Selective expansion of a peripheral blood CD8+ memory T cell subset expressing both granzyme B and l -selectin during primary viral infection in renal allograft recipients

Selective Expansion of a Peripheral Blood CD81 Memory T Cell Subset Expressing Both Granzyme B and L-Selectin During Primary Viral Infection in Renal Allograft Recipients I.J.M. ten Berge, P.C. Wever, R.J. Rentenaar, L.H.A. Spaeny, J. Surachno, P.M.E. Wertheim, P.T.A. Schellekens, and C.E. Hack

D

URING posttransplant primary cytomegalovirus (CMV) infection, expansion of the peripheral blood (PB) CD81 T cell subset is commonly observed.1 Granzyme A (GrA) has been detected in up to 90% of PB CD81 lymphocytes from renal allograft recipients experiencing CMV infection,2 whereas the presence of granzyme B (GrB)1 lymphocytes has been reported in the bronchoalveolar lavage fluid from lung and heart–lung allograft recipients suffering from CMV pneumonia.3 We have produced monoclonal antibodies (mAb) directed against native GrB, which are suitable for three-color flow cytometry. In the present study, we measured expression of GrB in PB CD81 T cells from renal allograft recipients during stable transplant function, acute rejection or primary viral infection. To analyze the naive or memory/effector phenotype of these cells, we analyzed co-expression of the lymph node (LN) homing receptor CD62L (L-selectin), CD45RO or Fas. Naive PB CD81 T cells uniformly express CD62L.4 During generation of memory/effector cells, CD62L expression can be differentially regulated, leading to the production of either CD62L2 or CD62L1 memory/effector T

cells.5 Furthermore, we analyzed co-expression of the activation markers HLA-DR or CD25 on PB CD81GrB1 T cells. PATIENTS AND METHODS Three groups of renal allograft recipients were evaluated. Patient characteristics as well as individual plasma creatinine levels and CD41/CD81 T cell ratios are presented in Table 1. Patients 7 and 11 were evaluated during an acute rejection episode as well as during a subsequent primary viral infection episode. Group I From the Academic Medical Center, Renal Transplant Unit, Department of Clinical Immunology and Rheumatology, Clinical and Laboratory Immunology Unit, Department of Internal Medicine, Department of Clinical Virology, and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, The Netherlands. This study was financially supported by the Dutch Kidney Foundation, grant C93.1278. Address reprint requests to Dr I.J.M. ten Berge, Academic Medical Center, Renal Transplant Unit, PO Box 22700, 1100 DE Amsterdam, Netherlands.

Table 1. Patient Characteristics

Group I

Group II

Group III

Patient

Sex

Age (y)

1 2 3 4 5 6 7 8 9 10 11 7 11 12 13

M M F M F M F M M M F F F M M

35 52 40 37 32 53 26 37 61 33 51 27 51 41 51

Time Posttransplantation

105 months 101 months 107 months 79 months 103 mohths 151 months 17 days 6 days 63 days 44 days 8 days 38 days 50 days 39 days 46 days

Immunosuppressive Treatment

P P P P P P P P P P P P P P

CsA CsA CsA CsA CsA CsA CsA CsA CsA CsA CsA CsA CsA CsA CsA

Plasma Creatinine (mmol/L)

CD41/CD81 T-Cell Ratio

Diagnosis

180 58 98 233 56 100 431 394 543 211 436 495 422 405 139

1.78 1.18 1.75 1.70 2.58 1.97 3.08 1.40 3.99 1.07 4.31 0.76 1.16 0.50 0.39

Stable Stable Stable Stable Stable Stable AR AR AR AR AR EBV CMV CMV CMV

Abbreviations: M, male; F, female; P, prednisolone; CsA, cyclosporin; AR, acute rejection; EBV, primary Epstein–Barr virus infection; CMV, primary cytomegalovirus infection.

© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0041-1345/98/$19.00 PII S0041-1345(98)01309-8

Transplantation Proceedings, 30, 3975–3977 (1998)

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Table 2. Percentages of CD62L1, CD45RO1, Fas1, HLA-DR1 and CD251 Cells Among Peripheral Blood CD81 Granzyme B1 T Cells From Renal Allograft Recipients During Stable Transplant Function (Group I), Acute Rejection (Group II), or Primary Viral Infection (Group III)

Group I Group II Group III

CD62L1

CD45RO1

Fas1

HLA-DR1

CD251

34 (15–37) 33 (18 –59) 81* (72–93)

77 (54 –95) 71 (47– 88) 94 (78 –97)

88 (61–92) 69 (46 –91) 85 (81–91)

46 (31– 81) 67 (19 – 89) 90 (84 –95)

0 (0 –1) 6† (2–19) 2 (0 – 6)

Data represent percentages of cells expressing a marker of interest among CD81 granzyme B1 T cells (median (range)). *P , .05 for group III vs group I and group II. † P , .05 for group II vs group I.

consisted of 6 patients during stable transplant function, over a period of at least 6 years. Group II consisted of 5 patients suffering from acute rejection, confirmed by histology. Group III consisted of 4 patients experiencing primary CMV or EBV infection, based on the appearance of specific IgM-antibodies and positive buffycoat cultures. Heparinized PB samples were obtained by venepuncture and PB mononuclear cells (PBMC) were isolated by Ficoll-Paque density gradient centrifugation.

Primary Antibodies Phycoerythrin (PE)-labeled IgG1 mAb GrB-11 was used for flow cytometric analysis of GrB expression.6 Other mAb used were fluorescein isothiocyanate (FITC)-labeled CD3 mAb (Becton Dickinson (BD), San Jose, Calif), FITC-labeled CD4 mAb (BD), PE-labeled CD8 mAb (BD), and RPE-Cy5–labeled CD8 mAb (DAKO, Glostrup, Denmark); FITC-labeled CD25 mAb (BD), FITC-labeled CD45RO mAb (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB), Amsterdam, The Netherlands), FITC-labeled CD62L mAb (BD), FITC-labeled anti-HLA-DR mAb (BD), FITC-labeled anti-TCR-gd-1 mAb (BD), and IgM anti-Fas mAb (Immunotech, Marseille, France). Antibodies used as isotype control were FITC- and PE-labeled IgG1 mAb and FITC-labeled IgG2a mAb directed against keyhole limpet hemocyanin (BD), RPE-Cy5-labeled IgG1 mAb directed against a nonbiological hapten (Immunotech) and purified myeloma IgM (ICN, Costa Mesa, Calif).

Flow Cytometric Analysis Isolated PBMC were washed and transferred to polystyrene roundbottom tubes. Cells were first incubated for 30 minutes with antibodies directed against surface markers or appropriate isotype control antibodies. Cells that had been incubated with unlabelled antibodies (eg, IgM anti-Fas mAb and irrelevant IgM antibodies) were additionally incubated for 30 minutes with FITC-labeled goat anti-mouse F(ab9)2 fragments (CLB). They were then washed and incubated for 10 minutes with 10% (v/v) normal mouse serum and then were washed again, followed by a separate incubation for 30 minutes with RPE-Cy5–labeled CD8 mAb, after which the cells were washed once again. Subsequently, all cells were fixed with 50 mL buffered formaldehyde acetone. Cells were gently mixed during fixation. After 90 seconds, cells were washed in saponin detergent composed of phosphate buffered saline (PBS) supplemented with 0.5% (w/v) bovine serum albumin, 0.01% (w/v) sodium azide (PBS-BSA-NaN3), 0.1% (w/v) saponin and 50 mmol/L D-glucose. Thereafter, cells were washed in saponin detergent containing 10% (v/v) normal human serum (NHS) and incubated with PE-labeled GrB-11 mAb and IgG1 isotype control mAb. After 1 hour, cells

were washed in saponin detergent. Data acquisition was performed on a FACScan flow cytometer (BD) and a number of 2.5 3 104 events were analyzed. PB lymphocytes (PBL) were gated by forward and side scatter parameters.

Statistical Analysis Data are presented as mean and range. Significance was assessed using the Mann–Whitney test for unpaired data. P , .05 was considered significant.

RESULTS

PBL were gated by forward and side scatter parameters. The CD81 T cell subset within PBL was discriminated on the basis of bright CD8 staining. This subset consisted for 100% of CD31 T cells and for 1% (0% to 2%) of TCR-gd1 T cells. No differences in PB CD81GrB1 T cell counts were observed between renal allograft recipients during either stable transplant function (107/mm3, 69 to 400) or acute rejection (60/mm3, 18 to 404). During both acute rejection and stable transplant function, CD62L was absent on the majority of PB CD81GrB1 T cells. In contrast, an increase in PB CD81GrB1 T-cell counts was observed during primary viral infection (413/mm3, 224 to 865), while the majority of these cells appeared to coexpress CD62L. Consequently, marked increases in the number of CD81GrB1CD62L1 T cells were observed, 359/mm3 (181 to 625); as compared to stable transplant function, 33 (15 to 157); and acute rejection, 24 (9 to 152). Table 2 shows the percentages of CD62L1, CD45RO1, Fas1, HLA-DR1, and CD251 cells among PB CD81GrB1 T cells in the different groups of renal allograft recipients. CD62L was the only marker of which expression among PB CD81GrB1 T cells during posttransplant primary viral infection did not overlap with those among PB CD81GrB1 T cells during either stable transplant function or acute rejection. DISCUSSION

In conclusion, a marked expansion of the PB CD81GrB1 T-cell subset during posttransplant primary viral infection was demonstrated. No difference in PB CD81GrB1 T-cell counts was observed between renal allograft recipients with either stable transplant function or acute rejection. Trapping of cells in the renal allograft presumably explains the

GRANZYME B AND L-SELECTIN IN INFECTION

absence of increased numbers of CD81GrB1 T cells in the PB compartment during acute rejection. In a mouse model, skin grafting induced a subpopulation of allospecific CD81GrA1CD62L2 T cells in draining LN, expressing high levels of adhesion molecules.7 This indicates that, once in the circulation, these cells are highly susceptible to extravasation into sites containing specific antigens. The majority of PB CD81GrB1 T cells during primary viral infection appeared to co-express CD62L, the homing receptor which was absent on the majority of PB CD81GrB1 T cells during either stable transplant function or acute rejection. The presence of CD45RO on the majority of PB CD81GrB1 T cells during both primary viral infection and acute rejection indicates that in both cases these cells are predominantly memory/effector T cells. Activation of naive T cells, which uniformly co-express CD45RA and CD62L,4 results in loss of the CD45RA epitope and gain of the CD45RO epitope.5 During this CD45RA/RO conversion, CD62L is initially upregulated, concomitant with blastogenesis, but is then heterogenously downregulated, leading to the production of both CD45RO1CD62L1 and CD45RO1CD62L2 memory/ef-

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fector T cells, concomitant with a return to small lymphocyte size. This differential expression of CD62L has been suggested to be determined by microenvironmental factors (e.g., local cytokine levels).5 The presence of CD62L on the majority of CD81GrB1 memory/effector T cells during posttransplant primary viral infection suggests that these cells can directly migrate from the PB compartment into peripheral LN. REFERENCES 1. Labalette M, Salez F, Pruvot FR, et al: Clin Exp Immunol 95:465, 1994 2. Sunder-Plassmann G, Wagner L, Hruby K, et al: Kidney Int 37:1350, 1990 3. Humbert M, Devergne O, Cerrina J, et al: Am Rev Respir Dis 145:1178, 1992 4. Roederer M, Dubs JG, Anderson MT, et al: J Clin Invest 95:2061, 1995 5. Picker LJ, Treer JR, Ferguson-Darnell B, et al: J Immunol 150:1105, 1993 6. Su B, Bochan MR, Hanna WL, et al: Eur J Immunol 24:2073, 1994 7. Mobley JL, Dailey MO: J Immunol 148:2348, 1992