correspondence
ZAP70 expression directly promotes chronic lymphocytic leukaemia cell adhesion to bone marrow stromal cells
ex vivo. Moreover, transient over-expression of ZAP70 in a B cell line or primary CLL cells led to increased stromal adhesion, suggesting a direct affect of ZAP70 in altering B cell biology to promote adhesion. In CLL, leukaemic B cells accumulate in the peripheral blood and the lymphoid organs where several other cell types (stromal cells, T cells, nurse-like cells) provide survival
Progressive chronic lymphocytic leukaemia (CLL) is associated with accumulation of leukaemic cells in lymphoid tissues and is frequently associated with poor prognostic markers, such as expression of the kinase ZAP70 [zeta-chain (TCR) associated protein kinase 70 kDa]. In our patient cohort, a highly significant correlation was found between ZAP70 expression in CLL cells and their stromal cell adhesion capacity assessed directly
ZAP70+
ZAP70–
(B)
Binding to stromal cells (%)
(A)
CLL
CLL Stroma
P < 0·0001
30
20
10
0
Binding to stromal cells (%)
(C)
(D) 30
R = 0·5878 P < 0·0001
ZAP70–
ZAP70+
(E)
20
293
30
209
15
20
20
290
348
308
10
397
10 5
126
0
20
40
60
80
100
10
0
20
498
489 472
266 492
0
0
527
62
0 40
60
80
100
0
20
40
60
80
100
ZAP70 expression (%) Fig 1. ZAP70+ chronic lymphocytic leukaemia cells are more adherent to stromal cells. (A) Fluorescent microscopy images of chronic lymphocytic leukaemia (CLL)-stromal cell co-cultures. CLL cells were stained with CellVue Claret prior to co-culture. After 1 day of co-culture, unbound CLL cells were washed away. Adherent CLL cells and S17 stromal cells were then fixed and stained with Alexa488-phalloidin. Cells were imaged on an Olympus IX71 inverted microscope equipped with an Ultraview LCI confocal imaging system (Perkin-Elmer, Waltham, MA). (B) Percentages of ZAP70+ and ZAP70 CLL adhering to stromal cells. After 1 day of co-culture, the percentage of adherent CLL cells was determined using flow cytometry. Horizontal lines represent medians. Statistical significance between the patient populations was analysed by Mann-Whitney unpaired test. (C) Correlation between ZAP70 expression and percent adhesion. Correlation significance was analysed by Spearman test. Results are from 56 patients. (D–E) Samples from 14 CLL patients assayed twice at different times. (D) CLL patients with unstable ZAP70 status during the study period. (E) CLL patients presenting a stable ZAP70 expression during the study period.
ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2015, 168, 139–159
Correspondence signals (Burger et al, 2009). CLL cells from patients can express different levels of ZAP70, whose expression is correlated to the disease progression (Crespo et al, 2003). As result, ZAP70 is now a widely-used prognostic biomarker. It remains unclear whether different prognostic groups of CLL have distinct interactions with their supportive microenvironment. This is particularly relevant as therapies that disrupt CLL-microenviroment interactions are emerging as viable treatment options (ten Hacken & Burger, 2014) and a better understanding is needed of which patient groups may benefit most. ZAP70 expression has been associated with changes in CLL signalling and response to factors, such as chemokines, which promote homing to lymphoid tissues (Stamatopoulos et al, 2009). Direct effects of ZAP70 in CLL signalling have been reported (Gobessi et al, 2007; Chen et al, 2008); however the functional impact on CLL interaction with tissue stromal cells is unknown. We have assessed ex vivo stromal cell adhesion capacity of a large cohort of CLL patients attending the Manitoba CLL
(A)
Clinic and also assessed ZAP70 status on the day of sample collection. Peripheral blood samples were obtained following informed consent, with the approval of the Research Ethics Board at the University of Manitoba, and CLL cells were isolated as described (Lafarge et al, 2014). For ZAP70 assessment by flow cytometry, cells were surface stained to identify CD19+CD5+CD3- cells and then fixed, permeabilized and further stained with anti-ZAP70 clone SBZAP, known to achieve the best resolution in flow cytometry assays (Vroblova et al, 2012). CLL cells can adhere to stromal cells, increasing survival and providing protection against cytotoxic drugs (Lagneaux et al, 1998). We performed CLL-stromal cell adhesion assays on freshly isolated cells as previously described (Lafarge et al, 2014). Using confocal microscopy, more B cells weer observed to attach to stromal cells in ZAP70+ patients compared to ZAP70 patients (Fig 1A; Supplementary Methods). Using flow cytometry, we quantified the CLL binding to stromal cells in each patient group and found that the
(B)
(C)
Empty vector Isotype control
Binding to stromal cells (%)
ZAP70 vector
ZAP70
GAPDH
ZAP70
P = 0·0024
30
20
10
0
Empty vector
(E)
(D)
(F)
100
Binding to stromal cells (%)
Isotype control
Percent live cells
80
Vector
ZAP70 vector
P = 0·012 20
ZAP70
60
40
20
0
ZAP70
40
Empty vector
ZAP70 vector
15
10
5
0
Empty vector
ZAP70 vector
Fig 2. Ectopic expression of ZAP70 increases adhesion to stromal cells. RAJI B cells or primary CLL B cells were transduced with ZAP70 expression vector using lentiviral transduction or nucleofection respectively. (A) ZAP70 expression in RAJI cells analysed by flow cytometry. The indicated lentiviral-transduced or control RAJI cells were fixed, permeabilized and then stained for ZAP70 expression. (B) ZAP70 expression analysed by Western Blot. The indicated lentiviral-transduced or control RAJI cells were lysed and subjected to Western Blot analysis. (C) ZAP70-expressing or control RAJI cells were cultured with S17 stromal cells. After 2 h of co-culture, the percentage of bound cells was determined. Dots represent 7 independent experiments. Statistical significance was analyzed by Wilcoxon paired test. (D) The indicated ZAP70-transfected or control CLL cells were fixed, permeabilized and then stained for ZAP70 expression. (E) After ZAP70 or control transfection, CLL cell viability was assessed by flow cytometry using AnnexinV-allophycocyanin and 7-aminoactinomycin D. Results are shown in box plot as median numbers of live cells, first and third quartiles, maximum and minimum values from 9 different patients. (F) ZAP70-expressing or control CLL cells were cultured with S17 stromal cells for 4 h. The percentage of bound cells was determined by flow cytometry as previously, using the green fluorescent green marker to gate on transfected cells. Dots represent 9 independent experiments. Statistical significance was analysed by Wilcoxon paired test.
140
ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2015, 168, 139–159
Correspondence median CLL adhesion was significantly increased in the ZAP70+ group (Fig 1B). We used a cutoff of 25% for ZAP70 positivity, as this provides optimal discrimination of progessive clinical disease. Moreover, we found that percent ZAP70 expression was highly correlated with the efficiency of CLL binding to stromal cells (Fig 1C). We obtained samples from the same CLL patients over time and submitted those samples to the same functional assays. Based on repeated assay of ZAP70 stable patients, the average variation of our adhesion assay is 41%. Interestingly, the patients unstable in their ZAP70 expression showed significantly different adhesion correlating with the change in ZAP70 (Fig 1D). Patients who were stable regarding their ZAP70 status appeared to be stable in their ability to adhere to stromal cells (Fig 1E). Most of the ZAP70 unstable patients were initially found in the “grey zone”, defined between 20% and 30% ZAP70 expression, and about 2/3 of unstable patients were treated over time whereas only 1/3 of stable patients were treated. In order to determine whether ZAP70 directly functions in CLL adhesion to stroma, we expressed ZAP70 in ZAP70 RAJI cells or primary CLL cells (see Supplementary Methods). RAJI cells were transduced with ZAP70 lentiviral expression vector or control vector and expression of ZAP70 was confirmed by flow cytometry (Fig 2A) or Western blot (Fig 2B). Using the stromal cell adhesion assay, we observed that ZAP70-expressing RAJI cells were more adherent (Fig 2C). ZAP70- CLL cells were transfected with ZAP70 expression vector or control vector using nucleofection and expression confirmed using flow cytometry (Fig 2D). ZAP70 and control transfected cells showed similar viability (Fig 2E). Using the stromal cell adhesion assay, we observed that ZAP70 expressing cells were more adherent to S17 stromal cells (Fig 2F) or HS-5 stromal cells (Figure S1). Together, these results suggest that ZAP70 can directly influence the process of stromal cell adhesion. Our results suggest that ZAP70 can act either via its kinase or adaptor function (Chen et al, 2008) to directly alter B cell signalling pathways affecting stable binding to stromal cells. This may occur via enhanced BCR signalling, which can activate integrins or via other unknown mechanisms. The direct functional linkage of ZAP70 to CLL interaction with stromal cells may partly explain why it remains a relatively robust prognostic marker, despite technical challenges in standardized measurement of intracellular proteins by flow cytometry. Variations of ZAP70 expression over time may predict altered course of disease and/or response to treatment. Given that ZAP70+ CLL cells strongly interact with stromal elements in lymphoid tissues, they may be differentially sensitive to therapies that interfere with CLL-microenvironment interactions, such as the new generation of kinase inhibitors antagonizing BCR signalling (Woyach et al, 2012; ten Hacken & Burger, 2014).
ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2015, 168, 139–159
Acknowledgements The authors would like to thank Michelle Queau for excellent technical help, Dr. Ferenc Boldizsar for providing ZAP70 lentiviral vector and all members of the Marshall, Gibson and Johnston laboratories for valuable discussions and support. We also acknowledge the Manitoba CLL clinic and Tumour Bank staff, especially Brenda Kuschak, Sadi Al Muktafi, Donna Hewitt and Courtney Edworthy, as well as all the patients who gave their blood samples to make this research possible. This research was funded by the Canadian Cancer Society (grant #700981) and supported in part by the CancerCare Manitoba Foundation, the Canadian Institutes of Health Research and the Canadian Tumour Repository Network (CTRNet). S.T.L. was funded by a Manitoba Health Research Council and CancerCare Manitoba post-doctoral fellowship. A.J.M. was funded by a Canada Research Chair. S.B.G. was funded by a Manitoba Health Research Chair.
Authorship and disclosures STL, HL, SDP and SH performed experiments. STL and AJM analysed results and created the figures. STL, JBJ, SBG and AJM designed the research. STL and AJM wrote the paper. The authors declare no competing financial interests. Sandrine T. Lafarge1,2 Hongzhao Li1 Samantha D. Pauls3 Sen Hou1 James B. Johnston2 Spencer B. Gibson1,2,3 Aaron J. Marshall1,3 1
Department of Immunology, University of Manitoba, 2Cancercare
Manitoba, Manitoba Institute of Cell Biology, and 3Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada E-mail: [email protected]
Keywords: chronic lymphocytic leukaemia, cell adhesion, stromal cells, signalling, prognostic factors First published online 4 Aug 2014 10.1111/bjh.13063
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig S1. Ectopic expression of ZAP70 increases adhesion to HS5 stromal cells. RAJI (A) or CLL (B) cells were transfected and assayed as described in Fig 2, except that HS-5 stromal cells were used. Data S1. Supplementary Methods.
141
Correspondence
References Burger, J.A., Ghia, P., Rosenwald, A. & CaligarisCappio, F. (2009) The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood, 114, 3367–3375. Chen, L., Huynh, L., Apgar, J., Tang, L., Rassenti, L., Weiss, A. & Kipps, T.J. (2008) ZAP-70 enhances IgM signaling independent of its kinase activity in chronic lymphocytic leukemia. Blood, 111, 2685–2692. Crespo, M., Bosch, F., Villamor, N., Bellosillo, B., Colomer, D., Rozman, M., Marce, S., Lopez-Guillermo, A., Campo, E. & Montserrat, E. (2003) ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. The New England Journal of Medicine, 348, 1764–1775.
Gobessi, S., Laurenti, L., Longo, P.G., Sica, S., Leone, G. & Efremov, D.G. (2007) ZAP-70 enhances B-cell-receptor signaling despite absent or inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cells. Blood, 109, 2032–2039. ten Hacken, E. & Burger, J.A. (2014) Molecular pathways: targeting the microenvironment in chronic lymphocytic leukemia–focus on the B-cell receptor. Clinical Cancer Research, 20, 548–556. Lafarge, S.T., Johnston, J.B., Gibson, S.B. & Marshall, A.J. (2014) Adhesion of ZAP-70 + chronic lymphocytic leukemia cells to stromal cells is enhanced by cytokines and blocked by inhibitors of the PI3-kinase pathway. Leukemia Research, 38, 109–115. Lagneaux, L., Delforge, A., Bron, D., De Bruyn, C. & Stryckmans, P. (1998) Chronic lymphocytic
leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells. Blood, 91, 2387– 2396. Stamatopoulos, B., Haibe-Kains, B., Equeter, C., Meuleman, N., Soree, A., De Bruyn, C., Hanosset, D., Bron, D., Martiat, P. & Lagneaux, L. (2009) Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA. Haematologica, 94, 790–799. Vroblova, V., Smolej, L. & Krejsek, J. (2012) Pitfalls and limitations of ZAP-70 detection in chronic lymphocytic leukemia. Hematology, 17, 268–274. Woyach, J.A., Johnson, A.J. & Byrd, J.C. (2012) The B-cell receptor signaling pathway as a therapeutic target in CLL. Blood, 120, 1175–1184.
Interleukin-22 in the diagnosis of active chronic graft-versus-host disease in paediatric patients
Chronic graft-versus-host disease (cGvHD) is a multisystem alloimmune disorder that occurs in 25–30% of paediatric patients after undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT), representing the major cause of late transplant-related mortality. The approach currently used to establish the diagnosis of cGvHD depends almost exclusively on physical examination and, when possible, histopathological confirmation. This approach is not adequate to distinguish disease activity from irreversible tissue damage. Improvements could come from biologically based indicators that could be used together with standard criteria. Manifestations of cGVHD show similarities with autoimmune diseases, but its pathogenesis is poorly understood. Although alloreactive donor T cells contained in the graft are essential in initiating cGvHD, B-cells and immune deregulation appear to be important. Standard therapy of cGvHD consists of corticosteroids with or without a calcineurin inhibitor, but mortality rate remains more than 40% (Ferrara et al, 2009). Treatment is limited by diverse factors including poor understanding of the immune targets for optimal treatment. Therefore, this study aimed to determine the expression levels of several cytokines involved in autoimmune and inflammatory diseases in a paediatric population subjected to allo-HSCT, in order to identify specific diagnostic biomarkers of active cGvHD that could also represent new drug targets. We retrospectively analysed 48 paediatric patients who underwent an allo-HSCT at the Centre of Paediatric Haema142
tology Oncology (Padova, Italy). The patient population was mixed for sex, age, original disease and source of HSCT (Table I, for more details please see Table SI, which also specifies the samples available for the study). Based on National Institutes of Health criteria (Pavletic et al, 2006), 16 patients were diagnosed with acute GvHD (aGvHD) only, 20 with cGvHD (always preceded by aGvHD) and 12 without GvHD. Peripheral blood (PB) was collected from recipients at 30-day intervals, where possible, from engraftment to day+360 after allo-HSCT. For 17 patients, the donor PB samples, taken at the time of transplantation, were also analysed. By Sybr Green Real Time Quantitative polymerase chain reaction (RQ-PCR) we evaluated the expression of IL2, IL4, IL10, IL12A, IL12B, IL17A, IL22, IL23A, IL23R, TRAF6, TNFSF13B, RORC, LTA (TNFB) and TNFRSF1A (Table SII) in peripheral blood mononuclear cells (PBMC) from patients and donors, using a pool of seven PBMC from healthy controls as calibrator. Only IL22 was more highly expressed in patients with active cGvHD (n = 6) than in patients whose cGvHD had resolved (n = 15) (P = 00005), patients with active aGvHD (n = 12) (P = 00001) and in patients who did not develop GvHD (n = 10) (P = 00012) (Fig 1A). These conclusions were unchanged when analysing only the subgroup of patients for whom both active cGvHD and resolved cGvHD samples (n = 4) were available (Figure S1). We were able to follow one patient over time, confirming our results (Figure S2). Using receiver-operating characteristic curve methods, we estimated an optimal cut-off point of IL22 expression of 25, ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2015, 168, 139–159
Copyright of British Journal of Haematology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
ZAP70 expression directly promotes chronic lymphocytic leukaemia cell adhesion to bone marrow stromal cells
ex vivo. Moreover, transient over-expression of ZAP70 in a B cell line or primary CLL cells led to increased stromal adhesion, suggesting a direct affect of ZAP70 in altering B cell biology to promote adhesion. In CLL, leukaemic B cells accumulate in the peripheral blood and the lymphoid organs where several other cell types (stromal cells, T cells, nurse-like cells) provide survival
Progressive chronic lymphocytic leukaemia (CLL) is associated with accumulation of leukaemic cells in lymphoid tissues and is frequently associated with poor prognostic markers, such as expression of the kinase ZAP70 [zeta-chain (TCR) associated protein kinase 70 kDa]. In our patient cohort, a highly significant correlation was found between ZAP70 expression in CLL cells and their stromal cell adhesion capacity assessed directly
ZAP70+
ZAP70–
(B)
Binding to stromal cells (%)
(A)
CLL
CLL Stroma
P < 0·0001
30
20
10
0
Binding to stromal cells (%)
(C)
(D) 30
R = 0·5878 P < 0·0001
ZAP70–
ZAP70+
(E)
20
293
30
209
15
20
20
290
348
308
10
397
10 5
126
0
20
40
60
80
100
10
0
20
498
489 472
266 492
0
0
527
62
0 40
60
80
100
0
20
40
60
80
100
ZAP70 expression (%) Fig 1. ZAP70+ chronic lymphocytic leukaemia cells are more adherent to stromal cells. (A) Fluorescent microscopy images of chronic lymphocytic leukaemia (CLL)-stromal cell co-cultures. CLL cells were stained with CellVue Claret prior to co-culture. After 1 day of co-culture, unbound CLL cells were washed away. Adherent CLL cells and S17 stromal cells were then fixed and stained with Alexa488-phalloidin. Cells were imaged on an Olympus IX71 inverted microscope equipped with an Ultraview LCI confocal imaging system (Perkin-Elmer, Waltham, MA). (B) Percentages of ZAP70+ and ZAP70 CLL adhering to stromal cells. After 1 day of co-culture, the percentage of adherent CLL cells was determined using flow cytometry. Horizontal lines represent medians. Statistical significance between the patient populations was analysed by Mann-Whitney unpaired test. (C) Correlation between ZAP70 expression and percent adhesion. Correlation significance was analysed by Spearman test. Results are from 56 patients. (D–E) Samples from 14 CLL patients assayed twice at different times. (D) CLL patients with unstable ZAP70 status during the study period. (E) CLL patients presenting a stable ZAP70 expression during the study period.
ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2015, 168, 139–159
Correspondence signals (Burger et al, 2009). CLL cells from patients can express different levels of ZAP70, whose expression is correlated to the disease progression (Crespo et al, 2003). As result, ZAP70 is now a widely-used prognostic biomarker. It remains unclear whether different prognostic groups of CLL have distinct interactions with their supportive microenvironment. This is particularly relevant as therapies that disrupt CLL-microenviroment interactions are emerging as viable treatment options (ten Hacken & Burger, 2014) and a better understanding is needed of which patient groups may benefit most. ZAP70 expression has been associated with changes in CLL signalling and response to factors, such as chemokines, which promote homing to lymphoid tissues (Stamatopoulos et al, 2009). Direct effects of ZAP70 in CLL signalling have been reported (Gobessi et al, 2007; Chen et al, 2008); however the functional impact on CLL interaction with tissue stromal cells is unknown. We have assessed ex vivo stromal cell adhesion capacity of a large cohort of CLL patients attending the Manitoba CLL
(A)
Clinic and also assessed ZAP70 status on the day of sample collection. Peripheral blood samples were obtained following informed consent, with the approval of the Research Ethics Board at the University of Manitoba, and CLL cells were isolated as described (Lafarge et al, 2014). For ZAP70 assessment by flow cytometry, cells were surface stained to identify CD19+CD5+CD3- cells and then fixed, permeabilized and further stained with anti-ZAP70 clone SBZAP, known to achieve the best resolution in flow cytometry assays (Vroblova et al, 2012). CLL cells can adhere to stromal cells, increasing survival and providing protection against cytotoxic drugs (Lagneaux et al, 1998). We performed CLL-stromal cell adhesion assays on freshly isolated cells as previously described (Lafarge et al, 2014). Using confocal microscopy, more B cells weer observed to attach to stromal cells in ZAP70+ patients compared to ZAP70 patients (Fig 1A; Supplementary Methods). Using flow cytometry, we quantified the CLL binding to stromal cells in each patient group and found that the
(B)
(C)
Empty vector Isotype control
Binding to stromal cells (%)
ZAP70 vector
ZAP70
GAPDH
ZAP70
P = 0·0024
30
20
10
0
Empty vector
(E)
(D)
(F)
100
Binding to stromal cells (%)
Isotype control
Percent live cells
80
Vector
ZAP70 vector
P = 0·012 20
ZAP70
60
40
20
0
ZAP70
40
Empty vector
ZAP70 vector
15
10
5
0
Empty vector
ZAP70 vector
Fig 2. Ectopic expression of ZAP70 increases adhesion to stromal cells. RAJI B cells or primary CLL B cells were transduced with ZAP70 expression vector using lentiviral transduction or nucleofection respectively. (A) ZAP70 expression in RAJI cells analysed by flow cytometry. The indicated lentiviral-transduced or control RAJI cells were fixed, permeabilized and then stained for ZAP70 expression. (B) ZAP70 expression analysed by Western Blot. The indicated lentiviral-transduced or control RAJI cells were lysed and subjected to Western Blot analysis. (C) ZAP70-expressing or control RAJI cells were cultured with S17 stromal cells. After 2 h of co-culture, the percentage of bound cells was determined. Dots represent 7 independent experiments. Statistical significance was analyzed by Wilcoxon paired test. (D) The indicated ZAP70-transfected or control CLL cells were fixed, permeabilized and then stained for ZAP70 expression. (E) After ZAP70 or control transfection, CLL cell viability was assessed by flow cytometry using AnnexinV-allophycocyanin and 7-aminoactinomycin D. Results are shown in box plot as median numbers of live cells, first and third quartiles, maximum and minimum values from 9 different patients. (F) ZAP70-expressing or control CLL cells were cultured with S17 stromal cells for 4 h. The percentage of bound cells was determined by flow cytometry as previously, using the green fluorescent green marker to gate on transfected cells. Dots represent 9 independent experiments. Statistical significance was analysed by Wilcoxon paired test.
140
ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2015, 168, 139–159
Correspondence median CLL adhesion was significantly increased in the ZAP70+ group (Fig 1B). We used a cutoff of 25% for ZAP70 positivity, as this provides optimal discrimination of progessive clinical disease. Moreover, we found that percent ZAP70 expression was highly correlated with the efficiency of CLL binding to stromal cells (Fig 1C). We obtained samples from the same CLL patients over time and submitted those samples to the same functional assays. Based on repeated assay of ZAP70 stable patients, the average variation of our adhesion assay is 41%. Interestingly, the patients unstable in their ZAP70 expression showed significantly different adhesion correlating with the change in ZAP70 (Fig 1D). Patients who were stable regarding their ZAP70 status appeared to be stable in their ability to adhere to stromal cells (Fig 1E). Most of the ZAP70 unstable patients were initially found in the “grey zone”, defined between 20% and 30% ZAP70 expression, and about 2/3 of unstable patients were treated over time whereas only 1/3 of stable patients were treated. In order to determine whether ZAP70 directly functions in CLL adhesion to stroma, we expressed ZAP70 in ZAP70 RAJI cells or primary CLL cells (see Supplementary Methods). RAJI cells were transduced with ZAP70 lentiviral expression vector or control vector and expression of ZAP70 was confirmed by flow cytometry (Fig 2A) or Western blot (Fig 2B). Using the stromal cell adhesion assay, we observed that ZAP70-expressing RAJI cells were more adherent (Fig 2C). ZAP70- CLL cells were transfected with ZAP70 expression vector or control vector using nucleofection and expression confirmed using flow cytometry (Fig 2D). ZAP70 and control transfected cells showed similar viability (Fig 2E). Using the stromal cell adhesion assay, we observed that ZAP70 expressing cells were more adherent to S17 stromal cells (Fig 2F) or HS-5 stromal cells (Figure S1). Together, these results suggest that ZAP70 can directly influence the process of stromal cell adhesion. Our results suggest that ZAP70 can act either via its kinase or adaptor function (Chen et al, 2008) to directly alter B cell signalling pathways affecting stable binding to stromal cells. This may occur via enhanced BCR signalling, which can activate integrins or via other unknown mechanisms. The direct functional linkage of ZAP70 to CLL interaction with stromal cells may partly explain why it remains a relatively robust prognostic marker, despite technical challenges in standardized measurement of intracellular proteins by flow cytometry. Variations of ZAP70 expression over time may predict altered course of disease and/or response to treatment. Given that ZAP70+ CLL cells strongly interact with stromal elements in lymphoid tissues, they may be differentially sensitive to therapies that interfere with CLL-microenvironment interactions, such as the new generation of kinase inhibitors antagonizing BCR signalling (Woyach et al, 2012; ten Hacken & Burger, 2014).
ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2015, 168, 139–159
Acknowledgements The authors would like to thank Michelle Queau for excellent technical help, Dr. Ferenc Boldizsar for providing ZAP70 lentiviral vector and all members of the Marshall, Gibson and Johnston laboratories for valuable discussions and support. We also acknowledge the Manitoba CLL clinic and Tumour Bank staff, especially Brenda Kuschak, Sadi Al Muktafi, Donna Hewitt and Courtney Edworthy, as well as all the patients who gave their blood samples to make this research possible. This research was funded by the Canadian Cancer Society (grant #700981) and supported in part by the CancerCare Manitoba Foundation, the Canadian Institutes of Health Research and the Canadian Tumour Repository Network (CTRNet). S.T.L. was funded by a Manitoba Health Research Council and CancerCare Manitoba post-doctoral fellowship. A.J.M. was funded by a Canada Research Chair. S.B.G. was funded by a Manitoba Health Research Chair.
Authorship and disclosures STL, HL, SDP and SH performed experiments. STL and AJM analysed results and created the figures. STL, JBJ, SBG and AJM designed the research. STL and AJM wrote the paper. The authors declare no competing financial interests. Sandrine T. Lafarge1,2 Hongzhao Li1 Samantha D. Pauls3 Sen Hou1 James B. Johnston2 Spencer B. Gibson1,2,3 Aaron J. Marshall1,3 1
Department of Immunology, University of Manitoba, 2Cancercare
Manitoba, Manitoba Institute of Cell Biology, and 3Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada E-mail: [email protected]
Keywords: chronic lymphocytic leukaemia, cell adhesion, stromal cells, signalling, prognostic factors First published online 4 Aug 2014 10.1111/bjh.13063
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig S1. Ectopic expression of ZAP70 increases adhesion to HS5 stromal cells. RAJI (A) or CLL (B) cells were transfected and assayed as described in Fig 2, except that HS-5 stromal cells were used. Data S1. Supplementary Methods.
141
Correspondence
References Burger, J.A., Ghia, P., Rosenwald, A. & CaligarisCappio, F. (2009) The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood, 114, 3367–3375. Chen, L., Huynh, L., Apgar, J., Tang, L., Rassenti, L., Weiss, A. & Kipps, T.J. (2008) ZAP-70 enhances IgM signaling independent of its kinase activity in chronic lymphocytic leukemia. Blood, 111, 2685–2692. Crespo, M., Bosch, F., Villamor, N., Bellosillo, B., Colomer, D., Rozman, M., Marce, S., Lopez-Guillermo, A., Campo, E. & Montserrat, E. (2003) ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. The New England Journal of Medicine, 348, 1764–1775.
Gobessi, S., Laurenti, L., Longo, P.G., Sica, S., Leone, G. & Efremov, D.G. (2007) ZAP-70 enhances B-cell-receptor signaling despite absent or inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cells. Blood, 109, 2032–2039. ten Hacken, E. & Burger, J.A. (2014) Molecular pathways: targeting the microenvironment in chronic lymphocytic leukemia–focus on the B-cell receptor. Clinical Cancer Research, 20, 548–556. Lafarge, S.T., Johnston, J.B., Gibson, S.B. & Marshall, A.J. (2014) Adhesion of ZAP-70 + chronic lymphocytic leukemia cells to stromal cells is enhanced by cytokines and blocked by inhibitors of the PI3-kinase pathway. Leukemia Research, 38, 109–115. Lagneaux, L., Delforge, A., Bron, D., De Bruyn, C. & Stryckmans, P. (1998) Chronic lymphocytic
leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells. Blood, 91, 2387– 2396. Stamatopoulos, B., Haibe-Kains, B., Equeter, C., Meuleman, N., Soree, A., De Bruyn, C., Hanosset, D., Bron, D., Martiat, P. & Lagneaux, L. (2009) Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA. Haematologica, 94, 790–799. Vroblova, V., Smolej, L. & Krejsek, J. (2012) Pitfalls and limitations of ZAP-70 detection in chronic lymphocytic leukemia. Hematology, 17, 268–274. Woyach, J.A., Johnson, A.J. & Byrd, J.C. (2012) The B-cell receptor signaling pathway as a therapeutic target in CLL. Blood, 120, 1175–1184.
Interleukin-22 in the diagnosis of active chronic graft-versus-host disease in paediatric patients
Chronic graft-versus-host disease (cGvHD) is a multisystem alloimmune disorder that occurs in 25–30% of paediatric patients after undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT), representing the major cause of late transplant-related mortality. The approach currently used to establish the diagnosis of cGvHD depends almost exclusively on physical examination and, when possible, histopathological confirmation. This approach is not adequate to distinguish disease activity from irreversible tissue damage. Improvements could come from biologically based indicators that could be used together with standard criteria. Manifestations of cGVHD show similarities with autoimmune diseases, but its pathogenesis is poorly understood. Although alloreactive donor T cells contained in the graft are essential in initiating cGvHD, B-cells and immune deregulation appear to be important. Standard therapy of cGvHD consists of corticosteroids with or without a calcineurin inhibitor, but mortality rate remains more than 40% (Ferrara et al, 2009). Treatment is limited by diverse factors including poor understanding of the immune targets for optimal treatment. Therefore, this study aimed to determine the expression levels of several cytokines involved in autoimmune and inflammatory diseases in a paediatric population subjected to allo-HSCT, in order to identify specific diagnostic biomarkers of active cGvHD that could also represent new drug targets. We retrospectively analysed 48 paediatric patients who underwent an allo-HSCT at the Centre of Paediatric Haema142
tology Oncology (Padova, Italy). The patient population was mixed for sex, age, original disease and source of HSCT (Table I, for more details please see Table SI, which also specifies the samples available for the study). Based on National Institutes of Health criteria (Pavletic et al, 2006), 16 patients were diagnosed with acute GvHD (aGvHD) only, 20 with cGvHD (always preceded by aGvHD) and 12 without GvHD. Peripheral blood (PB) was collected from recipients at 30-day intervals, where possible, from engraftment to day+360 after allo-HSCT. For 17 patients, the donor PB samples, taken at the time of transplantation, were also analysed. By Sybr Green Real Time Quantitative polymerase chain reaction (RQ-PCR) we evaluated the expression of IL2, IL4, IL10, IL12A, IL12B, IL17A, IL22, IL23A, IL23R, TRAF6, TNFSF13B, RORC, LTA (TNFB) and TNFRSF1A (Table SII) in peripheral blood mononuclear cells (PBMC) from patients and donors, using a pool of seven PBMC from healthy controls as calibrator. Only IL22 was more highly expressed in patients with active cGvHD (n = 6) than in patients whose cGvHD had resolved (n = 15) (P = 00005), patients with active aGvHD (n = 12) (P = 00001) and in patients who did not develop GvHD (n = 10) (P = 00012) (Fig 1A). These conclusions were unchanged when analysing only the subgroup of patients for whom both active cGvHD and resolved cGvHD samples (n = 4) were available (Figure S1). We were able to follow one patient over time, confirming our results (Figure S2). Using receiver-operating characteristic curve methods, we estimated an optimal cut-off point of IL22 expression of 25, ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2015, 168, 139–159
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