Expression of Activating KIR2DS2 and KIR2DS4 Genes after Hematopoietic Cell Transplantation: Relevance to Cytomegalovirus Infection Ghislaine M. Gallez-Hawkins,

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Expression of Activating KIR2DS2 and KIR2DS4 Genes after Hematopoietic Cell Transplantation: Relevance to Cytomegalovirus Infection Ghislaine M. Gallez-Hawkins, Anne E. Franck, Xiuli Li, Lia Thao, Arisa Oki, Ketevan Gendzekhadze, Andrew Dagis, Joycelynne Palmer, Ryotaro Nakamura, Stephen J. Forman, David Senitzer, John A. Zaia Biology of Blood and Marrow Transplantation Volume 17, Issue 11, Pages 1662-1672 (November 2011) DOI: 10.1016/j.bbmt.2011.04.008 Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 KIR2DS2 (circles) and KIR2DS4 (squares) expression by mRNA-based Q-PCR in donors. KIR2DS2 and KIR2DS4 expression is reported only in the presence of the corresponding genotype. The copy number for each subject was normalized to 1 × 106 β-actin copies, and the median copy number is shown at the top of each group. Null-expressers are represented as log 100 = 1 on the y-axis. Biology of Blood and Marrow Transplantation 2011 17, 1662-1672DOI: (10.1016/j.bbmt.2011.04.008) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 KIR2DS2 and KIR2DS4 levels in donor–recipient pairs. The aKIR levels are reported for the donor before infusion (gray) and for the recipient after infusion (black). The copy number for each subject was normalized to 1 × 106 β-actin copies, and the median copy number is shown at the top of each group. The mean values of all samples per recipient after HCT are shown in A and B, and the means of all samples per recipient before day 90 post-HCT are shown in C and D. (A) KIR2DS2 levels for 25 donor–recipient pairs analyzed using the Wilcoxon paired-rank test (P = .02). (B) KIR2DS4 levels for 51 donor–recipient pairs analyzed using the Wilcoxon paired-rank test (P < .0001). (C) KIR2DS2 levels for 22 donor–recipient pairs analyzed using the Wilcoxon paired-rank test (P = .21). (D) KIR2DS4 levels for 42 donor–recipient pairs analyzed using the Wilcoxon paired-rank test (P < .0025). Null-expressers are represented as log 100 = 1 on the y-axis. Biology of Blood and Marrow Transplantation 2011 17, 1662-1672DOI: (10.1016/j.bbmt.2011.04.008) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 KIR2DS2 and KIR2DS4 expression by mRNA-based Q-PCR in HCT recipients. The samples from NV subjects are represented by open symbols; samples from viremic subjects, by solid symbols. KIR2DS2 is represented by circles; KIR2DS4, by squares. The mean value for each patient is reported, with KIR expression values reported only if the corresponding donor KIR genotype was present. The copy number for each subject was normalized to 1 × 106 β-actin copies, and the median copy number is shown at the top of each group. The Mann-Whitney U test was performed to establish P values between the NV and viremic groups. (A) Overall expression of either KIR 2DS2 or KIR2DS4 in the NV and viremic groups. (B) Differentiation between expression of KIR2DS4 and of the deletion mutant KIR2DS4d. (C) Results for recipients receiving both genotypes together from donor cells. The null-expressers are represented as log 100 = 1 on the y-axis. Biology of Blood and Marrow Transplantation 2011 17, 1662-1672DOI: (10.1016/j.bbmt.2011.04.008) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Time course of KIR expression post-HCT in NV and viremic subjects. Cryopreserved peripheral blood lymphocytes were available at days 40, 90, 120, 150, 180, and 360 post-HCT. The presence of at least one full-length genotype KIR2DS4 allele was a condition for inclusion in B. (A) Results for KIR2DS2 expression in NV subjects and viremic subjects (Mann-Whitney P values are shown). (B) Expression of KIR2DS4 in NV subjects and viremic subjects (Mann-Whitney test). The median values are depicted above each column. Null-expressers are represented as log 100 = 1 on the y-axis. Biology of Blood and Marrow Transplantation 2011 17, 1662-1672DOI: (10.1016/j.bbmt.2011.04.008) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Effect of KIR expression status before CMV reactivation. (A) The percent cumulative CMV incidence in the presence or absence of KIR2DS2 expression before CMV reactivation (P = .45, log-rank test). (B) The same plot applied to KIR2DS4 (P = .51, log-rank test). (C) Expression (>0) for KIR2DS2, KIR2DS4, or KIR2DS2 + KIR2DS4 before CMV reactivation (P = .04, log-rank test). Biology of Blood and Marrow Transplantation 2011 17, 1662-1672DOI: (10.1016/j.bbmt.2011.04.008) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions