SDC (Supplemental Digital Content) Figure S1. Percentages of Granzyme B expressing CD8 + T lymphocytes

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1 Figure S1. Percentages of Granzyme expressing D8 + T lymphocytes 13.5% 1.4%.6% 77% 21% 24% 76% 8 8 Granzyme + (%) Granzyme + (%) D8 + Tnaive Tcm Tem Temra D8 + D8 + D28 + D8 + D28 null We analyzed percentages of Granzyme + cells within the total D8 compartment, D28 + and D28null D8 + T lymphocytes and within the different D8 + T cell subsets. For this purpose, the cells were thawed, left overnight to recover and subsequently the cell surface was stained with antibodies directed against D3, D8, D28, D45RO and R7. Upon permeabilization of the cells, we stained Granzyme intracellular. We acquired 1x1 5 D8 + T lymphocytes during each measurement to ensure reliable results upon dissection of the signal into the different fractions as mentioned above. Samples were analyzed on the D FS anto II using D FS Diva version software. typical example of the flowcytometric analysis with respect to the dissection of the D8 + T cells into fractions is depicted in panel. In panel, the Granzyme expressing D8 + T cells (left) are displayed. This signal is than backgated in the D8 subset plot (middle) and D28 + versus D28null D8 + T cell plot (right). In panel, the percentages Granzyme expressing D8 + T cells are displayed within the different T cells subsets (left) and D28 + and D28null T cells (right). The healthy controls are depicted in the white bars (N=14), the acute rejectors (N=7) in black bars and non-rejectors in grey bars (N=7). ll data are expressed as means with standard error of the mean and differences were analyzed by NOV with post-hoc analysis using onferroni's test for multiple comparisons. P-value<.5 for healthy controls vs. NTX-NR or NTX-R and P-value<.5 for NTX-R vs. NTX-NR.

2 Figure S2. Percentages of IL-1, IFN-γ and TNF-α expressing D8 + T lymphocytes.42% IL-1 + (%) 3 2 IL-1 + (%) D8 + Tnaive Tcm Tem Temra D8 + D8 + D28 + D8 + D28 null 25.33% IFN-γ + (%) 6 4 IFN-γ + (%) D8 + Tnaive Tcm Tem Temra D8 + D8 + D28 + D8 + D28 null 26.2% TNF-α + (%) 6 4 TNF-α + (%) D8 + Tnaive Tcm Tem Temra D8 + D8 + D28 + D8 + D28 null We analyzed percentages of IL-1, IFN-γ and TNF-α expressing cells within the total D8 compartment, D28 + and D28null D8 + T lymphocytes and within the different D8 + T cell subsets. For this purpose, the cells were thawed, left overnight to recover and either left unstimulated or stimulated with the combination of PM (5 ng/ml) and ionomycin (1 µg/ml) for 5 hours in the presence of the cytokine secretion inhibitors brefeldin and monensin. Subsequently, the cell surface was stained with antibodies directed against D3, D8, D28, D45RO and R7. Upon permeabilization of the cells, we stained intracellular for IL-1 (panel ), IFN-γ (panel ) and TNF-α (panel ). We acquired 1x1 5 D8 + T

3 lymphocytes during each measurement to ensure reliable results upon dissection of the signal into the different fractions as mentioned above. Samples were analyzed on the D FS anto II using D FS Diva version software. Each panel is preceded by the cytokine producing cells within the total D8 + T cell compartment and the insert in each graph depicts the backgating of the cytokine signal in the D8 subset plot (middle) and D28 + versus D28null D8 + T cell plot (right). Finally, the graphs represent the percentages cytokine expressing cells within each fraction. The healthy controls are depicted in the white bars (N=14), the acute rejectors (N=7) in black bars and non-rejectors in grey bars (N=7). ll data are expressed as means with standard error of the mean and differences were analyzed by NOV with post-hoc analysis using onferroni's test for multiple comparisons. P- value<.5 for healthy controls vs. NTX-NR or NTX-R and P-value<.5 for NTX-R vs. NTX-NR.

4 Figure S3. Direct suppressive capacity of D8 + Temra cells on D4 + T cell proliferation induced by αd3/d28 or allogenous stimulation presort post sort -D8 + Temra + 4x1 4 D8 + Temra PKH-67 In order to test direct suppressive capacity, highly pure (>95%) FS sorted D8 + Temra cells () were PKH26-labeled and used at different numbers in a proliferation assay. T cells depleted for this fraction were used as responder cells, labeled with PKH67 and used at 5x1 4 /well in triplicate. Proliferation of T cells depleted for D8 + Temra was induced by stimulation with plate-bound αd3/d28 (both at.1 µg/ml) or irradiated allogenous PM (4Gy, 5x1 4 /well). t day 6, cells were harvested, stained using monoclonal antibodies for D3, D4 and using 7D, we selected for viable T cells. typical example is shown of the flowcytometric analysis of T cell proliferation induced by αd3/d28 in the absence as well as in the presence of 4x1 4 D8 + Temra cells (). In panel, percentages of dividing T cells induced by αd3/d28 (left) or irradiated allogenous PM (right) are depicted on the Y-axis whereas on the X-axis, the different ratio s of suppressors added are depicted. The closed squares resemble T cells depleted for D8 + Temra. Values represent: mean and SEM of three independent experiments.

5 Table S1. Demographic and clinical characteristics of validation set of patients (n=53) Gender % male 64.2 % Ethnic background Western European/non-Western European 18/35 ge (mean ± SEM) 5.3 ± 2. MV seropositive 66% Previous transplant 8/45 % PR (median with IQR) (-4) Patients not on dialysis % 41.5 % Time on dialysis (median years with IQR) 1.2 (.8-3.5) Living kidney donation % 1 % Delayed graft function 7.5 % asiliximab induction therapy 1 % Number of HL- mismatches (mean ± SEM) 3.6 ±.18 HL class I mismatches (mean ± SEM) 2.5 ±.13 HL class II mismatches (mean ± SEM) 1.1 ±.8 Kidney disease - Hypertensive nephropathy - Glomerulonephritis - Diabetic nephropathy - Polycystic kidney disease - Reflux nephropathy - Other/Unknown 24.5 % 22.6 % 18.9 % 11.3 % 7.5 % 15.1 %

6 Figure S 4. nalysis of T cell differentiation status of a different cohort of ESRD patients D4 + T cells (1 6 /ml) 1..5 D8 + T cells (1 6 /ml) % of total D4 + T cells % of total D8 + T cells D naive M EM naive M EM EMR D4 + D28 null T cells (%) E D8 + D28 null T cells (%) F D4 + D28 null T cells (1 3 /ml) G D8 + D28 null T cells (1 3 /ml) H D4 + D28 null T cells (%) I D8 + D28 null T cells (%) J naive M EM naive M EM EMR

7 Using a whole blood staining, absolute numbers of circulating D4 + and D8 + T cells (-) were determined in a group of patients receiving a kidney transplantation (measurement is done before kidney transplantation) and age-matched healthy controls (N=52, white bars). The patient population is divided into a group that experienced acute rejection at follow-up (NTX-R, N=1, black bars) and a group that did not (NTX-NR, N=42, grey bars). The T cell differentiation within the NTX-R (black bars), NTX-NR (grey bars) and healthy control (white bars) group was expressed as the percentage of D4 + () and D8 + (D). Next, percentages (E-F) and absolute numbers (G-H) of circulating D4 + (E, G) and D8 + (F, H) T cells that do not express D28 (D28null T cells) were determined in these groups using a whole blood staining. The expression of D28null T cells within the different T cell subsets for the NTX-R, NTX-NR and healthy control group is expressed for D4 + (I) and D8 + (J) T cells. ll data are expressed as means with standard error of the mean and differences were analyzed by NOV with post-hoc analysis using onferroni's test for multiple comparisons. P-value<.5 for healthy controls vs. NTX-NR or NTX-R and p-value<.5 for NTX-R vs. NTX-NR.