Figure 3. Long-lasting circulating donor cells show GvH tolerance.

(A) Functional MLR using Pt15 pre-Tx donor splenocytes or post-Tx total or CD25⁻ T cells sorted from recipient PBMCs collected on POD83 and POD214 as responders, and irradiated recipient pre-Tx splenocytes or 3^rd party PBMCs as stimulators. CFSE proliferation profile of gated donor CD4 and CD8 T cells is shown. (B) Summary of %CFSE^low donor CD4 and CD8 T cells in functional MLRs using pre-Tx donor splenocytes (Pts15, 7 and 16) or post-Tx PBMCs (Pt15 POD214, Pt7 POD253) or splenocytes (Pt16 POD786) as responders against irradiated stimulators. Student t test was used to compare paired data as indicated (* p<0.05, **p<0.01, ***p<0.001; n.s.: no significant difference). Means and standard deviations (SDs) are shown in bar graphs. (C) Donor HLA⁺CD3⁺CFSE^low cells were sorted on Day 6 of a MLR in which CD25⁻ T cells sorted from POD83 PBMCs were used as responders, and irradiated recipient pre-Tx splenocytes as stimulators. TCRβ CDR3 DNA sequencing was performed on sorted donor HLA⁺CD3⁺CFSE^low cells, and the cumulative frequency of GvH clones as a percentage of all sequences within donor mappable clones is shown. “Donor mappable clones” refer to clones that were detectable in sequenced pre-Tx spleen, lymph node and/or MLR CFSE^low T cell populations from the donor. Four such clones were identified among a total of 124 unique sequences. (D) Total frequencies among all TCR sequences for representative GvH clones that were detected in pre-Tx donor spleen or MLN, post-Tx early (POD<70) and late (POD>140) ileum biopsies (Bx), and early (POD<70) and late (POD>250) PBMCs in Pts 4, 7, 13 and 15. (E) HLA typing (HLA-A, B, C, DRB1 and DQ) and number of HLA mismatches (red) of donor with recipient and 3^rd party is shown for Pt15. Pt 15’s donor had a greater number of HLA mismatches to the recipient (8/10) than to the 3^rd party (7/10). Pt7 and Pt16’s donors each had equal numbers of HLA mismatches to the recipient and the 3^rd party, 5/10 and 8/10, respectively (data not shown). See also Figure S1 and Table S1, S2.