Donor-specific indirect pathway analysis reveals a B-cell-independent signature which reflects outcomes in kidney transplant recipients

Abstract

To investigate the role of donor-specific indirect pathway T cells in renal transplant tolerance, we analyzed responses in peripheral blood of 45 patients using the trans-vivo delayed-type hypersensitivity assay. Subjects were enrolled into five groups-identical twin, clinically tolerant (TOL), steroid monotherapy (MONO), standard immunosuppression (SI) and chronic rejection (CR)-based on transplant type, posttransplant immunosuppression and graft function. The indirect pathway was active in all groups except twins but distinct intergroup differences were evident, corresponding to clinical status. The antidonor indirect pathway T effector response increased across patient groups (TOL < MONO < SI < CR; p < 0.0001) whereas antidonor indirect pathway T regulatory response decreased (TOL > MONO = SI > CR; p < 0.005). This pattern differed from that seen in circulating naïve B-cell numbers and in a cross-platform biomarker analysis, where patients on monotherapy were not ranked closest to TOL patients, but rather were indistinguishable from chronically rejecting patients. Cross-sectional analysis of the indirect pathway revealed a spectrum in T-regulatory:T-effector balance, ranging from TOL patients having predominantly regulatory responses to CR patients having predominantly effector responses. Therefore, the indirect pathway measurements reflect a distinct aspect of tolerance from the recently reported elevation of circulating naïve B cells, which was apparent only in recipients off immunosuppression.

Conflict of interest statement

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

© copyright 2011 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

Figure 1. Indirect pathway T-cell analysis

(A) The tvDTH responses to donor antigen (Teff responses) are significantly different between subject groups (p < 0.0001). PBMC from patients (n = 45) were co-injected into the mouse footpad with cell-free donor antigen. Swelling was measured before and 24 h after each injection. Net swelling was determined by subtracting the swelling response obtained from the injection of the same PBMC in PBS. Patient groups: TWIN, identical twin donor (n = 2); TOL, clinically tolerant, no immunosuppression for >1 year (n = 11); MONO, prednisone monotherapy (n = 7); SI, standard immunosuppression (n = 18) and CR, chronic rejection (n = 7). (B) Antibody blocking of indirect pathway T-cell responses. PBMC from patients with CR (n = 5) were injected into the footpad with cell-free donor antigen and blocking antibodies to IFNγ or IL-17 (right side), whereas PBMC from patients in the TOL group (n = 5) were injected with cell-free donor antigen and antibodies to block TGFβ or IL-10 (left side). The net swelling response to donor antigen was significantly elevated in the TOL patients when TGFβ was neutralized (p < 0.01) but not when IL10 was neutralized whereas blocking of either IFNγ or IL17 significantly decreased the swelling response to donor antigen in the CR patients tested (p < 0.01). Net swelling response for each subject with donor antigen and a given test antibody were compared to that with PBMC + donor antigen and isotype control Ab (−) using a paired t-test. (C) Indirect pathway Treg responses are significantly different between subject groups (p = 0.005). PBMC from patients in the same groups as described in (A) were injected with recall antigen (TT/DT or EBV) alone and with recall plus cell-free donor antigen into the mouse footpad. Net swelling was determined as in (A). The percentage inhibition of recall alone response in the presence of donor antigen was calculated as described in the methods. All panels: Individual results from each subject (individual symbols) and group median (horizontal line) with quartiles are shown. Each patient was tested one or two times. Significant differences between groups using pair-wise comparisons (Dunn’s correction for multiple comparisons shown in solid lines); *p < 0.05, **p < 0.01 and ***p < 0.001; (Mann–Whitney U test unadjusted for multiple comparisons shown with dotted lines) #p = 0.04, ##p = 0.03 and ###p = 0.008.

Figure 2. Naive B-cell counts and cross-platform probabilitÿ of being tolerant scores

(A) Naïve B-cell number (CD19+CD27-IgM+IgD+) in fresh peripheral blood of the 45 patients analyzed in Figure 1. (B) Probability of being tolerant, estimated according to the Indices of Tolerance model of 14 biomarkers, including B-cell parameters and direct pathway T-cell parameters (11). Subjects in the TWIN group were not tested (n.t.). All panels: Individual results from each subject (individual symbols) and group median (horizontal line) with quartiles (panel B only) are shown. Significant differences between groups using pair-wise comparisons, *p < 0.05 and **p < 0.01; Dunn’s correction for multiple comparisons shown in solid lines, unadjusted Mann–Whitney U test comparisons shown with dotted lines.

Figure 3. B cells do not impact tvDTH suppression via indirect pathway

PBMC from two patients with high circulating naive B-cell counts, TOL patient 002002 and SI patient 002055 were assayed directly (PBMC, filled squares), after being depleted of CD19+ B cells (open triangles) or after depletion and add-back of B-cell fraction (gray circles). Cell fractions were injected into the footpad with PBS (negative control, data not shown) with recall antigen (TT/DT or EBV, left panel), recall antigen and self-peptide (p37TE or HA-1R, middle panel) or recall antigen and donor peptide (p37MA, or HA-1H, right panel). Swelling was measured before and 24 h after each injection. Net swelling was determined by subtracting the swelling response obtained from the injection of the same cell fractions in PBS.

Figure 4. Donor-specific indirect pathway responses using HLA-single antigen beads

(A) PBMC of tolerant patient 002002 were mixed with varying amounts (0–10 μL) of donor type HLA-B62 (open circle, p37MA+), third party HLA–B57 (open square, p37MA+) or self HLA-A1 (filled triangle, p37MA−)-coated single antigen beads before addition of TT/DT and injected into the mouse footpad. Net swelling was determined as in Figure 1A. The maximum percentage inhibition of response with recall TT/DT (0 beads) versus the response with 10 μL of single antigen beads was calculated as described in the methods and is 60% with B57 beads and 63% with B62 beads. Peptides: PBMC were co-injected into the mouse footpad with recall antigen TT/DT and 1000 ng of HLA-B62 allopeptide p37MA (DSDAASPRMAPRAPWIEQ) or HLA-B37 self peptide p37TE (DSDAASPRTEPRAPWIEQ). The percentage inhibition of recall antigen response with donor peptide is 68%. Patient 002002 = HLA A1, 2; B37, 60; DR 4, 13; donor type: A1, 2; B44, 62; DR 4, 13. (B) Indirect pathway tvDTH response to single antigen-beads detects stronger sensitization to a DSA+ as compared with DSA− donor HLA antigens. PBMC from CR patient 002048 were mixed with donor HLA-A2 (circles), HLA-A1 (squares) or HLA-B57 (inverted triangle)-coated single antigen beads and injected into a CB17 SCID mouse footpad. Net swelling was determined as in Figure 1A. Mean fluorescent intensity (MFI) values for a contemporaneous serum sample of the same subject tested using HLA-A2 (gray circle)-, HLA-A1 (gray square)- or HLA-B57 (gray inverted triangle)-coated single antigen beads are shown on the right. Patient 002048 = HLA-A11, 25; B18, 51;DR 9, 15 Donor = HLA-A1, 2; B7, 57; DR 11,15 (mismatch in bold).

Figure 5. Proposed model of indirect pathway in renal transplant recipients

Red intensity indicates relative frequency of donor specific indirect pathway Teff cells, which may be Th1 or Th17 type; blue intensity denotes relative frequency of indirect pathway Treg cells, which may be Th3 or Tr1 type. Dendritic cells (DC) or germinal center (GC) B cells present donor-derived peptides (pink lines), which arise from processed soluble donor HLA (sHLA) or minor H antigens, to indirect pathway T cells. The black intensity (bottom) represents the relative amount of the immunosuppressive medication of patients, ranging from steroid monotherapy to standard immunosuppression (SI; middle), to chronic rejection (left). Rescue therapy refers to emergency measures such as plasmapheresis and high dose steroids applied in cases of unstable and decreasing graft function, along with increased Teff cells and increased DSA-secreting B cells. Decreased immunosuppression is associated with increased indirect pathway Treg cells and, once the patient is off of all immunosuppression, increased numbers of naïve B, possibly Breg, cells.