2018 Nov 15
Early Expansion of Donor-Specific Tregs in Tolerant Kidney Transplant Recipients
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Early Expansion of Donor-Specific Tregs in Tolerant Kidney Transplant Recipients
Allograft tolerance, in which a graft is accepted without long-term immunosuppression, could overcome numerous obstacles in transplantation. Human allograft tolerance has been intentionally induced across HLA barriers via combined kidney and bone marrow transplantation (CKBMT) with a regimen that induces only transient chimerism. Tregs are enriched early after CKBMT. While deletional tolerance contributes to long-term tolerance, the role of Tregs remains unclear. We have optimized a method for identifying the donor-specific Treg repertoire and used it to interrogate the fate of donor-specific Tregs after CKBMT. We expanded Tregs with several different protocols. Using functional analyses and T cell receptor sequencing, we found that expanding sorted Tregs with activated donor B cells identified the broadest Treg repertoire with the greatest potency and donor specificity of suppression. This method outperformed both alloantigen stimulation with CTLA4Ig and sequencing of CFSElo cells from the primary mixed lymphocyte reaction. In 3 tolerant and 1 nontolerant CKBMT recipients, we sequenced donor-specific Tregs before transplant and tracked them after transplant. Preexisting donor-specific Tregs were expanded at 6 months after CKBMT in tolerant patients and were reduced in the nontolerant patient. These results suggest that early expansion of donor-specific Tregs is involved in tolerance induction following CKBMT.
Immunology; Organ transplantation; T-cell receptor; Tolerance; Transplantation.
Conflict of interest statement
Conflict of interest: EB owns stock in ITB-MED AB Inc. MS is chairman of the scientific advisory board of ITB-MED AB Inc.
Figure 1. Methods to identify the donor-specific Treg repertoire.
A) TCRβ sequencing was performed on unstimulated Tregs and CD4 + non-Tregs from PBMCs to establish the unstimulated Treg and non-Treg repertoires. ( B) Bulk CFSE-MLRs were performed to identify the CD4 donor-reactive repertoire — defined as unique TCRβ sequences expanded ≥5-fold from unstimulated CD4 samples and with frequency ≥10 –4 in the CFSE lo sample — which was compared with the unstimulated Treg repertoire from A to determine the “CFSE-MLR Treg” repertoire. ( C) Activated (act.) donor B cells were generated, with representative flow cytometry staining demonstrating activation, then irradiated to culture with sorted Tregs. ( D) Primary and restimulated CTLA4Ig MLRs were performed as shown.
Figure 2. Comparison of donor-specific TCRβ repertoires in healthy controls.
A) Proportional Venn diagrams of the CFSE-MLR Treg repertoire (yellow), culture of Tregs with activated donor B cells (blue), and restimulated (Restim.) CTLA4Ig MLR (red), where n denotes the number of unique TCRβ sequences identified via each method; for the CFSE-MLR Tregs, n is stated among the total number of CD4 donor-reactive sequences. ( B) Number and ( C) sum frequency of sequences in stated methods mapping to the unstimulated (Unstim.) Treg and CD4 + non-Treg repertoires. ( B and C) “Donor-reactive” refers to the total CD4 donor-reactive repertoire identified through the CFSE-MLR. ( D) Clonality and ( E) R20 of each method. Clonality ranges from 0 (each sequence has equal frequency) to 1 (a single clone). R20 is the proportion of clones that occupy 20% of the sum frequency when the clones are ordered from highest to lowest frequency; a low R20 indicates clonal dominance. ( F– J) Jenson-Shannon divergence (JSD) of the top 100 unique TCRβ sequences ranked by frequency in each method compared with the TCR repertoire identified via ( F) unstimulated Tregs, ( G) CFSE-MLR Treg repertoire, ( H) culture of Tregs with activated donor B cells, ( I) primary CTLA4Ig MLR, and ( J) restimulated CTLAIg MLR. ( A–J) Data from 3 independent healthy controls.
Figure 3. Tregs cultured with activated donor B cells have higher Foxp3 expression and are more suppressive of donor-specific responses in vitro.
A) Representative Foxp3 stain of an unstimulated Treg sample prior to FACS sorting, gated on CD3 +CD4 +CD8 – cells. ( B) CD25 +CD127 – percentage among CD4 + cells and Foxp3 expression among CD4 +CD25 +CD127 – cells from 3 independent healthy controls at the end of each assay assessed by flow cytometry. ( C) With either donor or HLA-mismatched third-party T cell–depleted PBMC stimulation, inhibition of CFSE-labeled fourth-party CD3 + T cell proliferation by sorted CD4 +CD25 +CD127 – cells from healthy control 1 at the end of each expansion. ( D) Comparison of potency of anti-donor suppression as in C, with unstimulated Treg data summarizing 3 experiments, including from healthy control 1. ( C and D) Mean ± SEM. Experiment performed in triplicate. ( D) * P < 0.05, ** P < 0.01, *** P < 0.001 for comparison of Tregs with activated (w/ act.) donor B cells to restimulated CTLA4Ig MLR (2-tailed t test).
Figure 4. Repertoire comparisons in CKBMT patients.
A) Proportional Venn diagrams of the repertoires, where n = the number of unique TCRβ sequences in each method; for the CFSE-MLR Tregs, n is stated among the total CD4 donor-reactive repertoire. ( B) Number and ( C) sum frequency of sequences in stated methods mapping to the unstimulated Treg and CD4 + non-Treg repertoires. ( B and C) “Donor-reactive” refers to the total CD4 donor-reactive repertoire identified through the CFSE-MLR. ( D) CD25 +CD127 – percentage among CD4 + cells and Foxp3 expression among CD4 +CD25 +CD127 – cells at the end of each assay. ( E) Clonality and ( F) R20 of each method. ( G–J) JSD of the top 100 unique TCRβ sequences ranked by frequency comparing each method.
Figure 5. Preexisting donor-specific Tregs expand in tolerance.
A) Tregs detected with activated donor B cells among the total CD4 repertoire as a fraction of all clones (Clone Fraction) and by cumulative frequency. Clone fraction and cumulative frequency are defined as the number of unique TCRβ sequences and the number of reads corresponding to the method in the total CD4 sample, respectively, divided by the total number of unique sequences and the total number of reads at the given time point, respectively. Labeled n corresponds to the number of unique TCRβ sequences identified as donor-specific Tregs in the subject. * P < 0.05, ** P < 0.01, *** P < 0.001, reduction or increase in clone fraction compared with before transplant (2-sided Fisher’s exact test). ( B) Fold change in the clone fraction and cumulative frequency relative to before transplant. Open symbols, statistically significant reduction or increase by clone fraction compared with before transplant ( P < 0.05, 2-sided Fisher’s exact test). ( C) Sequences in the circulation from the repertoire of Tregs expanded with activated donor B cells that map to the pre-transplant unstimulated Tregs (left) and pre-transplant unstimulated non-Tregs (right) as a proportion of the cumulative frequency of all sequences mapping to this method in the circulation at the indicated time after transplant. ( D) Fold expansion of preexisting donor-specific Tregs (repertoire identified via culture of Tregs with activated donor B cells that maps to pre-transplant unstimulated Treg repertoire) among CD4 repertoire by clone fraction and cumulative frequency compared with fold expansion of pre-transplant-identified unstimulated Tregs at 6 months after transplant relative to before transplant.
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Research Support, N.I.H., Extramural
B-Lymphocytes / immunology
B-Lymphocytes / transplantation
Bone Marrow Transplantation
CTLA-4 Antigen / immunology
Lymphocyte Culture Test, Mixed
Receptors, Antigen, T-Cell / metabolism
T-Lymphocytes, Regulatory / immunology
T-Lymphocytes, Regulatory / transplantation
Receptors, Antigen, T-Cell