Research ArticleTransplantation

Tracking donor-reactive T cells: Evidence for clonal deletion in tolerant kidney transplant patients

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Science Translational Medicine  28 Jan 2015:
Vol. 7, Issue 272, pp. 272ra10
DOI: 10.1126/scitranslmed.3010760
  • Fig. 1. MLR experimental design and schematic of TCR sequencing analysis strategy to identify and track donor-reactive T cells.

    (A) CD3+violet cells, representing the responder T cells, were selected and further separated into CD4+ and CD8+ subgroups. Within each subgroup, the CFSE-low cells were isolated for DNA extraction and TCRβ CDR3 deep sequencing. Sorting strategy is indicated with boxes on the dot plots and bars on the histograms; CFSE staining in unstimulated control sample shown in fig. S1A. (B) Pretransplant fingerprint of anti-donor T cell repertoire defined as all clones detected at a frequency greater than 10−4 in the stimulated condition (CFSE-low cells in MLR) that have expanded at least fivefold relative to their frequency in the unstimulated T cell population (unstimulated repertoire defined via TCR sequencing of CD3+CD4+ or CD3+CD8+ T cells isolated via FACS sorting of PBMCs from the same sample used for the CFSE MLR). Each donor-reactive clone identified by its unique CDR3 nucleotide sequence could then be tracked in posttransplant unstimulated peripheral blood samples.

  • Fig. 2. Overlap of the alloreactive T cell repertoire via TCR sequencing in repeat MLR assays in healthy controls.

    (A) Top panel: Linear correlation of log frequencies of alloreactive clones detected in MLRs performed at time point 1 [stimulated (T1)] and time point 2 [stimulated (T2)] for healthy control #1 (HC#1) from blood draws separated by a 2-week interval. Second panel from top: No such correlation is observed when clonal frequencies in the stimulated populations are plotted against frequencies in the unstimulated population [unstimulated (T1)]; high-frequency clones in the unstimulated populations highlighted in yellow. These results are representative of similar analyses in HC#2 and HC#3 (fig. S1D). Bottom two panels: Linear correlation persists in MLRs performed from blood draws separated by a year interval (HC#2, HC#3). Same allogeneic stimulator for each HC at T1 and T2. Overlapping number of sequences detected: HC#1 T1 and T2 stim CD4 = 2944, CD8 = 465; HC#1 T1 stim and T1 unstim: CD4 = 3011, CD8 = 478; HC#2 T1 and T2 stim: CD4 =1162, CD8 = 642; HC#3 T1 and T2 stim: CD4 =2850, CD8 = 652. (B) Pie charts showing relative overlap of the summed frequencies of alloreactive clones (fingerprint as defined in Fig. 1) over time in three healthy controls. Each circle represents the cumulative frequency of all alloreactive clones identified in the sample; red segment shows the percentage of that total frequency arising from alloreactive clones identified at both T1 and T2 (tabulated values table S2). (C) Boxplot comparing the clonality of the unstimulated CD4 and CD8 T cell repertoires (n = 5; *P = 0.0062, two-tailed paired t test; tabulated values in table S1). (D) Cumulative frequencies of all alloreactive clones for each pair of stimulated and unstimulated HC at all time points.

  • Fig. 3. Clinical course, tracking of donor-reactive T cell clones, MLR, and CML results in CKBMT subjects.

    (A) Overview of clinical course. (B) Number of donor-reactive TRB CDR3 clones (y axis) detected in the unstimulated CD4 (black) and CD8 (white) repertoire at the indicated time points (x axis). The fingerprint of the anti-donor T cell repertoire was defined for each subject as clones in the pretransplant MLR with ≥10−4 frequency that were expanded at least fivefold relative to their frequency in the pretransplant unstimulated sample (total number indicated above the relevant panel). Sufficient power was obtained to consider a frequency of 10−5 as detectable in all unstimulated populations for subjects 1, 2, and 4, and 5 × 10−5 for subject 5. *P < 0.05 compared to pretransplant (P values in table S4, two-sided Fisher’s exact test). (C) MLR: proliferative responses to recipient (anti-self), donor (anti-donor), and third party (anti-3rd party) relative to proliferation of unstimulated PBMCs are shown at the indicated time points. Each bar represents the mean ± SD stimulation index of triplicate cultures. (D) CML: responses at the indicated time points are shown. MLR and CML data have been previously summarized by Kawai et al. (8).

  • Fig. 4. Tracking of donor-reactive T cell clones in conventional kidney transplant recipients and summary of clonal analysis results for all six subjects.

    (A) Clinical course and number of donor-reactive TRB CDR3 clones (y axis) detected in the unstimulated CD4 (black) and CD8 (white) repertoire at the indicated time points (x axis). Sufficient power was obtained to consider a frequency of 10−5 as detectable in all unstimulated populations for IS#1 and 5 × 10−5 for IS#2. *P < 0.05 compared to pretransplant (P values in table S4, two-sided Fisher’s exact test). (B) Change over time in detection of unstimulated T cell populations of donor-reactive CD4 and CD8 clones (defined in Fig. 3B legend). Fold change is the odds ratio of the number of donor-reactive clones detected in unstimulated posttransplant CD4 and CD8 populations relative to the number detected in unstimulated pretransplant populations (pretransplant = 1). Open symbol, statistically significant reduction or increase (P < 0.05). Tabulated results are shown in tables S4 and S5.

  • Fig. 5. TCR repertoire turnover in kidney allograft recipients.

    (A) JSD comparing pre- and posttransplantation (last posttransplantation time point) TCR repertoires. JSD on top 1000 nucleotide clones ranked by frequency (0 denotes identical repertoires; 1 denotes complete repertoire divergence). Healthy controls: average JSD on top 1000 nucleotide clones of two healthy controls in whom TCR sequencing was performed at two time points 1 year apart. (B) Anti-donor CD4 and CD8 clonal analysis relative to overall repertoire turnover. Relative numbers of donor-reactive (as defined in Fig. 3B legend) versus non–donor-reactive clones (all other clones detected in unstimulated pretransplant sample) detected at any level (threshold detection of assay 10−6) in posttransplant samples. Relative change is the odds ratio of the relative (post/pre) number of donor-reactive clones divided by the relative (post/pre) number of non–donor-reactive clones in unstimulated samples at the same time. A value of 1 indicates that the proportion of donor-reactive clones (defined before transplant as above) detected at a given time point was equal to that of all clones (detected before transplant) detected at the same time point. A value <1 indicates lower rate of detection of donor-reactive versus all clones, and a value >1 indicates greater rate of detection of donor-reactive versus all clones. Open symbols, statistically significant reduction or increase (P < 0.05, two-sided Fisher’s exact test); tabulated data and P values in table S7. (C) Anti-donor CD4 and CD8 clonal analysis relative to overall repertoire turnover in subject 1: effect of varying definition of donor reactivity by different fold expansion criteria (frequency in pretransplant anti-donor MLR/frequency in unstimulated pretransplant sample >5, 7, or 10). Red points are statistically significant (P < 0.05); tabulated data and P values in table S9.

  • Fig. 6. Recovery of CD4 repertoire diversity in tolerant recipients.

    (A) CD4 T cell repertoire diversity (measured by Simpson’s index D) over time after transplantation (D of 1 indicates that all clones are identical; smaller D indicates clones are more unique and therefore the repertoire is more diverse). (B) Comparison of CD4 Simpson’s index (D) in tolerant (subjects 1, 2, and 4) and nontolerant (subjects 5, IS#1, and IS2) subjects near 1 year after transplant (10 months: IS#2; 12 months: subjects 2, 4, and IS#1; 14 months: subject 5; 18 months: subject 1). *P = 0.017, two-sided Student’s t test on logarithm of D (n = 6).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/272/272ra10/DC1

    Fig. S1. Validation study design and analysis details of the tracking of alloreactive clones in healthy controls.

    Fig. S2. Anti-donor clonal analysis at increasing fold expansion criteria.

    Fig. S3. Frequencies of donor-reactive clones in unstimulated PBMCs before and after transplant in CKBMT recipients (CD4).

    Fig. S4. Frequencies of donor-reactive clones in unstimulated PBMCs before and after transplant in the CKBMT recipients (CD8).

    Fig. S5. LDA (CTLp and HTL) for subjects 1, 2, 4, and 5.

    Fig. S6. Comparison of pre- and posttransplant anti-donor MLR in subject 4.

    Fig. S7. Frequencies of donor-reactive clones before and after transplant in the conventional kidney transplant recipients.

    Fig. S8. Detection of third party–reactive TCRs before and after transplant.

    Table S1. Cell counts, entropy, and clonality for healthy control experiments.

    Table S2. Sum frequency of alloreactive clones in healthy controls (%).

    Table S3. Cell numbers, number of unique clones, and total number of reads for each patient sample.

    Table S4. Tabulated data from clonal analysis in subjects 1, 2, 4, 5, IS#1, and IS#2 (Figs. 3B and 4).

    Table S5. Tabulated data from clonal analysis in healthy controls (Fig. 4B).

    Table S6. Sum frequency of alloreactive clones in subject 4 (%).

    Table S7. Relative turnover analysis in tolerant subjects (Fig. 5B).

    Table S8. Cell numbers, number of unique clones, and total number of reads for anti–third party responses.

    Table S9. Relative turnover analysis at increasing fold expansion criteria: Subject 1 (Fig. 5C).

  • Supplementary Material for:

    Tracking donor-reactive T cells: Evidence for clonal deletion in tolerant kidney transplant patients

    Heather Morris, Susan DeWolf, Harlan Robins, Ben Sprangers, Samuel A. LoCascio, Brittany A. Shonts, Tatsuo Kawai, Waichi Wong, Suxiao Yang, Julien Zuber, Yufeng Shen,* Megan Sykes*

    *Corresponding author. E-mail: megan.sykes{at}columbia.edu (M.S.); ys2411{at}columbia.edu (Y.S.)

    Published 28 January 2015, Sci. Transl. Med. 7, 272ra10 (2015)
    DOI: 10.1126/scitranslmed.3010760

    This PDF file includes:

    • Fig. S1. Validation study design and analysis details of the tracking of alloreactive clones in healthy controls.
    • Fig. S2. Anti-donor clonal analysis at increasing fold expansion criteria.
    • Fig. S3. Frequencies of donor-reactive clones in unstimulated PBMCs before and after transplant in CKBMT recipients (CD4).
    • Fig. S4. Frequencies of donor-reactive clones in unstimulated PBMCs before and after transplant in the CKBMT recipients (CD8).
    • Fig. S5. LDA (CTLp and HTL) for subjects 1, 2, 4, and 5.
    • Fig. S6. Comparison of pre- and posttransplant anti-donor MLR in subject 4.
    • Fig. S7. Frequencies of donor-reactive clones before and after transplant in the conventional kidney transplant recipients.
    • Fig. S8. Detection of third party–reactive TCRs before and after transplant.
    • Table S1. Cell counts, entropy, and clonality for healthy control experiments.
    • Table S2. Sum frequency of alloreactive clones in healthy controls (%).
    • Table S3. Cell numbers, number of unique clones, and total number of reads for each patient sample.
    • Table S4. Tabulated data from clonal analysis in subjects 1, 2, 4, 5, IS#1, and IS#2 (Figs. 3B and 4).
    • Table S5. Tabulated data from clonal analysis in healthy controls (Fig. 4B).
    • Table S6. Sum frequency of alloreactive clones in subject 4 (%).
    • Table S7. Relative turnover analysis in tolerant subjects (Fig. 5B).
    • Table S8. Cell numbers, number of unique clones, and total number of reads for anti–third party responses.
    • Table S9. Relative turnover analysis at increasing fold expansion criteria: Subject 1 (Fig. 5C).

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