Volume 64, Issue 5, Pages 1685-1694 (November 2003) Regulation by CD25+ lymphocytes of autoantigen-specific T-cell responses in Goodpasture's (anti-GBM) disease  Alan D. Salama, Afzal N. Chaudhry, Kathryn A. Holthaus, Karen Mosley, Raghu Kalluri, Mohamed H. Sayegh, Robert I. Lechler, Charles D. Pusey, Liz Lightstone  Kidney International  Volume 64, Issue 5, Pages 1685-1694 (November 2003) DOI: 10.1046/j.1523-1755.2003.00259.x Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 1 Limiting dilution analysis (LDA) reveals that convalescent but not acute patients demonstrate responses to collagenase-solubilized glomerular basement membrane (CS-GBM) and collagen α3(IV)NC1 that follow nonlinear kinetics. Plots of the logarithm of the fraction of nonresponding wells against the cell number in the well were graphed. In acute patients (N = 4), the CS-GBM- or α3(IV)NC1-specific responses all followed first-order kinetics, evidenced by a straight line (A). By contrast, in convalescent patients (N = 3), the responses followed zigzag lines, characteristic of multiple-hit kinetics, shown for patients 2 and 3 (in patient 6, the overall response to CS-GBM was poor making analysis difficult) (B). In patient 2, the zigzag response was confined to the CS-GBM and α3(IV)NC1 responses, while response to tetanus toxoid followed a straight line. A similar pattern demonstrating regulation was seen in healthy control individuals (N = 4) (C). Kidney International 2003 64, 1685-1694DOI: (10.1046/j.1523-1755.2003.00259.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 2 Differences in the effector: responder (E:R) ratio in patients. The differences in E:R ratio for (A) collagenase-solubilized glomerular basement membrane (CS-GBM)-reactive cells between acute (N = 4) and convalescent patients (N = 3) and healthy controls (N = 3) and (B) α3(IV)NC1-reactive cells (N = 1, N = 2, and N = 2, respectively, for acute, convalescent, and control samples) is shown. A value less than unity suggests that regulatory responses predominate, while a value greater than unity suggests that proliferative effector responses predominate. It can be seen that only in acute patients does the ratio approach unity and is statistically higher than the values for convalescent patients [P = 0.026, by one-way analysis of variance (ANOVA)]. The changes in the E:R ratio in two patients (C) to CS-GBM (patient 2) or α3(IV)NC1 (patient 3) over time. There is a dramatic decline in the E:R ratio from the time of presentation with acute disease to the convalescent period, which is apparent as early as 3 months. Kidney International 2003 64, 1685-1694DOI: (10.1046/j.1523-1755.2003.00259.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 3 Changes in the frequencies of collagenase-solubilized glomerular basement membrane (CS-GBM)- and collagen α3(IV)NC1-specific interferon-gamma (IFN-γ)-producing T cells following CD25+ cell depletion. (A) The increase in CS-GBM-specific IFN-γ frequencies following CD25+ depletion is shown for patient 4. Of note, there is little change in the frequencies of tetanus-specific IFN-γ–producing cells, demonstrating that the regulation is antigen-specific. (B) The increase in α3(IV)NC1-specific and CS-GBM-specific T-cell responses following CD25 depletion is shown for patient 7. The response to the CS-GBM and the recombinant α3(IV)NC1 protein demonstrate that these responses are autoantigen-specific. (C) The change in CS-GBM- and α3(IV)NC1-specific IFN-γ frequencies for each patient following CD25 depletion is shown. There is a significant increase in CS-GBM- and α3(IV)NC1-specific IFN-γ frequencies following CD25 cell depletion (left panel, P = 0.031, by paired t test comparing pre-and postdepletion frequencies). Regulation, demonstrated by a positive slope, can be seen in five out of seven convalescent patients, while in two convalescent patients there is little change (patients 2 and 5) and in one acute patient there is a decrease in the frequency to both CS-GBM and α3(IV)NC1 following CD25 depletion (patient 8) (right panel). Solid lines represent response to CS-GBM and dotted lines response to α3(IV)NC1. Kidney International 2003 64, 1685-1694DOI: (10.1046/j.1523-1755.2003.00259.x) Copyright © 2003 International Society of Nephrology Terms and Conditions