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Phenotypic and Functional Characteristics of NK Cells Associated with CMV Infection after Allogeneic Hematopoietic Stem Cell Transplantation (HCT)
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Abstracts / Biol Blood Marrow Transplant 22 (2016) S19eS481
Interestingly in contrast to the overall high mortality rate of 93.7% (32/35) in non-HSCT cases reported, death occurred in only 38.5% (5/13) of the HDT/HSCT patients that developed PML (p<0.01). Conclusion: We reviewed published literature of case reports on PML in non-HIV patients and found that most oncologic cases were associated with lymphoproliferative disorders and/or HSCT. We found that PML cases related to HSCT had significantly better rates of survival compared to cases associated with biologic treatments or purine analogues. Supported by observations of improved PML outcomes in HIV patients in the era of cART, immune reconstitution in HSCT patients with PML appears to be a plausible mechanism behind their favorable survival. Timely withdrawal of immune suppression, donor lymphocyte infusion leading to reconstitution of the immune system in HSCT associated PML may be important, while in PML associated with monoclonal antibodies it may be important to consider novel therapies like adoptive T cell therapy.
CMV-IgG increased the risk of unexpected diseases, such as recurrent pregnancy loss and cardiovascular disease. Longterm survivors after allo-HSCT would be increased accompanied with secondary disorders, some of which might be related to CMV. Therefore, we should carefully monitor the long-term survivors of allo-HSCT showing abnormal serological tests against CMV and clarify the meaning of this phenomenon.
193 Frequently Observed Long-Lasting Abnormal Serological Tests Against Cytomegaloviruses after Allogeneic Hematopoietic Stem Cell Transplantation Hideaki Maeba 1, Kazuhiro Noguchi 1, Masaki Fukuda 1, Toshihiro Fujiki 1, Shintaro Mase 1, Rie Kuroda 1, Raita Araki 1, Yasuhiro Ikawa 1, Ryosei Nishimura 2. 1 Pediatrics, Kanazawa University, Kanazawa, Japan; 2 Pediatrics, Kanazawa Univ., Kanazawa, Japan We have recently noticed that not a few patients had both CMV specific IgM and IgG antibody for a long time after hematopoietic stem cell transplantation (HSCT). Aim of this study is to clarify how often unusual production of antibodies against CMV were observed after allo-HSCT and to discuss the role of them. We performed a retrospective study of 24 evaluable childhood patients who survived more than 2 years and were monitored CMV-IgM and CMV-IgG over time after allo-HSCT without receiving intravenous immunoglobulin more than 3 months. Serological tests for CMV were measured by EIA. Titer of CMV specific IgM and IgG were plotted as shown in the figure below. Six out of 24 patients (25%) showed the long lasting positivity of both CMV specific IgM and IgG. Only one patient, the titer of CMV-IgM returned to be negative during follow-up. In all but one, CMV-IgM titer did not returned to be negative during follow-up (range, 2.7-11.4 years, average 6.0 years). The patients showing long lasting positivity of serological test against CMV had a history of temporal CMV antigenemia and all of these patients had cleared CMVantigenemia during follow-up without any symptoms. In addition, 5 out of 24 patients (21%) showed high-titer of CMV-IgG ( 32) with negative CMV-IgM. Significant proportion of patients receiving allo-HSCT showed persisting abnormal CMV serological tests, though definite CMV related disease has not been developed. To exclude the possibility that frequent detection of CMVIgM is due to the cross reaction against other kinds of herpes viruses, HSV-IgM, VZV-IgM and EBV-VCA-IgM were also measured. All patients with CMV-IgM had been negative for these antibodies. Moreover, only 2 out of 6 patients with CMV IgM (+) and IgG (+) showed chronic GVHD requiring immunosuppressive therapy. These results suggested that unusual appearance of antibodies against CMV was not due to the immune dysregulation by chronic GVHD and/or immunosuppressant and might have a particular meaning. In fact, some reports have described recently that high-titer of
Figure. Association between CMV-IgG antibody levels and CMV-IgM antibody levels after allo-HSCT.
194 Phenotypic and Functional Characteristics of NK Cells Associated with CMV Infection after Allogeneic Hematopoietic Stem Cell Transplantation (HCT) Ryotaro Nakamura 1, John A. Zaia 2, Stephen J. Forman 1, Ghislaine Gallez-hawkins 3, Anne Franck 4, Laetitia Jeannet 4, Xiuli Li 5, Andy Dagis 6, Carolyn Behrendt 7. 1 Hematology/ Hematopoietic Cell Transplant, City of Hope National Medical Center, Duarte, CA; 2 Virology, Beckman Research Institute of City of Hope, Duarte, CA; 3 Department of Virology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA; 4 Department of Virology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA; 5 Department of Virology, City of Hope, Duarte, CA; 6 Information Sciences, City of Hope National Medical Center, Duarte, CA; 7 Biostatistics, City of Hope National medical Center, Duarte, CA Background: Cytomegalovirus (CMV) infection represents a major complication in hematopoietic stem cell transplantation (HCT). There is accumulating evidence that immune responses to CMV infection involve the expansion of specific subsets of NK cells largely driven by the activating receptor NKG2C. Methods/Patients: We prospectively examined the reconstitution of NK cells post-HCT (days 21, 30, 45, 80 and 120) for their immunophenotypes and functions. Of the total 111 patients enrolled, the current analyses focus on 82 CMV seropositive recipients with complete and longitudinal immunologic data available (median age: 55, range 19-70) who received HCT (PBSC: n¼75, BM: n¼4, UCB: n¼3) from related (n¼36) or unrelated donors (n¼46) after fully ablative (n¼22) or reduced-intensity (n¼60) conditioning. Donor CMV serostatus was positive in 56, negative in 26. CMV reactivations occurred in 19 patients (23.2%). % of NK cells expressing the following markers/cytokines were evaluated
Abstracts / Biol Blood Marrow Transplant 22 (2016) S19eS481
in multivariate analyses: NKG2C, granzyme B (GrB), Ki67, CD107, CD137, IFNg, and PD1. P-values are unadjusted unless specified otherwise. To evaluate multiple test parameters, we used “q-value” which is a false discovery rate incurred when calling the finding significant. Results: Consistent with earlier reports, CMV reactivation was significantly associated with increased %NK cells expressing NKG2C (6.1%, p¼0.034, q¼0.017) as well as GrB (25.5% increase, p¼0.002, q<0.001) and Ki67 (5.6%, p¼0.004, q<0.01). When we examined characteristics of NKG2C+NK cells in samples on day +80, activation markers, CD107, IFNg, CD137, and Ki67, were significantly over-expressed compared with NKG2C-NK cells (by 15.2%, [p<0.001], 11.0% [p<0.001], 5.0% [p<0.001], 1.7% [p¼0.002], respectively) while GrB expression was lower (-8.4%, p¼0.025) (adjusted p). We further explored possible relationship between in vitro T cell responses to CMVpp65 Ag and NK cell phenotypes. In day +80 samples, % CMVpp65-induced IFNg+CD3+T cells significantly associated with NK cells expressing NKG2C (p¼0.007, q<0.001), suggesting a coordinated response to CMV between innate and adaptive immune responses. CMVpp65-specific IFNg+CD3+ T cells were also associated with IFNg+ response in the NKG2C- population (p<0.001, q<0.001) while the association was less significant in the NKG2C+ population (p¼0.091, q¼0.029). Similarly, pp65reactive IFNg+CD3+ T cells associated with CD137 expression in NKG2C-NK cells (p¼0.007, q¼0.003) but not in NKG2C+NK cells (p¼0.8, q¼0.19). Conclusion: In summary, our data support that CMV reactivation is associated with expansion (Ki67) and cytotoxic functions (GrB, IFNg) of NK cells expressing NKG2C following HCT. The data also suggest that there are coordinated immune responses between T cells and NK cells with NKG2Cpopulation showing a greater reactivity with in-vitro CMVAg stimulation.
195 Immune Reconstitution after Autologous Hematopoietic Cell Transplant (AHCT) in B-Cell Non-Hodgkin Lymphoma (B-NHL) Patients Mobilized with Granulocyte Colony Stimulating Factor (G-CSF), with or without Plerixafor Pallawi Torka 1, Paul K. Wallace 2, Yali Zhang 1, George L. Chen 1, Christine M. Ho 1, Sophia R. Balderman 1, Maureen Ross 1, Bruno Paiva 3, Francisco J. Hernandez-Ilizaliturri 1, Philip L. McCarthy 1, Theresa E. Hahn 1. 1 Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY; 2 Department of Flowcytometry, Roswell Park Cancer Institute, Buffalo, NY; 3 Clínica Universidad de Navarra, Pamplona, Spain AHCT is important in treatment of B-NHL, both in the upfront and relapsed/refractory settings. In B-NHL patients mobilized with G-CSF, early absolute lymphocyte count recovery (ALC> 500 cells/mL by day +15 post AHCT) and high CD16+/ 56+/CD3- NK cell count are prognostic factors for improved overall survival (OS, Porrata et al, BBMT, 2008). We instituted plerixafor-based mobilization in all B-NHL patients in 04/ 2010 in order to improve HSC mobilization and collection. We report the first prospective study of immune cell subsets and recovery patterns with AHCT outcomes in B-NHL patients. A comprehensive immunophenotyping panel including 35 blood cell subsets was performed in 110 consecutive B-NHL patients [59% diffuse large B-cell lymphoma (DLBCL), 41% mantle cell lymphoma (MCL)] who received AHCT from
S155
01/2008 to 11/2014, at a median of 28 days pre-AHCT (N¼110) and at day +100 (N¼86) post-AHCT. Baseline characteristics and outcomes in all 110 patients have been reported previously (Torka et al, ASH 2015). Due to significant differences in immune cell subsets and clinical outcomes by disease status pre-AHCT, this report focuses on 81 patients in complete remission (CR) pre-AHCT. Those mobilized with G-CSF+ plerixafor (80%) were older and more frequently received BuCy as the designated high dose regimen as compared to those mobilized with G-CSF alone (20%). There were no differences in race, KPS, BMI and NHL histology by mobilization agent (G-CSF+/- plerixafor). Time to platelet and neutrophil recovery was similar. In comparison to patients mobilized with G-CSF+ plerixafor, G-CSF mobilized patients had a higher percentage of CD4+ naïve, CD8+ naïve, CD8+ central memory (CM) and CD4+ thymic emigrants at day +100 post-AHCT, despite an equal number of total CD4+ and CD8+ T-cells suggesting a relative delay in maturation of the T-cell compartment. All patients survived to day +100; median follow-up was 20.5 (3-74) months. PFS and OS did not significantly differ by plerixafor use, NHL histology, age, KPS, BMI, conditioning regimen or pre-AHCT vitamin D status. We did not find a significant association of early ALC recovery with improved OS in our study population (Figure 1). A higher proportion of CD8+ CM T-cells at day +100 post-AHCT was associated with significantly worse PFS and OS (Figure 2). The frequencies of other immune cell subsets (including NK cells) at day +100 post-AHCT were not associated with outcomes. Similar associations were seen when the study population was restricted to G-CSF+ plerixafor mobilized patients (n¼65). Plerixafor leads to early maturation of the T-cell compartment which may in turn, improve outcomes. The impact of CD8+ CM T-cell proportions on outcomes may be related to differential antigen threshold requirements for recall proliferation between naïve and memory CD8+ T cells. Longer follow-up is needed to assess durability of disease response and survival.
Figure 1. Early ALC recovery is not associated with improved PFS or OS in B-NHL patients in CR pre-AHCT
Figure 2. Higher proportions of CD8+ central memory (CM) T-cells are associated with worse PFS and OS in B-NHL patients in CR pre-AHCT
Abstracts / Biol Blood Marrow Transplant 22 (2016) S19eS481
Interestingly in contrast to the overall high mortality rate of 93.7% (32/35) in non-HSCT cases reported, death occurred in only 38.5% (5/13) of the HDT/HSCT patients that developed PML (p<0.01). Conclusion: We reviewed published literature of case reports on PML in non-HIV patients and found that most oncologic cases were associated with lymphoproliferative disorders and/or HSCT. We found that PML cases related to HSCT had significantly better rates of survival compared to cases associated with biologic treatments or purine analogues. Supported by observations of improved PML outcomes in HIV patients in the era of cART, immune reconstitution in HSCT patients with PML appears to be a plausible mechanism behind their favorable survival. Timely withdrawal of immune suppression, donor lymphocyte infusion leading to reconstitution of the immune system in HSCT associated PML may be important, while in PML associated with monoclonal antibodies it may be important to consider novel therapies like adoptive T cell therapy.
CMV-IgG increased the risk of unexpected diseases, such as recurrent pregnancy loss and cardiovascular disease. Longterm survivors after allo-HSCT would be increased accompanied with secondary disorders, some of which might be related to CMV. Therefore, we should carefully monitor the long-term survivors of allo-HSCT showing abnormal serological tests against CMV and clarify the meaning of this phenomenon.
193 Frequently Observed Long-Lasting Abnormal Serological Tests Against Cytomegaloviruses after Allogeneic Hematopoietic Stem Cell Transplantation Hideaki Maeba 1, Kazuhiro Noguchi 1, Masaki Fukuda 1, Toshihiro Fujiki 1, Shintaro Mase 1, Rie Kuroda 1, Raita Araki 1, Yasuhiro Ikawa 1, Ryosei Nishimura 2. 1 Pediatrics, Kanazawa University, Kanazawa, Japan; 2 Pediatrics, Kanazawa Univ., Kanazawa, Japan We have recently noticed that not a few patients had both CMV specific IgM and IgG antibody for a long time after hematopoietic stem cell transplantation (HSCT). Aim of this study is to clarify how often unusual production of antibodies against CMV were observed after allo-HSCT and to discuss the role of them. We performed a retrospective study of 24 evaluable childhood patients who survived more than 2 years and were monitored CMV-IgM and CMV-IgG over time after allo-HSCT without receiving intravenous immunoglobulin more than 3 months. Serological tests for CMV were measured by EIA. Titer of CMV specific IgM and IgG were plotted as shown in the figure below. Six out of 24 patients (25%) showed the long lasting positivity of both CMV specific IgM and IgG. Only one patient, the titer of CMV-IgM returned to be negative during follow-up. In all but one, CMV-IgM titer did not returned to be negative during follow-up (range, 2.7-11.4 years, average 6.0 years). The patients showing long lasting positivity of serological test against CMV had a history of temporal CMV antigenemia and all of these patients had cleared CMVantigenemia during follow-up without any symptoms. In addition, 5 out of 24 patients (21%) showed high-titer of CMV-IgG ( 32) with negative CMV-IgM. Significant proportion of patients receiving allo-HSCT showed persisting abnormal CMV serological tests, though definite CMV related disease has not been developed. To exclude the possibility that frequent detection of CMVIgM is due to the cross reaction against other kinds of herpes viruses, HSV-IgM, VZV-IgM and EBV-VCA-IgM were also measured. All patients with CMV-IgM had been negative for these antibodies. Moreover, only 2 out of 6 patients with CMV IgM (+) and IgG (+) showed chronic GVHD requiring immunosuppressive therapy. These results suggested that unusual appearance of antibodies against CMV was not due to the immune dysregulation by chronic GVHD and/or immunosuppressant and might have a particular meaning. In fact, some reports have described recently that high-titer of
Figure. Association between CMV-IgG antibody levels and CMV-IgM antibody levels after allo-HSCT.
194 Phenotypic and Functional Characteristics of NK Cells Associated with CMV Infection after Allogeneic Hematopoietic Stem Cell Transplantation (HCT) Ryotaro Nakamura 1, John A. Zaia 2, Stephen J. Forman 1, Ghislaine Gallez-hawkins 3, Anne Franck 4, Laetitia Jeannet 4, Xiuli Li 5, Andy Dagis 6, Carolyn Behrendt 7. 1 Hematology/ Hematopoietic Cell Transplant, City of Hope National Medical Center, Duarte, CA; 2 Virology, Beckman Research Institute of City of Hope, Duarte, CA; 3 Department of Virology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA; 4 Department of Virology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA; 5 Department of Virology, City of Hope, Duarte, CA; 6 Information Sciences, City of Hope National Medical Center, Duarte, CA; 7 Biostatistics, City of Hope National medical Center, Duarte, CA Background: Cytomegalovirus (CMV) infection represents a major complication in hematopoietic stem cell transplantation (HCT). There is accumulating evidence that immune responses to CMV infection involve the expansion of specific subsets of NK cells largely driven by the activating receptor NKG2C. Methods/Patients: We prospectively examined the reconstitution of NK cells post-HCT (days 21, 30, 45, 80 and 120) for their immunophenotypes and functions. Of the total 111 patients enrolled, the current analyses focus on 82 CMV seropositive recipients with complete and longitudinal immunologic data available (median age: 55, range 19-70) who received HCT (PBSC: n¼75, BM: n¼4, UCB: n¼3) from related (n¼36) or unrelated donors (n¼46) after fully ablative (n¼22) or reduced-intensity (n¼60) conditioning. Donor CMV serostatus was positive in 56, negative in 26. CMV reactivations occurred in 19 patients (23.2%). % of NK cells expressing the following markers/cytokines were evaluated
Abstracts / Biol Blood Marrow Transplant 22 (2016) S19eS481
in multivariate analyses: NKG2C, granzyme B (GrB), Ki67, CD107, CD137, IFNg, and PD1. P-values are unadjusted unless specified otherwise. To evaluate multiple test parameters, we used “q-value” which is a false discovery rate incurred when calling the finding significant. Results: Consistent with earlier reports, CMV reactivation was significantly associated with increased %NK cells expressing NKG2C (6.1%, p¼0.034, q¼0.017) as well as GrB (25.5% increase, p¼0.002, q<0.001) and Ki67 (5.6%, p¼0.004, q<0.01). When we examined characteristics of NKG2C+NK cells in samples on day +80, activation markers, CD107, IFNg, CD137, and Ki67, were significantly over-expressed compared with NKG2C-NK cells (by 15.2%, [p<0.001], 11.0% [p<0.001], 5.0% [p<0.001], 1.7% [p¼0.002], respectively) while GrB expression was lower (-8.4%, p¼0.025) (adjusted p). We further explored possible relationship between in vitro T cell responses to CMVpp65 Ag and NK cell phenotypes. In day +80 samples, % CMVpp65-induced IFNg+CD3+T cells significantly associated with NK cells expressing NKG2C (p¼0.007, q<0.001), suggesting a coordinated response to CMV between innate and adaptive immune responses. CMVpp65-specific IFNg+CD3+ T cells were also associated with IFNg+ response in the NKG2C- population (p<0.001, q<0.001) while the association was less significant in the NKG2C+ population (p¼0.091, q¼0.029). Similarly, pp65reactive IFNg+CD3+ T cells associated with CD137 expression in NKG2C-NK cells (p¼0.007, q¼0.003) but not in NKG2C+NK cells (p¼0.8, q¼0.19). Conclusion: In summary, our data support that CMV reactivation is associated with expansion (Ki67) and cytotoxic functions (GrB, IFNg) of NK cells expressing NKG2C following HCT. The data also suggest that there are coordinated immune responses between T cells and NK cells with NKG2Cpopulation showing a greater reactivity with in-vitro CMVAg stimulation.
195 Immune Reconstitution after Autologous Hematopoietic Cell Transplant (AHCT) in B-Cell Non-Hodgkin Lymphoma (B-NHL) Patients Mobilized with Granulocyte Colony Stimulating Factor (G-CSF), with or without Plerixafor Pallawi Torka 1, Paul K. Wallace 2, Yali Zhang 1, George L. Chen 1, Christine M. Ho 1, Sophia R. Balderman 1, Maureen Ross 1, Bruno Paiva 3, Francisco J. Hernandez-Ilizaliturri 1, Philip L. McCarthy 1, Theresa E. Hahn 1. 1 Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY; 2 Department of Flowcytometry, Roswell Park Cancer Institute, Buffalo, NY; 3 Clínica Universidad de Navarra, Pamplona, Spain AHCT is important in treatment of B-NHL, both in the upfront and relapsed/refractory settings. In B-NHL patients mobilized with G-CSF, early absolute lymphocyte count recovery (ALC> 500 cells/mL by day +15 post AHCT) and high CD16+/ 56+/CD3- NK cell count are prognostic factors for improved overall survival (OS, Porrata et al, BBMT, 2008). We instituted plerixafor-based mobilization in all B-NHL patients in 04/ 2010 in order to improve HSC mobilization and collection. We report the first prospective study of immune cell subsets and recovery patterns with AHCT outcomes in B-NHL patients. A comprehensive immunophenotyping panel including 35 blood cell subsets was performed in 110 consecutive B-NHL patients [59% diffuse large B-cell lymphoma (DLBCL), 41% mantle cell lymphoma (MCL)] who received AHCT from
S155
01/2008 to 11/2014, at a median of 28 days pre-AHCT (N¼110) and at day +100 (N¼86) post-AHCT. Baseline characteristics and outcomes in all 110 patients have been reported previously (Torka et al, ASH 2015). Due to significant differences in immune cell subsets and clinical outcomes by disease status pre-AHCT, this report focuses on 81 patients in complete remission (CR) pre-AHCT. Those mobilized with G-CSF+ plerixafor (80%) were older and more frequently received BuCy as the designated high dose regimen as compared to those mobilized with G-CSF alone (20%). There were no differences in race, KPS, BMI and NHL histology by mobilization agent (G-CSF+/- plerixafor). Time to platelet and neutrophil recovery was similar. In comparison to patients mobilized with G-CSF+ plerixafor, G-CSF mobilized patients had a higher percentage of CD4+ naïve, CD8+ naïve, CD8+ central memory (CM) and CD4+ thymic emigrants at day +100 post-AHCT, despite an equal number of total CD4+ and CD8+ T-cells suggesting a relative delay in maturation of the T-cell compartment. All patients survived to day +100; median follow-up was 20.5 (3-74) months. PFS and OS did not significantly differ by plerixafor use, NHL histology, age, KPS, BMI, conditioning regimen or pre-AHCT vitamin D status. We did not find a significant association of early ALC recovery with improved OS in our study population (Figure 1). A higher proportion of CD8+ CM T-cells at day +100 post-AHCT was associated with significantly worse PFS and OS (Figure 2). The frequencies of other immune cell subsets (including NK cells) at day +100 post-AHCT were not associated with outcomes. Similar associations were seen when the study population was restricted to G-CSF+ plerixafor mobilized patients (n¼65). Plerixafor leads to early maturation of the T-cell compartment which may in turn, improve outcomes. The impact of CD8+ CM T-cell proportions on outcomes may be related to differential antigen threshold requirements for recall proliferation between naïve and memory CD8+ T cells. Longer follow-up is needed to assess durability of disease response and survival.
Figure 1. Early ALC recovery is not associated with improved PFS or OS in B-NHL patients in CR pre-AHCT
Figure 2. Higher proportions of CD8+ central memory (CM) T-cells are associated with worse PFS and OS in B-NHL patients in CR pre-AHCT