Volume 86, Issue 1, Pages 175-183 (July 2014) The immune phenotype may relate to cancer development in kidney transplant recipients  Christopher M. Hope, Blair S. Grace, Katherine R. Pilkington, Patrick T. Coates, Ivo P. Bergmann, Robert P. Carroll  Kidney International  Volume 86, Issue 1, Pages 175-183 (July 2014) DOI: 10.1038/ki.2013.538 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Panel of receiver operator characteristic (ROC) curves for the development of squamous cell carcinoma (SCC) in kidney transplant recipients (KTRs). A panel of ROC curves for the detection of SCC development in KTRs within 100 days of measurement. Top left depicts the regulatory T cell (Treg) ROC, with the area under the curve (AUC)=0.78, specificity=68%, and sensitivity=71% at a trade-off value of 16 Tregs/μl. Top right is the memory B-cell curve with AUC=0.68 along with a specificity=85%, and sensitivity=56%, at a trade-off value of 3 memory B cells/μl. Bottom left shows the CD8 γδ T-cell curve with AUC=0.64, specificity=55%, and sensitivity=69% at a trade-off value of 27 CD8 γδ T cell/μl. Bottom right shows the NK cell ROC with AUC=0.55, specificity=38%, and sensitivity=72% at a trade-off value of 64 NK cells/μl. Kidney International 2014 86, 175-183DOI: (10.1038/ki.2013.538) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Percentage squamous cell carcinoma (SCC)-free survival of dichotomized data. High levels (>16 cells/μl) of CD4+FOXP3+CD127LoCD25Hi regulatory T cells (red line) indicate less SCC-free survival compared to low levels (<16 cells/μl, blue line). With a hazard ratio of 3.4, 95% confidence intervals of 1.6–7.5 and a log-ranked chi-squared P-value of <0.01. Kidney International 2014 86, 175-183DOI: (10.1038/ki.2013.538) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Absolute numbers of peripherally circulating regulatory T cells (Tregs, i.e., CD4+FOXP3+CD127LoCD25Hi) in kidney transplant recipients (KTRs) with various categories of cancer. Absolute numbers of CD4+FOXP3+CD127LoCD25Hi T cell (Tregs) in the peripheral blood of KTRs determined by flow cytometry: current solid-organ cancer (SOC, n=11); current squamous cell carcinoma (SCC, n=21); past solid-organ cancer (SOC)/SCC (n=33) represents KTRs who did not have a current cancer in situ; and no cancer represents those KTRs who did not have a history of SCC or SOC (n=51). Current SOC versus no cancer (P=0.053); current SCC versus no cancer (P<0.001); and past SCC/SOC compared with no cancer (P=0.155) using the Mann–Whitney two-tailed test. Kidney International 2014 86, 175-183DOI: (10.1038/ki.2013.538) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Helios coexpression in gated Tregs of kidney transplant recipients (KTRs) with cancer and with no cancer. A box and whisker plot depicting the percentage coexpression of the Ikaros transcription factor, Helios, in regulatory T cells (Tregs) in KTRs with no cancer versus KTRs with cancer with median (range) (82% (50–96%) and 85% (45–95%), respectively). Kidney International 2014 86, 175-183DOI: (10.1038/ki.2013.538) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Changes in regulatory T-cell (Treg) number in kidney transplant recipients. Boxplots (median and range) of changes in Treg numbers in kidney transplant recipients who subsequently developed a cancer (n=9, P=0.022). Kidney International 2014 86, 175-183DOI: (10.1038/ki.2013.538) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 The effect of cancer resection on immune phenotype. (a) Cancer resection immune phenotype. Boxplots (median and range) change in cell types with resection of squamous cell carcinoma (SCC) or solid-organ cancer (SOC; n=23). Left-hand boxplot represents cell value at the time of cancer diagnosis and right hand side represents cell value post cancer resection. Prospective follow-up between time points was a median (inter quartile range (IQR)) of 6 (1.5–14) months. (b) Phenotypes of kidney transplant recipients (KTRs) with no cancer over two time points. Boxplots of cell types recorded at two time points in 18 KTRs who did not develop any SCC or SOC. Prospective follow-up between observation point 1 (Obs. 1) and observation point 2 (Obs. 2) time points, median (IQR) 12 (3–16) months. NK, natural killer; Treg, regulatory T cell. Kidney International 2014 86, 175-183DOI: (10.1038/ki.2013.538) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Lesion resection of post-biopsy-proven basal cell carcinoma (BCC) tumors. Box and whisker of enumerated regulatory T-cell (Treg) number of kidney transplant recipients (n=12) who had suspect lesions removed that were subsequently classified as BCC tumors. Kidney International 2014 86, 175-183DOI: (10.1038/ki.2013.538) Copyright © 2014 International Society of Nephrology Terms and Conditions