are efficient APC,6
Fig 1. CD28/CTLA-4 and B7 co-stimulatory molecule expression on PBL in the pre- and posttransplant periods. (A) Absolute number of PBL cell subsets CD4⫹CD28⫹ and CD4⫹CD28⫹CTLA-4⫹ (Cell/L) of the AR and NAR rejection groups from liver recipients. (B) Mean fluorescence intensity (MFI) of B7 molecules on CD19⫹ and CD28 on CD4⫹CD28⫹ lymphocytes of AR and NAR rejection groups from liver recipients. *Indicates statistical differences between groups (P ⬍ .05). 0041-1345/01/$–see front matter PII S0041-1345(00)02520-3
whereas B7-negative resting B cells and some T lymphocytes acting as nonefficient APCs could induce anergy.7 The present study investigated changes in the expression of B7 co-stimulatory molecules in potential APCs from peripheral blood and tissues of liver transplantations, and their counter-receptors (CD28/CTLA-4) on T cells, as well as their possible roles in the acceptance or rejection of liver grafts. MATERIALS AND METHODS Both B7 (CD80/CD86) expression and CD28/CTLA-4 expression on peripheral blood cells were monitored in 74 liver recipients with different diagnoses (27 had alcoholic cirrhosis; 19 had hepatitis B or C virus cirrhosis; 10 had carcinomas; 7 had primary biliary cirrhosis; 7 were retransplants; and 4 had amyloidosis), classified into acute rejection (AR, n ⫽ 27) and nonacute rejection (NAR, n ⫽ 47) groups. The AR diagnosis was based on conventional clinical, biochemical, and histological criteria.8 Immunosuppression consisted of standard triple-drug therapy with methylprednisolone, azathioprine, and cyclosporine A (CyA). Additional individual regimens with a bolus of methylprednisolone (1 g) were used in case of AR episodes. Cytometric analysis was performed on EDTA-anticoagulated peripheral blood samples collected on preoperative (day 0) and postoperative periods: days 1 to 3, 4 to 6, 7 to 9, 10 to 13, 14 to 17, 18 to 21, 22 to 25, throughout the first month posttransplant. Blood samples were immediately stained by a standard immunofluorescence method with different monoclonal antibodies (MAbs) (antiCD4, BD; anti-CD19 Caltag, San Francisco, CA; anti-CD28, Immunotech, Marseille, France; anti-CD80, anti-CD86, and antiCTLA-4 (CD152), Pharmingen, San Diego, CA), and acquired in a FACScan flow cytometer (Becton Dickinson [BD], San Jose, CA). Mean fluorescence intensity (MFI), used as a relative moleculedensity measurement of CD80-CD86 on CD19⫹ cells or monocytes and of CD28 on CD4⫹ lymphocytes, was calculated as described From the Immunology Section (A.M., L.M., M.M., A.T., A.M.G.-A., R.R., M.R.A.-L.), Surgery Service (F.S.-B., P.P., P.R.), and Digestive Medicine Service (M.M., J.A.P.), University Hospital “Virgen de la Arrixaca,” 30120 Murcia, Spain. This work was supported by grants from the Fondo de Investigacio´n Sanitaria (F.I.S. projects 94/0382 and 97/0397), Ministerio de Sanidad y Consumo, and by “Caja Murcia.” Alfredo Minguela was a fellow of F.I.S. (B.A.E. 96/5028). Address reprint requests to Dr Maria R. A´lvarez-Lo´pez, Hospital U. Virgen de la Arrixaca, Seccio´n de Immunologı´a, El Palmar, 30120-Murcia, Spain. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1384
Transplantation Proceedings, 33, 1384–1385 (2001)
CO-STIMULATORY MOLECULES
1385
Table 1. PBL and Intragraft Expression of Co-stimulatory Molecules CD28
CTLA-4
Histologic diagnoses
PBL (⌬MFI)*
PBL (%)†
Acute rejection Colostasis Nonspecific inflammation
⫹22 ⫾ 12 ⫺7.2 ⫾ 3.5 ⫺3.0 ⫾ 15
1.8 ⫾ 1.2 0.5 ⫾ 0.3 0.5 ⫾ 0.2
‡
‡
B7 Molecules ⫹ Biopsies
PBL (⌬MFI)*
⫹ Biopsies
5 out of 5 3 out of 6 1 out of 5
⫹28 ⫾ 18 ⫺2.1 ⫾ 1.8 ⫺5 ⫾ 4.8
0 out of 5 0 out of 6 0 out of 5
‡
*⌬MFI: Mean fluorescence intensity increment on PBL. Data are expressed as posttransplant percentage changes (mean ⫾ SD) observed on the diagnosis day with respect to pretransplant values, which were considered as 0% of change. † Percentage of CD4⫹CD28⫹CTLA-4⫹ in PBLs on diagnosis day (mean ⫾ SD). ‡ Statistical differences between patients with AR and NAR diagnoses (P ⬍ .05).
previously.9 The absolute number of CD4⫹CD28⫹, CD4⫹ CD28⫹CTLA-4⫹, CD19⫹CD80⫹, and CD19⫹CD86⫹ cells was calculated from the total number of lymphocytes obtained by routine leukocyte count (Coulter T-540, Northwell Drive, UK), together with their estimated cytometric percentage values. Additionally, B7 and CTLA-4 expression was simultaneously analyzed, by standard flow-cytometric and immunohistochemical methods, on peripheral blood lymphocytes (PBLs) and 16 percutaneous liver graft biopsies with different histologic diagnoses: AR (n ⫽ 5), portal nonspecific inflammation (n ⫽ 5), and nonspecific cholestasis (n ⫽ 6). Percutaneous liver biopsies were immediately embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan) and snap-frozen in liquid nitrogen, and later stained with anti-CD80, anti-CD86, or anti-CTLA-4 (Pharmingen) MAb by using a standard immunoperoxidase protocol (Supersensitive Detection Kit, Biogenex, San Ramon, CA), as previously described.10
RESULTS
Clear differences were observed in the number of CD19⫹B7⫹ and CD4⫹CD28⫹CTLA-4⫹ cells between the AR and NAR groups in the posttransplant period (Fig 1A). Thus, in the NAR group the absolute number of these peripheral blood cell subsets remained at pretransplant levels, whereas in the AR group they significantly increased from days 7 to 17 after the graft (P ⬍ .05). In this period, AR was detected in 13 of 16 AR patients. Additionally, in the NAR group the MFI of CD80/CD86 on CD19⫹ cells as well as that of CD28 on CD4⫹CD28⫹ lymphocytes remained at basal levels. In contrast, all these molecules were significantly up-regulated in patients from the AR group on days with the highest frequency of AR (P ⬍ .01), peaking on the day of rejection diagnosis (Fig 1B). After antirejection treatment, CD28 and CTLA-4 recovered to the pretransplant level, but CD80 and CD86 up-regulation persisted 4 to 6 days beyond diagnosis. The expression of CD86 in monocytes declined in the posttransplant period while that of CD80 was undetectable, but in no case were significant differences found between the AR and NAR groups. CTLA-4 expression was observed in the infiltrating lymphocytes of the portal tracts of all sample biopsies from patients with AR and in 4 of 11 biopsies of those without rejection. Neither CD80 nor CD86 expression was undetected in any of these cases (Table 1). DISCUSSION
The AR of human liver transplants is associated with an up-regulation of CD28/CTLA-4 –B7 molecules on T and B
from PBLs.9,11,12 These findings suggest that Th allospecific clones are activated in peripheral tissues by alloantigen recognition on autologous APCs (possibly B7-positive B lymphocytes). In liver transplants, immunosuppressive agents and the high posttransplant IL-10 serum concentration10,13 might hinder the ability of macrophages and dendritic cells to stimulate Th lymphocytes.14,15 Therefore, it is tempting to speculate that, depending on their costimulatory molecule levels,6,7 B lymphocytes can be the APCs that modulate the host alloresponse that leads to graft rejection or acceptance. Thus, resting-B lymphocytes (B7 negatives) from NAR recipients would present alloantigens to Th cells in a tolerogenic manner favoring graft acceptance, whereas activated-B lymphocytes (B7 positives) from the AR patients, using the CD28 pathway, might be efficient APCs.
REFERENCES 1. Kamada N: In Tilney NL, Strom TB, Paul LC (eds): Lippincott-Raven; 1996; p 531 2. Leon MP, Bassendine MF, Wilson JL, et al: Hepatology 24:561, 1996 3. Bachmann MF, McKall-Faienza K, Schmits R, et al: Immunity 7:549, 1997 4. Boussiotis VA, Freeman GJ, Gribben JG, et al: Immunol Rev 153:5, 1996 5. Malajoni ER, Cinti P, Orlandini A, et al: Hum Immunol 53:57, 1997 6. Wilson JL, Cunningham AC, Kirby JA: Immunology 86:325, 1995 7. Croft M, Joseph SB, Miner KT: J Immunol 159:3257, 1997 8. Mor E, Solomon H, Gibbs JF, et al: Semin Liver Dis 12:28, 1992 9. Minguela A, Marı´n L, Muro M, et al: Hum Immunol 61:658, 2000 10. Minguela A, Torı´o A, Marı´n L, et al: Hum Immunol 60:500, 1999 11. Garcı´a-Alonso AM, Minguela A, Muro M, et al: Hum Immunol 53:64, 1997 12. Minguela A, Garcı´a-Alonso AM, Marı´n L, et al: Transplant Proc 29:499, 1997 13. Minguela A, Torı´o A, Marı´n L, et al: Transplant Proc 31:516, 1999 14. Peguet-Navarro J, Moulon C, Caux C, et al: Eur J Immunol 24:884, 1994 15. Willems F, Marchant A, Delville JP, et al: Eur J Immunol 24:1007, 1994
A
MONG other factors,1 a low expression of MHC and co-stimulatory molecules in potential antigen-presenting cells (APCs) from liver grafts makes these organs show a special tolerogenic behavior,2 probably by inducing specific anergy of recipient Th lymphocytes.3,4 However, other nonprofessional APCs in the periphery can indirectly present allo-antigens and trigger a strong alloresponse depending on their co-stimulatory molecule expression and therefore of their capacity to efficiently stimulate Th lymphocytes.5 It is well known that dendritic cells, macrophages, and activated B lymphocytes with optimal expression of B7 molecules (CD80 and CD86) are efficient APC,6
Fig 1. CD28/CTLA-4 and B7 co-stimulatory molecule expression on PBL in the pre- and posttransplant periods. (A) Absolute number of PBL cell subsets CD4⫹CD28⫹ and CD4⫹CD28⫹CTLA-4⫹ (Cell/L) of the AR and NAR rejection groups from liver recipients. (B) Mean fluorescence intensity (MFI) of B7 molecules on CD19⫹ and CD28 on CD4⫹CD28⫹ lymphocytes of AR and NAR rejection groups from liver recipients. *Indicates statistical differences between groups (P ⬍ .05). 0041-1345/01/$–see front matter PII S0041-1345(00)02520-3
whereas B7-negative resting B cells and some T lymphocytes acting as nonefficient APCs could induce anergy.7 The present study investigated changes in the expression of B7 co-stimulatory molecules in potential APCs from peripheral blood and tissues of liver transplantations, and their counter-receptors (CD28/CTLA-4) on T cells, as well as their possible roles in the acceptance or rejection of liver grafts. MATERIALS AND METHODS Both B7 (CD80/CD86) expression and CD28/CTLA-4 expression on peripheral blood cells were monitored in 74 liver recipients with different diagnoses (27 had alcoholic cirrhosis; 19 had hepatitis B or C virus cirrhosis; 10 had carcinomas; 7 had primary biliary cirrhosis; 7 were retransplants; and 4 had amyloidosis), classified into acute rejection (AR, n ⫽ 27) and nonacute rejection (NAR, n ⫽ 47) groups. The AR diagnosis was based on conventional clinical, biochemical, and histological criteria.8 Immunosuppression consisted of standard triple-drug therapy with methylprednisolone, azathioprine, and cyclosporine A (CyA). Additional individual regimens with a bolus of methylprednisolone (1 g) were used in case of AR episodes. Cytometric analysis was performed on EDTA-anticoagulated peripheral blood samples collected on preoperative (day 0) and postoperative periods: days 1 to 3, 4 to 6, 7 to 9, 10 to 13, 14 to 17, 18 to 21, 22 to 25, throughout the first month posttransplant. Blood samples were immediately stained by a standard immunofluorescence method with different monoclonal antibodies (MAbs) (antiCD4, BD; anti-CD19 Caltag, San Francisco, CA; anti-CD28, Immunotech, Marseille, France; anti-CD80, anti-CD86, and antiCTLA-4 (CD152), Pharmingen, San Diego, CA), and acquired in a FACScan flow cytometer (Becton Dickinson [BD], San Jose, CA). Mean fluorescence intensity (MFI), used as a relative moleculedensity measurement of CD80-CD86 on CD19⫹ cells or monocytes and of CD28 on CD4⫹ lymphocytes, was calculated as described From the Immunology Section (A.M., L.M., M.M., A.T., A.M.G.-A., R.R., M.R.A.-L.), Surgery Service (F.S.-B., P.P., P.R.), and Digestive Medicine Service (M.M., J.A.P.), University Hospital “Virgen de la Arrixaca,” 30120 Murcia, Spain. This work was supported by grants from the Fondo de Investigacio´n Sanitaria (F.I.S. projects 94/0382 and 97/0397), Ministerio de Sanidad y Consumo, and by “Caja Murcia.” Alfredo Minguela was a fellow of F.I.S. (B.A.E. 96/5028). Address reprint requests to Dr Maria R. A´lvarez-Lo´pez, Hospital U. Virgen de la Arrixaca, Seccio´n de Immunologı´a, El Palmar, 30120-Murcia, Spain. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1384
Transplantation Proceedings, 33, 1384–1385 (2001)
CO-STIMULATORY MOLECULES
1385
Table 1. PBL and Intragraft Expression of Co-stimulatory Molecules CD28
CTLA-4
Histologic diagnoses
PBL (⌬MFI)*
PBL (%)†
Acute rejection Colostasis Nonspecific inflammation
⫹22 ⫾ 12 ⫺7.2 ⫾ 3.5 ⫺3.0 ⫾ 15
1.8 ⫾ 1.2 0.5 ⫾ 0.3 0.5 ⫾ 0.2
‡
‡
B7 Molecules ⫹ Biopsies
PBL (⌬MFI)*
⫹ Biopsies
5 out of 5 3 out of 6 1 out of 5
⫹28 ⫾ 18 ⫺2.1 ⫾ 1.8 ⫺5 ⫾ 4.8
0 out of 5 0 out of 6 0 out of 5
‡
*⌬MFI: Mean fluorescence intensity increment on PBL. Data are expressed as posttransplant percentage changes (mean ⫾ SD) observed on the diagnosis day with respect to pretransplant values, which were considered as 0% of change. † Percentage of CD4⫹CD28⫹CTLA-4⫹ in PBLs on diagnosis day (mean ⫾ SD). ‡ Statistical differences between patients with AR and NAR diagnoses (P ⬍ .05).
previously.9 The absolute number of CD4⫹CD28⫹, CD4⫹ CD28⫹CTLA-4⫹, CD19⫹CD80⫹, and CD19⫹CD86⫹ cells was calculated from the total number of lymphocytes obtained by routine leukocyte count (Coulter T-540, Northwell Drive, UK), together with their estimated cytometric percentage values. Additionally, B7 and CTLA-4 expression was simultaneously analyzed, by standard flow-cytometric and immunohistochemical methods, on peripheral blood lymphocytes (PBLs) and 16 percutaneous liver graft biopsies with different histologic diagnoses: AR (n ⫽ 5), portal nonspecific inflammation (n ⫽ 5), and nonspecific cholestasis (n ⫽ 6). Percutaneous liver biopsies were immediately embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan) and snap-frozen in liquid nitrogen, and later stained with anti-CD80, anti-CD86, or anti-CTLA-4 (Pharmingen) MAb by using a standard immunoperoxidase protocol (Supersensitive Detection Kit, Biogenex, San Ramon, CA), as previously described.10
RESULTS
Clear differences were observed in the number of CD19⫹B7⫹ and CD4⫹CD28⫹CTLA-4⫹ cells between the AR and NAR groups in the posttransplant period (Fig 1A). Thus, in the NAR group the absolute number of these peripheral blood cell subsets remained at pretransplant levels, whereas in the AR group they significantly increased from days 7 to 17 after the graft (P ⬍ .05). In this period, AR was detected in 13 of 16 AR patients. Additionally, in the NAR group the MFI of CD80/CD86 on CD19⫹ cells as well as that of CD28 on CD4⫹CD28⫹ lymphocytes remained at basal levels. In contrast, all these molecules were significantly up-regulated in patients from the AR group on days with the highest frequency of AR (P ⬍ .01), peaking on the day of rejection diagnosis (Fig 1B). After antirejection treatment, CD28 and CTLA-4 recovered to the pretransplant level, but CD80 and CD86 up-regulation persisted 4 to 6 days beyond diagnosis. The expression of CD86 in monocytes declined in the posttransplant period while that of CD80 was undetectable, but in no case were significant differences found between the AR and NAR groups. CTLA-4 expression was observed in the infiltrating lymphocytes of the portal tracts of all sample biopsies from patients with AR and in 4 of 11 biopsies of those without rejection. Neither CD80 nor CD86 expression was undetected in any of these cases (Table 1). DISCUSSION
The AR of human liver transplants is associated with an up-regulation of CD28/CTLA-4 –B7 molecules on T and B
from PBLs.9,11,12 These findings suggest that Th allospecific clones are activated in peripheral tissues by alloantigen recognition on autologous APCs (possibly B7-positive B lymphocytes). In liver transplants, immunosuppressive agents and the high posttransplant IL-10 serum concentration10,13 might hinder the ability of macrophages and dendritic cells to stimulate Th lymphocytes.14,15 Therefore, it is tempting to speculate that, depending on their costimulatory molecule levels,6,7 B lymphocytes can be the APCs that modulate the host alloresponse that leads to graft rejection or acceptance. Thus, resting-B lymphocytes (B7 negatives) from NAR recipients would present alloantigens to Th cells in a tolerogenic manner favoring graft acceptance, whereas activated-B lymphocytes (B7 positives) from the AR patients, using the CD28 pathway, might be efficient APCs.
REFERENCES 1. Kamada N: In Tilney NL, Strom TB, Paul LC (eds): Lippincott-Raven; 1996; p 531 2. Leon MP, Bassendine MF, Wilson JL, et al: Hepatology 24:561, 1996 3. Bachmann MF, McKall-Faienza K, Schmits R, et al: Immunity 7:549, 1997 4. Boussiotis VA, Freeman GJ, Gribben JG, et al: Immunol Rev 153:5, 1996 5. Malajoni ER, Cinti P, Orlandini A, et al: Hum Immunol 53:57, 1997 6. Wilson JL, Cunningham AC, Kirby JA: Immunology 86:325, 1995 7. Croft M, Joseph SB, Miner KT: J Immunol 159:3257, 1997 8. Mor E, Solomon H, Gibbs JF, et al: Semin Liver Dis 12:28, 1992 9. Minguela A, Marı´n L, Muro M, et al: Hum Immunol 61:658, 2000 10. Minguela A, Torı´o A, Marı´n L, et al: Hum Immunol 60:500, 1999 11. Garcı´a-Alonso AM, Minguela A, Muro M, et al: Hum Immunol 53:64, 1997 12. Minguela A, Garcı´a-Alonso AM, Marı´n L, et al: Transplant Proc 29:499, 1997 13. Minguela A, Torı´o A, Marı´n L, et al: Transplant Proc 31:516, 1999 14. Peguet-Navarro J, Moulon C, Caux C, et al: Eur J Immunol 24:884, 1994 15. Willems F, Marchant A, Delville JP, et al: Eur J Immunol 24:1007, 1994