American Journal of Transplantation 2015; 15: 715–722 Wiley Periodicals Inc.
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Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13012
Factors Related to the Development of CMV-Specific CD8þ T cell Response in CMV-Seropositive Solid Organ Transplant Candidates S. Cantisa´n1,2,*, C. Rodelo-Haad3, A. Pa´ez-Vega1,2, A. Nieto4, J. M. Vaquero2,5, ~ as2,8, A. Poyato6, M. Montejo2,7, M. C. Farin 1,9 1,2,10 , A. Martı´n-Malo1,3 A. Rivero , R. Solana 1,2,9 and J. Torre-Cisneros 1 Instituto Maimo´nides de Investigacio´n Biome´dica de Co´rdoba (IMIBIC)/Reina Sofia University Hospital/ University of Cordoba, Cordoba, Spain 2 Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain 3 Department of Nephrology, Reina Sofia University Hospital, Cordoba, Spain 4 Department of Immunology, Puerta del Mar University Hospital, Cadiz, Spain 5 Department of Neumology, Reina Sofia University Hospital, Cordoba, Spain 6 Department of Hepatology, Reina Sofia University Hospital, Cordoba, Spain 7 Infectious Diseases Unit, Cruces University Hospital, Bilbao, Spain 8 Infectious Diseases Unit, Marque´s de Valdecilla Hospital, University of Cantabria, IDIVAL, Santander, Spain 9 Infectious Diseases Unit, Reina Sofia University Hospital, Cordoba, Spain 10 Immunology Department, Reina Sofia University Hospital, Cordoba, Spain Corresponding author: Sara Cantisa´n, [email protected]
This cross-sectional study analyzes factors associated with the development of CMV-specific CD8þ response, measured by IFNg production after cytomegalovirus (CMV) peptide stimulation, in CMV-seropositive solid organ transplantation candidates. A total of 114 candidates were enrolled, of whom 22.8% (26/114) were nonreactive (IFNg < 0.2 IU/mL). Multivariate logistic regression analysis showed that age, HLA alleles and organ to be transplanted were associated with developing CMV-specific CD8þ immunity (reactive; IFNg 0.2 IU/mL). The probability of being reactive was higher in candidates over 50 than in those under 50 (OR 6.33, 95%CI 1.93–20.74). Candidates with HLA-A1 and/or HLA-A2 alleles had a higher probability of being reactive than those with non-HLA-A1/non-HLA-A2 alleles (OR 10.97, 95%CI 3.36–35.83). Renal candidates had a higher
probability of being reactive than lung (adjusted OR 8.85, 95%CI 2.24–34.92) and liver candidates (OR 4.87, 95%CI 1.12–21.19). The AUC of this model was 0.84 (p < 0.001). Positive and negative predictive values were 84.8% and 76.9%, respectively. In renal candidates longer dialysis was associated with an increased frequency of reactive individuals (p ¼ 0.040). Therefore, although the assessment of CMV-specific CD8þ response is recommended in all Rþ candidates, it is essential in those with a lower probability of being reactive, such as non-renal candidates, candidates under 50 or those with non-HLA-A1/non-HLA-A2 alleles. Abbreviations: AUC, area under the curve; CMV, cytomegalovirus; IFNg, interferon-gamma; QF-CMV, QuantiFERON-CMV; Rþ, CMV-seropositive recipient; R-, CMV-seronegative recipient Received 11 June 2014, revised 10 September 2014 and accepted for publication 11 September 2014
Introduction The risk of cytomegalovirus (CMV) infection or disease in solid organ transplant recipients is mainly defined by donor and recipient CMV serostatus (highest risk in seropositive donor/seronegative recipients), although other risk factors such as the transplanted organ and the type of immunosuppressive therapy are also considered (1–2). However, while CMV serology (anti-CMV IgG), or humoral immunity, has traditionally been used to determine the risk of CMV infection after transplantation, cellular immunity mediated by T lymphocytes is more relevant in controlling CMV infection (3). For this reason, cellular immune response, which is measured as the in vitro production of IFNg by T lymphocytes in response to CMV peptides, has recently been defined as a biomarker associated with the risk of CMV infection and disease (4–7). In this same line, recent studies indicate that a variable percentage of CMV-seropositive (Rþ) solid organ transplant candidates lack functional T cell response to CMV (8–10). That is, although these candidates have humoral immunity to the virus they do not produce IFNg when T cells are stimulated with CMV antigens. These patients with anti-CMV 715
Cantisa´n et al
IgG but without T cell response against the virus have a higher incidence of CMV replication and disease after transplantation than patients with anti-CMV IgG and specific cellular immunity (8–10). It is not known, however, what factors are associated with the development of specific cellular response against CMV in Rþ candidates. In the present study we analyzed anti-CMV CD8þ T cell response in Rþ solid organ transplant candidates, using the QuantiFERON-CMV (QF-CMV) assay (11), to determine the factors associated with being QF-CMV reactive/nonreactive. Knowledge of these factors can aid in identifying which Rþ solid organ transplant candidates are more likely to have or not have cellular immunity against CMV and thus associate it to the risk of CMV replication or disease after transplantation.
Material and Methods Study design and population This cross-sectional study was carried out with solid organ transplant candidates from three centers of the REIPI network (Reina Sofı´a Hospital, Co´rdoba, Marque´s de Valdecilla Hospital, Santander and Cruces Hospital, Bilbao). Candidates older than 18 years old who were awaiting a solid organ transplant (lung, kidney or liver) were eligible for the study. Lung candidates were recruited from March 2009 to June 2010, kidney candidates from February 2013 to April 2014, and liver candidates from February 2013 to March 2014. Written informed consent was obtained from all candidates and the study was approved by the ethics committee of the Reina Sofı´a Hospital.
Determination of anti-CMV IgG antibodies Pretransplant anti-CMV IgG antibodies were determined in donors and recipients by chemoluminiscence (Diasorin SA, Madrid, Spain).
HLA class I typing The HLA typing of solid organ transplant candidates was carried out by PCR-sequence-specific oligonucleotide (PCR-SSO) using the Luminex platform (Lifecodes HLA typing kits; Immucor Transplant Diagnostics, Inc., Stamford, CT).
QuantiFERON-CMV assay The QuantiFERON-CMV1 (QF-CMV) test was performed according to the manufacturer’s instructions (Cellestis, a QIAGEN company, Melbourne, Australia) (11). In brief, 1 mL of heparinized whole blood was collected in three QF-CMV blood collection tubes. The tubes contained either (i) a mix of 21 CMV peptides (for candidates recruited before June 2010) or 22 CMV peptides (for candidates recruited beyond February 2013) from a variety of proteins; (ii) no antigens (negative control); or (iii) phytohemagglutinin (positive mitogen control). All candidates enrolled in the study had HLA class I alleles capable of binding CMV peptides independently of the QuantiFERON-CMV kit format (21 or 22 CMV peptides). The 22-peptide kit includes a pp65 peptide (TRATKMQVI) that is restricted by HLA Cw6, HLA-A30 and/or HLA-B13. After collection, the tubes were shaken vigorously and incubated for 16–24 h at 378C. Supernatants were subsequently harvested and analyzed for IFNg (IU/mL) by standard ELISA. A result for the CMV antigen was considered ‘‘reactive’’ when the CMV antigen response minus the negative control response was greater than 0.2 IU/mL of IFNg. Following the manufacturer’s instructions, a result was considered ‘‘Indeterminate’’ when the IFNg level in the CMV antigen tube minus the negative control was less
716
than 0.2 IU/mL and the IFNg level in the mitogen tube (once the negative control was subtracted) was less than 0.5 IU/mL.
Statistical analysis Statistical analysis was performed using IBM SPSS Statistics 18.0 software (IBM Corporation, New York, NY). We studied the association between the QF-CMV test result and the following variables: age, gender, CMV serology, organ to be transplanted, dialysis-related factors, retransplantation, inflammatory markers and immunomodulatory treatments (Vitamin D and others). For the bivariate analysis, categorical variables were compared with the Chisquare test or with Fisher’s exact test. Quantitative variables were analyzed with the Student’s t-test or the Mann–Whitney test according to the normality test result. The Kruskal–Wallis non-parametric test was used to compare IFNg production or age according to the organ to be transplanted. Variables found to be significant in the bivariate analysis and others that were considered clinically important were included in the multiple logistic regression model. Multivariate logistic regression was used to evaluate the association between various factors and pretransplant IFNg production. Goodness-of-fit (calibration and discrimination ability) was performed using the Hosmer-Lemeshow test and the ROC curve. The area under the curve (AUC), sensitivity, specificity, positive and negative predictive values and the accuracy of the model were also assessed. Values were considered statistically significant when the p-value was 50 77 (54.6) Sex, n (%) Female 44 (31.2) Male 97 (68.8) CMV serology, n (%) R 27 (19.1) Rþ 114 (80.9) Organ to be transplanted, n (%) Lung 45 (31.9) Liver 30 (21.3) Kidney 66 (46.8)
QF-CMV nonreactive (n ¼ 51)
QF-CMV reactive (n ¼ 90)
42.10 (14.71)
53.12 (12.30)
34 (66.7) 17 (33.3)
30 (33.3) 60 (66.7)
15 (29.4) 36 (70.6)
29 (32.2) 61 (67.8)
25 (49.0) 26 (51.0)
2 (2.2) 88 (97.8)
25 (49.0) 7 (13.7) 19 (37.3)
20 (22.2) 23 (25.6) 47 (52.2)
QF-CMV (QuantiFERON-CMV) reactive (IFNg 0.2 IU/mL); R/Rþ, CMV-seronegative/CMV-seropositive. 1 Age stratified in the median as younger/older than 50.
Table 2: Agreement between CMV QuantiFERON-CMV (QF-CMV) test result Candidates
QF-CMV nonreactive (n ¼ 51)
All (n ¼ 141) R Rþ Lung (n ¼ 45) R Rþ Liver (n ¼ 30) R Rþ Kidney (n ¼ 66) R Rþ
serology
QF-CMV reactive (n ¼ 90)
and
the
Factors associated with the QuantiFERON-CMV test result in CMV-Seropositive transplant candidates In the bivariate analysis (Table 3) we observed that age was associated with the development of CMV-specific CD8þ immunity (QF-CMV reactive). The mean age of candidates in the QF-CMV reactive group was higher than the mean age of candidates in the QF-CMV nonreactive group (53.15 years old vs. 46.31 years old; p ¼ 0.018). Age as a binary variable (stratified in the median, as younger/older than 50) remained statistically significant. Specifically, 84.3% of the candidates above 50 years old developed CMV-specific CD8þ response (were QF-CMV reactive), while 65.9% of candidates younger than 50 were QF-CMV reactive (p ¼ 0.023). The variable ‘organ to be transplanted’ was also associated with being QF-CMV reactive, with the group of kidney candidates having the highest frequency of QF-CMV reactive individuals. The percentage of Rþ QF-CMV reactive individuals was 61.3% (19/31) in the group of lung candidates, 76.7% (23/30) in the group of liver candidates and 86.8% (46/53) in the group of renal candidates (p ¼ 0.027). However, when we analyzed the mean quantitative IFNg production in Rþ QF-CMV reactive candidates according to the ‘organ to be transplanted’, we did not find significant differences, since the mean IFNg production in lung and liver candidates was very similar: 27.88 IU/mL (SD 33.41 IU/mL) for lung candidates,
x2 p
25 (92.6) 26 (22.8)
2 (7.4) 88 (77.2)
C
Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13012
Factors Related to the Development of CMV-Specific CD8þ T cell Response in CMV-Seropositive Solid Organ Transplant Candidates S. Cantisa´n1,2,*, C. Rodelo-Haad3, A. Pa´ez-Vega1,2, A. Nieto4, J. M. Vaquero2,5, ~ as2,8, A. Poyato6, M. Montejo2,7, M. C. Farin 1,9 1,2,10 , A. Martı´n-Malo1,3 A. Rivero , R. Solana 1,2,9 and J. Torre-Cisneros 1 Instituto Maimo´nides de Investigacio´n Biome´dica de Co´rdoba (IMIBIC)/Reina Sofia University Hospital/ University of Cordoba, Cordoba, Spain 2 Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain 3 Department of Nephrology, Reina Sofia University Hospital, Cordoba, Spain 4 Department of Immunology, Puerta del Mar University Hospital, Cadiz, Spain 5 Department of Neumology, Reina Sofia University Hospital, Cordoba, Spain 6 Department of Hepatology, Reina Sofia University Hospital, Cordoba, Spain 7 Infectious Diseases Unit, Cruces University Hospital, Bilbao, Spain 8 Infectious Diseases Unit, Marque´s de Valdecilla Hospital, University of Cantabria, IDIVAL, Santander, Spain 9 Infectious Diseases Unit, Reina Sofia University Hospital, Cordoba, Spain 10 Immunology Department, Reina Sofia University Hospital, Cordoba, Spain Corresponding author: Sara Cantisa´n, [email protected]
This cross-sectional study analyzes factors associated with the development of CMV-specific CD8þ response, measured by IFNg production after cytomegalovirus (CMV) peptide stimulation, in CMV-seropositive solid organ transplantation candidates. A total of 114 candidates were enrolled, of whom 22.8% (26/114) were nonreactive (IFNg < 0.2 IU/mL). Multivariate logistic regression analysis showed that age, HLA alleles and organ to be transplanted were associated with developing CMV-specific CD8þ immunity (reactive; IFNg 0.2 IU/mL). The probability of being reactive was higher in candidates over 50 than in those under 50 (OR 6.33, 95%CI 1.93–20.74). Candidates with HLA-A1 and/or HLA-A2 alleles had a higher probability of being reactive than those with non-HLA-A1/non-HLA-A2 alleles (OR 10.97, 95%CI 3.36–35.83). Renal candidates had a higher
probability of being reactive than lung (adjusted OR 8.85, 95%CI 2.24–34.92) and liver candidates (OR 4.87, 95%CI 1.12–21.19). The AUC of this model was 0.84 (p < 0.001). Positive and negative predictive values were 84.8% and 76.9%, respectively. In renal candidates longer dialysis was associated with an increased frequency of reactive individuals (p ¼ 0.040). Therefore, although the assessment of CMV-specific CD8þ response is recommended in all Rþ candidates, it is essential in those with a lower probability of being reactive, such as non-renal candidates, candidates under 50 or those with non-HLA-A1/non-HLA-A2 alleles. Abbreviations: AUC, area under the curve; CMV, cytomegalovirus; IFNg, interferon-gamma; QF-CMV, QuantiFERON-CMV; Rþ, CMV-seropositive recipient; R-, CMV-seronegative recipient Received 11 June 2014, revised 10 September 2014 and accepted for publication 11 September 2014
Introduction The risk of cytomegalovirus (CMV) infection or disease in solid organ transplant recipients is mainly defined by donor and recipient CMV serostatus (highest risk in seropositive donor/seronegative recipients), although other risk factors such as the transplanted organ and the type of immunosuppressive therapy are also considered (1–2). However, while CMV serology (anti-CMV IgG), or humoral immunity, has traditionally been used to determine the risk of CMV infection after transplantation, cellular immunity mediated by T lymphocytes is more relevant in controlling CMV infection (3). For this reason, cellular immune response, which is measured as the in vitro production of IFNg by T lymphocytes in response to CMV peptides, has recently been defined as a biomarker associated with the risk of CMV infection and disease (4–7). In this same line, recent studies indicate that a variable percentage of CMV-seropositive (Rþ) solid organ transplant candidates lack functional T cell response to CMV (8–10). That is, although these candidates have humoral immunity to the virus they do not produce IFNg when T cells are stimulated with CMV antigens. These patients with anti-CMV 715
Cantisa´n et al
IgG but without T cell response against the virus have a higher incidence of CMV replication and disease after transplantation than patients with anti-CMV IgG and specific cellular immunity (8–10). It is not known, however, what factors are associated with the development of specific cellular response against CMV in Rþ candidates. In the present study we analyzed anti-CMV CD8þ T cell response in Rþ solid organ transplant candidates, using the QuantiFERON-CMV (QF-CMV) assay (11), to determine the factors associated with being QF-CMV reactive/nonreactive. Knowledge of these factors can aid in identifying which Rþ solid organ transplant candidates are more likely to have or not have cellular immunity against CMV and thus associate it to the risk of CMV replication or disease after transplantation.
Material and Methods Study design and population This cross-sectional study was carried out with solid organ transplant candidates from three centers of the REIPI network (Reina Sofı´a Hospital, Co´rdoba, Marque´s de Valdecilla Hospital, Santander and Cruces Hospital, Bilbao). Candidates older than 18 years old who were awaiting a solid organ transplant (lung, kidney or liver) were eligible for the study. Lung candidates were recruited from March 2009 to June 2010, kidney candidates from February 2013 to April 2014, and liver candidates from February 2013 to March 2014. Written informed consent was obtained from all candidates and the study was approved by the ethics committee of the Reina Sofı´a Hospital.
Determination of anti-CMV IgG antibodies Pretransplant anti-CMV IgG antibodies were determined in donors and recipients by chemoluminiscence (Diasorin SA, Madrid, Spain).
HLA class I typing The HLA typing of solid organ transplant candidates was carried out by PCR-sequence-specific oligonucleotide (PCR-SSO) using the Luminex platform (Lifecodes HLA typing kits; Immucor Transplant Diagnostics, Inc., Stamford, CT).
QuantiFERON-CMV assay The QuantiFERON-CMV1 (QF-CMV) test was performed according to the manufacturer’s instructions (Cellestis, a QIAGEN company, Melbourne, Australia) (11). In brief, 1 mL of heparinized whole blood was collected in three QF-CMV blood collection tubes. The tubes contained either (i) a mix of 21 CMV peptides (for candidates recruited before June 2010) or 22 CMV peptides (for candidates recruited beyond February 2013) from a variety of proteins; (ii) no antigens (negative control); or (iii) phytohemagglutinin (positive mitogen control). All candidates enrolled in the study had HLA class I alleles capable of binding CMV peptides independently of the QuantiFERON-CMV kit format (21 or 22 CMV peptides). The 22-peptide kit includes a pp65 peptide (TRATKMQVI) that is restricted by HLA Cw6, HLA-A30 and/or HLA-B13. After collection, the tubes were shaken vigorously and incubated for 16–24 h at 378C. Supernatants were subsequently harvested and analyzed for IFNg (IU/mL) by standard ELISA. A result for the CMV antigen was considered ‘‘reactive’’ when the CMV antigen response minus the negative control response was greater than 0.2 IU/mL of IFNg. Following the manufacturer’s instructions, a result was considered ‘‘Indeterminate’’ when the IFNg level in the CMV antigen tube minus the negative control was less
716
than 0.2 IU/mL and the IFNg level in the mitogen tube (once the negative control was subtracted) was less than 0.5 IU/mL.
Statistical analysis Statistical analysis was performed using IBM SPSS Statistics 18.0 software (IBM Corporation, New York, NY). We studied the association between the QF-CMV test result and the following variables: age, gender, CMV serology, organ to be transplanted, dialysis-related factors, retransplantation, inflammatory markers and immunomodulatory treatments (Vitamin D and others). For the bivariate analysis, categorical variables were compared with the Chisquare test or with Fisher’s exact test. Quantitative variables were analyzed with the Student’s t-test or the Mann–Whitney test according to the normality test result. The Kruskal–Wallis non-parametric test was used to compare IFNg production or age according to the organ to be transplanted. Variables found to be significant in the bivariate analysis and others that were considered clinically important were included in the multiple logistic regression model. Multivariate logistic regression was used to evaluate the association between various factors and pretransplant IFNg production. Goodness-of-fit (calibration and discrimination ability) was performed using the Hosmer-Lemeshow test and the ROC curve. The area under the curve (AUC), sensitivity, specificity, positive and negative predictive values and the accuracy of the model were also assessed. Values were considered statistically significant when the p-value was 50 77 (54.6) Sex, n (%) Female 44 (31.2) Male 97 (68.8) CMV serology, n (%) R 27 (19.1) Rþ 114 (80.9) Organ to be transplanted, n (%) Lung 45 (31.9) Liver 30 (21.3) Kidney 66 (46.8)
QF-CMV nonreactive (n ¼ 51)
QF-CMV reactive (n ¼ 90)
42.10 (14.71)
53.12 (12.30)
34 (66.7) 17 (33.3)
30 (33.3) 60 (66.7)
15 (29.4) 36 (70.6)
29 (32.2) 61 (67.8)
25 (49.0) 26 (51.0)
2 (2.2) 88 (97.8)
25 (49.0) 7 (13.7) 19 (37.3)
20 (22.2) 23 (25.6) 47 (52.2)
QF-CMV (QuantiFERON-CMV) reactive (IFNg 0.2 IU/mL); R/Rþ, CMV-seronegative/CMV-seropositive. 1 Age stratified in the median as younger/older than 50.
Table 2: Agreement between CMV QuantiFERON-CMV (QF-CMV) test result Candidates
QF-CMV nonreactive (n ¼ 51)
All (n ¼ 141) R Rþ Lung (n ¼ 45) R Rþ Liver (n ¼ 30) R Rþ Kidney (n ¼ 66) R Rþ
serology
QF-CMV reactive (n ¼ 90)
and
the
Factors associated with the QuantiFERON-CMV test result in CMV-Seropositive transplant candidates In the bivariate analysis (Table 3) we observed that age was associated with the development of CMV-specific CD8þ immunity (QF-CMV reactive). The mean age of candidates in the QF-CMV reactive group was higher than the mean age of candidates in the QF-CMV nonreactive group (53.15 years old vs. 46.31 years old; p ¼ 0.018). Age as a binary variable (stratified in the median, as younger/older than 50) remained statistically significant. Specifically, 84.3% of the candidates above 50 years old developed CMV-specific CD8þ response (were QF-CMV reactive), while 65.9% of candidates younger than 50 were QF-CMV reactive (p ¼ 0.023). The variable ‘organ to be transplanted’ was also associated with being QF-CMV reactive, with the group of kidney candidates having the highest frequency of QF-CMV reactive individuals. The percentage of Rþ QF-CMV reactive individuals was 61.3% (19/31) in the group of lung candidates, 76.7% (23/30) in the group of liver candidates and 86.8% (46/53) in the group of renal candidates (p ¼ 0.027). However, when we analyzed the mean quantitative IFNg production in Rþ QF-CMV reactive candidates according to the ‘organ to be transplanted’, we did not find significant differences, since the mean IFNg production in lung and liver candidates was very similar: 27.88 IU/mL (SD 33.41 IU/mL) for lung candidates,
x2 p
25 (92.6) 26 (22.8)
2 (7.4) 88 (77.2)
