Expression of T cell activation antigen CD134 (OX40) has no ...

Bone Marrow Transplantation, (1999) 23, 1013–1017  1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt

Expression of T cell activation antigen CD134 (OX40) has no predictive value for the occurrence or response to therapy of acute graft-versus-host disease in partial T cell-depleted bone marrow transplantation APA Gadisseur1, JW Gratama2, C Lamers2, JWJ van Esser1, RLH Bolhuis2 and JJ Cornelissen1 Departments of 1Hematology and 2Immunology, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands

Summary: CD134 (OX40) is a member of the tumor necrosis factor family which is expressed by activated T lymphocytes. CD134 expression on T cells was monitored during the first 35 days post-transplant in 14 patients, receiving either an HLA-identical sibling bone marrow transplant (BMT), a matched unrelated transplant (MUD-BMT) or an autologous peripheral blood progenitor cell transplant (PBPCT). The sibling and unrelated grafts were partially depleted of T cells. CD134 expression on CD4+ T cells peaked between 7 and 14 days after BMT, with a mean peak value of 45% of CD4+ cells (range 26–70%) over all three patient groups. The observed pattern of CD4+CD134+ expression, an increase during the first 2 weeks post-BMT followed by a gradual decline towards values of 15–40%, was similar in all groups. No difference in the kinetics of CD134 expression by CD4+ T cells was observed between the patients that did or did not develop graft-versus-host disease (GVHD), nor did the clinical effect of any treatment given for GVHD correlate with alterations in CD134 expression by CD4+ T cells. Absolute CD4+,CD134+ T cell numbers showed a more rapid increment after autologous PBPCT than after sibling or MUD transplants. We conclude that expression of CD134+ by CD4+ T lymphocytes cannot serve as a surrogate marker for allo-reactivity. CD134+ expression may reflect lymphocyte regeneration, rather than alloreactivity. Keywords: graft-versus-host disease; T cell; CD134 antigen; bone marrow transplantation; reconstitution

Graft-versus-host disease (GVHD) is still a major and potentially lethal complication of allogeneic bone marrow transplantation (BMT).1 It has been shown that GVHD is initiated by donor-derived, alloreactive cytotoxic T lymphocytes (CTLs, CD8+) and helper T lymphocytes (CD4+), which is then further amplified by elaboration of cytokines.2,3 The ‘cytokine storm’ concept of GVHD is Correspondence: Dr JJ Cornelissen, Department of Hematology, Daniel den Hoed Cancer Center, Groene Hilledijk 301, 3075 EA Rotterdam, The Netherlands Received 21 October 1998; accepted 3 December 1998

defined as an outpouring of endogenous cytokines resulting in an attack of various tissues by antigen-specific and non- specific effector leukocyte populations.3,4 Acute GVHD is preferably diagnosed on the basis of clinical signs together with biochemical data (liver enzymes, bilirubin).5 Other ways of monitoring and predicting acute GVHD have been proposed, such as the repopulation pattern of the T lymphocytes.6 The rate of CD8+ T cell repopulation and the expression of T cell activation markers such as CD25 have been shown to predict GVHD in some patient cohorts.7,8 CD134 (OX40) is a 50-kDa type I transmembrane protein that is expressed on activated CD4+ T lymphocytes and to a lesser extent on CD8+ T lymphocytes.9 It is a member of the tumor necrosis factor (TNF)/nerve growth factor (NGF) family. CD134 is rarely expressed by lymphocytes under normal conditions and, if it is, mainly by T lymphocytes residing in the lymph nodes. The ligand (L) for CD134 is a gp34 membrane bound protein, structurally similar to other ligands in the TNF receptor family with a still incompletely documented tissue distribution (activated B cells, vascular endothelial cells, heart, skeletal muscle, testis).10 The CD134/CD134L (gp34) system is involved in inducing T cell-dependent antibody responses, differentiation of activated B cells into immunoglobin producing plasma cells, and ‘direct’ adhesion of activated T cells to vascular endothelial cells.11–13 Studies in animal models have shown enhanced expression of CD134 by lymphocytes in the blood after bone marrow transplantation as early as 5 days post-BMT and peaking at 12 days post-BMT.14 As the increment in CD134 expression ran parallel to the development of GVHD in the rat model, it was suggested that CD134+ T lymphocytes may serve as a possible target for the therapy of acute GVHD. Preliminary data in partially mismatched human bone marrow transplants indicated that CD134 expression, while not predictive of acute GVHD, may be predictive of the response to anti-GVHD therapy.15 The current study was set out to investigate in human recipients of HLA-matched allogeneic T cell-depleted marrow grafts whether the kinetics of repopulation of CD4+ and CD8+ lymphocytes and their CD134 expression would be predictive of clinical acute GVHD and/or its response to immunosuppressive therapy.

CD134 expression does not predict GVHD APA Gadisseur et al

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Patients and methods Patients CD134 expression by T cells was followed in 14 patients with hematological malignancies, who received either an autologous or allogeneic stem cell graft. Three patients received an autologous peripheral blood stem cell transplant (auto-PBSCT). Seven patients received an allogeneic HLA-identical sibling stem cell transplantation. Stem cells were harvested from the bone marrow in six patients (alloBMT) and from the peripheral blood in one patient (alloPBSCT). Four patients received a graft from an HLA-A, B, DRB1 identical unrelated donor (MUD). The mean age of the patients was 43 years (range 22–65), eight were female, six male. Indications for the BMT were non-Hodgkin lymphoma (NHL, n = 4), acute myeloid leukemia (AML, n = 3), chronic myeloid leukemia (CML, n = 2), myelodysplastic syndrome (MDS, n = 2), aplastic anemia (AA, n = 2) and chronic lymphocytic leukemia (CLL, n = 1) (Table 1). Blood samples were taken at day 0, +7, +10, +14, +21, +28 and +35 after transplantation, and if possible, at longer intervals thereafter. Transplant procedures Different conditioning regimens were used depending on diagnosis and donor type (related or unrelated). Eleven patients (11/14) received myelo-ablative conditioning by cyclophosphamide and total body irradiation (cyclo/TBI). Two non-Hodgkin’s lymphoma patients were treated with BCNU, etoposide (VP16), cytosine arabinoside and cyclophosphamide (BEAC regimen). One patient (1/14) was conditioned with busulfan and melphalan. Patients with an unrelated donor received additional lymphocyte depletion by anti-thymocyte globulin (ATG-Merieux, Pasteur Merieux, Lyon, France) before BMT. All allogeneic grafts were administered after in vitro partial T cell depletion. T cell

Table 1

depletion was performed either by sheep E-rosetting or CD34+ selection using the CellPro device (CellPro, Wezembeek-Oppem, Belgium). Acute GVHD was graded according to standard criteria.5 Flow cytometry Heparin anticoagulated whole blood samples were stained directly using fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAbs). The following mAb mixtures were used: CD4 (antiLeu3ab/FITC) + CD134 (OX40/PE), CD8 (antiLeu2a/FITC) + CD134 (OX40/PE), and CD4/FITC or CD8/FITC + mIgG1/PE as isotype controls. All mAbs were obtained from Becton Dickinson (BDIS, San Jose, CA, USA). In short, 200 ␮l aliquots of whole blood were incubated with mAb mixtures for 20 min at room temperature in the dark. After lysing the erythrocytes using FACS lysing buffer (BDIS), the remaining leukocytes were washed once and resuspended in PBS containing 1% paraformaldehyde and 0.04 ␮g/ml LDS-751 (Exciton, Dayton, OH, USA). During flow cytometric data acquisition a live gate was set on nucleated cells (LDS-751+), and sufficient nucleated cells were acquired so as to contain at least 1000 CD4+ or CD8+ lymphocytes. During list mode data analysis the CD4+ or CD8+ lymphocytes were selected on the basis of their low sideward scatter signals in combination with their CD4 or CD8 positivity. The expression of CD134 by the CD4+ or CD8+ lymphocytes was analyzed by placing the marker to discriminate between positive and negative signals at the foot of the negative peak in the isotype control histogram. With this marker setting the CD134-stained cells were analyzed and from the %CD134+ lymphocytes (ie fraction of CD4+ or CD8+ lymphocytes) the % lymphocytes exceeding the marker setting on each isotype control was subtracted. The detection limit was 0.5% of CD4+ or CD8+ lymphocytes expressing the CD134 antigen.

Patient characteristics

UPN

Sex

Disease

Stem cell transplantation

Conditioning regimen

GVHD maximum grade

Steroidsa

379 381 394 380 382 385 395 396 397 398 386 387 388 392

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

NHL NHL CML AML CLL/NHL ALL MDS-RA CLL NHL MDS-RA AML CML AA-PNH AML

auto-PBSCT auto-PBSCT auto-PBSCT allo-BMT allo-BMT allo-PBSCT allo-BMT allo-BMT allo-BMT allo-BMT MUD-BMT MUD-BMT MUD-BMT MUD-BMT

BEAC BEAC Bu/Melp Cyclo/TBI Cyclo/TBI Cyclo/TBI Cyclo/TBI Cyclo/TBI Cyclo/TBI Cyclo/TBI Cyclo/TBI/ATG Cyclo/TBI/ATG Cyclo/TBI/ATG Cyclo/TBI/ATG

— — — — I (skin) II (skin) III (GI) I (skin) II (skin) — I (skin) II (skin) III (skin, GI) I (skin)

— — — — + + ++ topical + — topical ++ ++ topical

NHL = non-Hodgkin lymphoma; CML = chronic myeloid leukemia; AML = acute myeloid leukemia; CLL = chronic lymphocytic leukemia; MDS-RA = myelodysplastic syndrome, type refractory anemia; AA-PNH = aplastic anemia-paroxysmal nocturnal hemoglobinuria; BEAC = BCNU, etoposide, arabinoside-C, cyclophosphamide; Bu/Melp = busulfan/melphalan; Cyclo = cyclophosphamide; TBI = total body irradiation; ATG = anti-thymocyte globulin; GI = gastro-intestinal tract. a Steroids were administered topically or prednisolone intravenously (+: 1 mg/kg/day; ++: 2 × 1 mg/kg/day).


Assessment of absolute CD4+CD134+ and CD8+CD134+ counts +

80

a

70

+

Absolute CD4 or CD8 lymphocyte counts were obtained by multiplying the proportion of CD4+ or CD8+ lymphocytes (fraction of nucleated cells) with the absolute leukocyte counts as assessed using a Technicon H1 automated cell counter (Technicon, Tarrytown, NY, USA). Blood samples from apparently healthy donors served as a reference source for CD134 expression by the CD4+ and CD8+ lymphocytes.

1015

UPN 380 UPN 382 UPN 385 UPN 395 UPN 396 UPN 397 UPN 398

60 50 40 30 20 10 0 5

10

15

20

25

30

35

40

35

40

35

40

Days after BMT 80

Results

CD134 expression on CD4+ T lymphocytes of healthy donors was invariably low with a mean percentage of 8.1% (range: 2.3–14.7; s.d., 3.2). The CD134 expression by CD8+ T lymphocytes of healthy donors was undetectable (ie ⬍0.5% of CD8+ lymphocytes). In contrast, the expression of CD134 by CD4+ T lymphocytes of BMT recipients was strongly increased, ranging between 5 and 70%. CD134 expression by the CD8+ lymphocytes of BMT recipients remained low and in many cases below the detection limit (ie ⬍0.5% of the CD8+ lymphocytes). The expression of CD134 became detectable on the CD8+ subset (mean 4.6%, range 0.6–25%) in 55/86 (64%) of the samples. The results of CD134 expression on CD4+ lymphocytes were evaluated further in the three patient groups. HLA-matched sibling transplants: The expression of CD134 as a percentage of the CD4+ cells showed peak levels up to 58% (mean 40.4%, range 26–58%) in this group of patients (Figure 1a). Peak values were reached between day +7 and +21. Five out of seven patients developed GVHD. Maximum grade and time of onset of GVHD are presented in Table 2. Four patients experienced skin GVHD and one patient developed gastro-intestinal GVHD. No correlation between the levels or the pattern in time of CD134 expression on CD4+ lymphocytes and the severity or clinical course of GVHD could be determined. Two patients showed a steep rise before GVHD could be observed clinically, while two other patients showed a moderate increase before the onset of GVHD. However, both patients without GVHD also showed steep rises of CD134+ expression as a percentage of CD4+ lymphocytes. Although the percentage of CD134+CD4+ expression showed a decline after initial peaking there was no evident correlation between the initiation of systemic steroids for GVHD and that decline. All patients with GVHD responded to steroid therapy. The absolute CD134+CD4+ values remained relatively low with levels ranging between 1–10/␮l and peak values between 20–35/␮l (Figure 2a). Absolute CD134+CD4+ values showed a clear increase from day 0 onwards. One patient with GVHD grade III (UPN 395) showed a steep rise just before GVHD was diagnosed but showed a further rise after therapy with systemic steroids had been initiated, upon which these symptoms quickly resolved.

70 CD134% of CD4+

Expression of CD134 by CD4+ and CD8+ lymphocytes

UNP 386 UPN 387 UPN 388 UPN 392

b

60 50 40 30 20 10 0 5

10

15

20

25

30

Days after BMT 80

c

70

UPN 379 UPN 381 UPN 394

60 50 40 30 20 10 0 5

10

15

20

25

30

Days after BMT

Figure 1 CD134 expression (%) by CD4+ T cells in recipients of (a) an allogeneic HLA-matched sibling stem cell transplantation (SCT), in recipients of an HLA-A, B, DRB1 matched unrelated SCT (b), and in recipients of an autologous SCT (c).

HLA-matched unrelated transplants: Patients who received an unrelated donor graft showed similar levels of CD134 expression as patients who received a sibling graft (mean 49.75%, range 5–70%). Peak values were reached between days +7 to +14 after transplantation. Two patients showed an early rise of CD134+CD4+ cells followed by a decline (Figure 1b). All four patients showed some evidence of GVHD during the study period (Table 2). Three patients experienced acute GVHD limited to the skin (grade I to II) and one patient showed extensive GVHD grade III of the skin, gastro-intestinal tract and liver. No correlation was observed between the severity and course of GVHD and the levels and pattern in time of CD134 expression as a percentage of CD4+ lymphocytes. The highest levels were observed in patient UPN 387 showing acute GVHD grade II while patient UPN 388 with grade III GVHD showed a low and decreasing CD134+CD4+ level when the picture of GVHD worsened. Absolute CD134+CD4+ numbers rose around day +14 with peak levels 10–18/␮l (Figure 2b). The levels of CD134+CD4+ T cells showed an initial peak but became much lower thereafter.


Table 2

CD134 expression and acute GVHD

UPN

BMT

379 381 394 380 382 385 395 396 397 398 386 387 388 392

1000

CD134 as % CD4+

GVHD

auto-PBSCT auto-PBSCT auto-PBSCT allo-sib-BMT allo-sib-BMT allo-sib-BMT allo-sib-BMT allo-sib-BMT allo-sib-BMT allo-sib-BMT MUD-BMT MUD-BMT MUD-BMT MUD-BMT

a

Maximum grade

Peak day

Peak level

Peak day

Peak level (/␮l)

— — — — +26 +21 +21 +21 +14 — +36 +11 +12 +28

— — — — II II III I II — I II III I

+29 +9 +7 +14 +21 +10 +21 +21 +7 +14 +48 +11 +14 +36

30% 45% 63% 53% 36% 26% 58% 28% 47% 35% 39% 70% 55% 26%

+29 +14 +14 +14 +10 +31 +35 +28 +35 +21 +38 +14 +14 +36

113 68 152 3 17 20 32 18 15 6 18 11 17 3

10 1 5

10

0.1

1000

15 20 25 Days after BMT

b

30

35

40

UPN 386 UPN 387 UPN 388 UPN 392

100

1 5

10

1000


30

35

40

35

40

UPN 379 UPN 381 UPN 394

c

100 10 1 5 0.1

10


30

Autologous transplants: These control patients were treated with high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Expression of CD134 as a percentage of CD4+ cells was high. Peak values (mean 46%, range 30–63%) were detected around 7 to 10 days after transplantation, thereafter levels declined (Figure 1c). The absolute CD134+CD4+ levels also peaked relatively early (ie day +15) and declined subsequently (Figure 2c). Peak values ranged between 70 and 160/␮l. None of the patients experienced severe infections. The highest value for CD134 expression as a percentage of CD4+ lymphocytes was detected in patient UPN 394 who recovered quickly after transplantation and experienced no complications. Discussion

10

0.1

Absolute CD134+CD4+

Onset (day)

UPN 380 UPN 382 UPN 385 UPN 395 UPN 396 UPN 397 UPN 398

100

CD134+ CD4+/mm3

1016

Figure 2 Absolute numbers of CD134+ CD4+ T cells in recipients of an allogeneic sibling SCT (a), recipients of a MUD-SCT (b) and in recipients of an autologous SCT (c).

CD134 (OX40) is a member of the NGFR/TNFR superfamily of receptors expressed on activated T cells, which, upon cross-binding, results in T cell proliferation, cytokine secretion, differentiation of B cells, and adhesion of activated T cells to vascular endothelial cells.9–13 Experimental studies in animals have suggested an association between CD134 expression on peripheral blood T lymphocytes and onset and course of acute GVHD.14 No such correlation was observed in the present study. The appearance of CD134+CD4+ T lymphocytes was not associated with the onset of acute GVHD in patients receiving a bone marrow graft from either an HLA genotypically matched sibling donor or a HLA-A, B, DR matched unrelated donor. Furthermore, a decline of CD134+CD4+ T lymphocytes was not associated with GVHD responding to therapy, neither was the persistence of GVHD associated with a continued presence of CD134+CD4+ T lymphocytes. Most strikingly, high levels of CD134+CD4+ T lymphocytes were detected after autologous transplantation and relative high values were also observed in patients who received an allogeneic graft without any GVHD. Our observations do not support


the view that CD134 expression is predictive of GVHD or is a useful tool for monitoring response to GVHD. In addition, earlier suggestions to develop a therapeutic approach aimed at the elimination of CD134+CD4+ lymphocytes are not supported by our findings. Prevention of GVHD following allogeneic BMT can be performed by anti-T cell therapy combining cyclosporine and methotrexate and alternatively, by T cell depletion (TCD) of the graft, which is often combined with cyclosporine.1,16–18 Depletion of virtually all T cells from the graft is the most effective way of GVH prevention. However, it is associated with an increased risk of graft failure and an increased relapse rate. Therefore, partial instead of complete TCD has been developed in order to prevent severe GVHD, while preserving an anti-leukemic effect and avoiding the risk of graft failure.19–21 It has been shown that the outcome of allo-BMT following partial TCD is associated with a low incidence of GVHD and a relapse rate for patients with acute leukemia, which seems comparable to non-TCD BMT.21 However, a major disadvantage of either complete or partial TCD is a delayed immune recovery, which may be associated with opportunistic infections. The low numbers of CD4+ T cells and the CD134+CD4+ subset observed in the present study in patients who received an allogeneic graft may reflect a slow immune recovery. In addition, the absence of any correlation between GVH and the presence of CD134+CD4+ T cells may be due to T cell depletion of the graft. It does not exclude the possibility that the pattern of CD134+CD4+ T cells in patients receiving an unmanipulated graft may be associated with onset and course of GVHD. Enhanced expression of CD134 of CD4+ T cells is considered to reflect activation of T cells as part of an inflammatory reaction. Opportunistic infections, as well as GVH, may induce inflammation and induction of CD134+CD4+ T cells following allogeneic BMT. In the present study, high numbers of CD134+CD4+ T cells were observed following autologous transplantation in patients, who did not experience any infection at that time. It suggests that CD134+ expression by CD4+ cells comes early in lymphocyte repopulation and may be a marker for T cell repopulation in general. Further experimental and clinical studies are needed to define the role of CD134+/CD4+ T cells in lymphocyte repopulation and lymphocyte development following stem cell transplantation. References 1 Lazarus H, Vogelsang G, Rowe J. Prevention and treatment of acute graft-versus-host disease: the old and the new. A report from the Eastern Cooperative Oncology Group (ECOG). Bone Marrow Transplant 1997; 19: 577–600. 2 Theobald M. Allorecognition and graft-versus-host disease. Bone Marrow Transplant 1995; 15: 489–498. 3 Antin J, Ferrara J. Cytokine dysregulation and acute graftversus-host disease. Blood 1992; 80: 2964–2968. 4 Ferrara J. Cytokine dysregulation as a mechanism of graftversus-host disease. Curr Opin Immun 1993; 3: 794–799.

5 Glucksberg H, Storb R, Fefer A et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HLA-matched sibling donors. Transplantation 1974; 18: 295–304. 6 Gratama J, Wuersch A, Nissen C et al. Influence of graftversus-host disease prophylaxis on early T lymphocyte repopulation in allogeneic bone marrow transplantation. Exp Hematol 1986; 14: 133–137. 7 Gratama J, Naipal A, Oljans P et al. T lymphocyte repopulation and differentiation after marrow transplantation. Early shifts in the ration between T4+ and T8+ T lymphocytes correlate with the occurrence of acute graft-versus-host disease. Blood 1984; 63: 1416–1423. 8 Miyamoto T, Akashi K, Hayashi S et al. Serum concentration of the soluble interleukin-2 receptor for monitoring acute graft-versus-host disease. Bone Marrow Transplant 1996; 17: 185–190. 9 Durkop H, Lanza U, Himmelreich P, Stein H. Expression of the human OX40 antigen in normal and neoplastic tissues. Br J Haematol 1995; 91: 927–931. 10 Baum P, Gayle R, Ramsdell F et al. Molecular characterization of murine and human OX40/OX40 ligand systems: identification of a human OX40 ligand as the HTLV-1 regulated protein gp34. EMBO J 1994; 13: 3992–4001. 11 Imura A, Hori T, Imada K et al. The human Ox40/gp34 system directly mediates adhesion of activated T cells to vascular endothelial cells. J Exp Med 1996; 183: 2185–2195. 12 Stuber E, Strober W. The T cell–B cell interaction via OX40OX40L is necessary for the T cell-dependent humoral immune response. J Exp Med 1996; 183: 979–989. 13 Higashimura N, Takasawa N, Tanaka Y et al. Induction of OX40, a receptor of gp34, on T cells by trans-acting transcriptional activator, Tax, of human T cell leukemia virus type I. Jpn J Cancer Res 1996; 87: 227–231. 14 Tittle T, Weinberg A, Steinkeler C, Maziarz R. Expression of the T cell activation antigen OX40 identifies alloreactive T cells in acute graft-versus-host disease. Blood 1997; 89: 4652–4658. 15 Lamb L, Abhyankar S, O’ Neal W et al. Expression of CD134 (OX40) on T cells during the first 100 days following T celldepleted bone marrow transplantation from partially mismatched related donors: correlation with incidence and severity on graft-versus-host disease. Blood 1996; 90: 542a. 16 Poynton CH. T cell depletion in bone marrow transplantation. Bone Marrow Transplant 1988; 3: 265–269. 17 Marmont A, Horowitz MM, Gale RP et al. T cell depletion of HLA-identical transplants in leukemia. Blood 1991; 78: 2120–2124. 18 Van Bekkum D. Graft-versus-host disease. Biological mechanisms and clinical practice. In: van Bekkum DW, Lo¨wenberg B (eds). Bone Marrow Transplant. Marcel Dekker: New York, 1985, pp 147–153. 19 Lo¨wenberg B, Wagemaker G, Van Bekkum DW et al. Graftversus-host disease following transplantation of ‘one log’ vs ‘two log’ T lymphocyte-depleted bone marrow from HLAidentical donors. Bone Marrow Transplant 1986; 1: 133–137. 20 Verdonck LF, De Gast GC, Van Heugten HG, Dekker A. A fixed low number of T cells in HLA-identical allogeneic bone marrow transplantation. Blood 1990; 75: 776–780. 21 Verdonck LF, Dekker AW, De Gast GC et al. Allogeneic bone marrow transplantation with fixed low number of T cells in the marrow graft. Blood 1994; 83: 3090–3093.

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