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. Author manuscript; available in PMC: 2011 Jun 1.

Published in final edited form as: Hum Immunol. 2010 Mar 11;71(6):566–576. doi: 10.1016/j.humimm.2010.02.008

(A & B) Cell Mediated Lympholysis (CML) in DBMC infused recipients: 4×10⁶ recipient responder PMBC were cultured with 4×10⁶ irradiated donor or third party PMBC and standard 4 hr ⁵¹Cr-release assays against the respective stimulators were performed on day 8. The data on the left are shown as percent specific lysis for E: T ratios of 50:1 in individual recipients, which segregated into two patterns of reactivity to the donor, i.e. the majority showing unresponsiveness (A) and a minority showing residual responses (B) after 1–3 years post-transplant. On the right is represented the mean ± SD responses of the two patterns A & B to the donor and third party. Percent specific lysis of 5 was considered the cut-off value for a positive response [11], represented by the dashed lines.

(C&D) Limiting Dilution Analysis (LDA) for the enumeration of CTL-precursors in DBMC infused Kidney transplant recipients: Serial dilutions of recipient responder T cells (0 – 20,000 cells/well) were stimulated with 1×10⁵ irradiated donor or third party PMBC in presence of 5×10⁴ autologous non-T cells. On day 8, 5×10³ ⁵¹Cr-labeled PHA blast target cells from the corresponding stimulator were added and 4-hr chromium release assays were performed. Data were plotted as log fraction non-responding cultures vs. T cell dose, the frequencies calculated and depicted here as CTLp per 1×10⁶ T cells. CTLp frequency of 50 per 1×10⁶ T cells was considered the cut-off value for a positive response, represented by the dashed lines. Two patterns of reactivity (C & D) were observed with the majority showing deletion of donor specific CTLp after 1–3 years (C). On the left are depicted the CTLp frequencies of individual recipients; and on the right are the mean ± SD frequencies of the groups (C or D).

E: Micro-CML responses in three groups of LRD-Kidney transplant recipients at 49–134 months: Micro-CML were performed as described under Materials and Methods. Percent specific lysis of 5 was considered the cut-off value shown by the dashed lines and the percentage of recipients demonstrating donor specific unresponsiveness are indicated below data symbols.

(A & B) Cell Mediated Lympholysis (CML) in DBMC infused recipients: 4×10⁶ recipient responder PMBC were cultured with 4×10⁶ irradiated donor or third party PMBC and standard 4 hr ⁵¹Cr-release assays against the respective stimulators were performed on day 8. The data on the left are shown as percent specific lysis for E: T ratios of 50:1 in individual recipients, which segregated into two patterns of reactivity to the donor, i.e. the majority showing unresponsiveness (A) and a minority showing residual responses (B) after 1–3 years post-transplant. On the right is represented the mean ± SD responses of the two patterns A & B to the donor and third party. Percent specific lysis of 5 was considered the cut-off value for a positive response [11], represented by the dashed lines.

(C&D) Limiting Dilution Analysis (LDA) for the enumeration of CTL-precursors in DBMC infused Kidney transplant recipients: Serial dilutions of recipient responder T cells (0 – 20,000 cells/well) were stimulated with 1×10⁵ irradiated donor or third party PMBC in presence of 5×10⁴ autologous non-T cells. On day 8, 5×10³ ⁵¹Cr-labeled PHA blast target cells from the corresponding stimulator were added and 4-hr chromium release assays were performed. Data were plotted as log fraction non-responding cultures vs. T cell dose, the frequencies calculated and depicted here as CTLp per 1×10⁶ T cells. CTLp frequency of 50 per 1×10⁶ T cells was considered the cut-off value for a positive response, represented by the dashed lines. Two patterns of reactivity (C & D) were observed with the majority showing deletion of donor specific CTLp after 1–3 years (C). On the left are depicted the CTLp frequencies of individual recipients; and on the right are the mean ± SD frequencies of the groups (C or D).

E: Micro-CML responses in three groups of LRD-Kidney transplant recipients at 49–134 months: Micro-CML were performed as described under Materials and Methods. Percent specific lysis of 5 was considered the cut-off value shown by the dashed lines and the percentage of recipients demonstrating donor specific unresponsiveness are indicated below data symbols.