Cryptic B cell response to renal transplantation
R J Lynch, I A Silva, B J Chen, J D Punch, M Cascalho, J L Platt, R J Lynch, I A Silva, B J Chen, J D Punch, M Cascalho, J L Platt
Abstract
Transplantation reliably evokes allo-specific B cell and T cell responses in mice. Yet, human recipients of kidney transplants with normal function usually exhibit little or no antibody specific for the transplant donor during the early weeks and months after transplantation. Indeed, the absence of antidonor antibodies is taken to reflect effective immunosuppressive therapy and to predict a favorable outcome. Whether the absence of donor-specific antibodies reflects absence of a B cell response to the donor, tolerance to the donor or immunity masked by binding of donor-specific antibodies to the graft is not known. To distinguish between these possibilities, we devised a novel ELISPOT, using cultured donor, recipient and third-party fibroblasts as targets. We enumerated donor-specific antibody-secreting cells in the blood of nine renal allograft recipients with normal kidney function before and after transplantation. Although none of the nine subjects had detectable donor-specific antibodies before or after transplantation, all exhibited increases in the frequency of donor-specific antibody-secreting cells eight weeks after transplantation. The responses were directed against the donor HLA-class I antigens. The increase in frequency of donor-specific antibody-secreting cells after renal transplantation indicates that B cells respond specifically to the transplant donor more often than previously thought.
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.
© 2013 The Authors. American Journal of Transplantation Published by Wiley Periodicals Inc.
Figures
Samples of perinephric fascia from donors and Scarpa’s fascia from recipients of renal transplants were explanted and used to establish primary cultures of fibroblasts. The fibroblasts generally reached confluence at day 30. PBMCs collected at the time of transplantation and 6 to 8 weeks later were cultured for 5 days prior to overlaying onto the fixed fibroblast layer and incubated for 24 hours. Antibodies bound to the fibroblasts were detected with goat anti-human IgM or IgG, AP conjugated, and developed as with a regular ELISPOT. A typical cellular ELISPOT well is shown.
(A)Expression of HLA-class I on cultured human fibroblasts. The figure shows that cultured fibroblasts express HLA-class I detected by binding of a murine monoclonal antibody specific for a non-polymorphic domain of HLA–A, B and C (W6/32) and measured measured by ELISA. Standard errors of triplicate measurements are indicated. (B) Flow cytometry analysis of HLA-class I and class II expression by cultured fibroblasts. Shown is the light scatter plot with the lymphocyte gate depicted in red. Dot plots show staining with antibody isotype control, anti-HLA class I and anti-HLA class II antibodies. The fractions of positively stained cells are noted. (C)Use of cultured fibroblasts for assay of donor-specific antibodies in the serum of renal transplant recipients. Cultured donor fibroblasts were incubated sequentially with serial dilutions of serum from a highly sensitized subject and with serum obtained before and two months after renal transplantation and with AP labeled anti-human IgG. Donor-specific IgG was measured by detecting the optical density at 405 nm (Y-axis). The assay detects binding of IgG from a highly sensitized subject but minimal or no binding of IgG from the renal transplant recipient or pooled IgG used a control. Standard errors of triplicate measurements are indicated.
(A)Expression of HLA-class I on cultured human fibroblasts. The figure shows that cultured fibroblasts express HLA-class I detected by binding of a murine monoclonal antibody specific for a non-polymorphic domain of HLA–A, B and C (W6/32) and measured measured by ELISA. Standard errors of triplicate measurements are indicated. (B) Flow cytometry analysis of HLA-class I and class II expression by cultured fibroblasts. Shown is the light scatter plot with the lymphocyte gate depicted in red. Dot plots show staining with antibody isotype control, anti-HLA class I and anti-HLA class II antibodies. The fractions of positively stained cells are noted. (C)Use of cultured fibroblasts for assay of donor-specific antibodies in the serum of renal transplant recipients. Cultured donor fibroblasts were incubated sequentially with serial dilutions of serum from a highly sensitized subject and with serum obtained before and two months after renal transplantation and with AP labeled anti-human IgG. Donor-specific IgG was measured by detecting the optical density at 405 nm (Y-axis). The assay detects binding of IgG from a highly sensitized subject but minimal or no binding of IgG from the renal transplant recipient or pooled IgG used a control. Standard errors of triplicate measurements are indicated.
(A)Expression of HLA-class I on cultured human fibroblasts. The figure shows that cultured fibroblasts express HLA-class I detected by binding of a murine monoclonal antibody specific for a non-polymorphic domain of HLA–A, B and C (W6/32) and measured measured by ELISA. Standard errors of triplicate measurements are indicated. (B) Flow cytometry analysis of HLA-class I and class II expression by cultured fibroblasts. Shown is the light scatter plot with the lymphocyte gate depicted in red. Dot plots show staining with antibody isotype control, anti-HLA class I and anti-HLA class II antibodies. The fractions of positively stained cells are noted. (C)Use of cultured fibroblasts for assay of donor-specific antibodies in the serum of renal transplant recipients. Cultured donor fibroblasts were incubated sequentially with serial dilutions of serum from a highly sensitized subject and with serum obtained before and two months after renal transplantation and with AP labeled anti-human IgG. Donor-specific IgG was measured by detecting the optical density at 405 nm (Y-axis). The assay detects binding of IgG from a highly sensitized subject but minimal or no binding of IgG from the renal transplant recipient or pooled IgG used a control. Standard errors of triplicate measurements are indicated.
Serum was obtained from nine subjects before and two months after renal transplantation. The serum diluted 1:16 was assayed for binding of IgM (A) or IgG (B) to donor fibroblasts and expressed as absorbance at 405nm (Y-axis). The results show that renal transplant recipients had no detectable donor-specific antibodies two months after transplantation. The average ODs and standard deviations for each population were 0.04±0.003, IgM pre-TX; 0.04±0.004, IgM post-TX; 0.03±0.003, IgG pre-TX; 0.03±0.004, IgG post-TX.
Serum was obtained from nine subjects before and two months after renal transplantation. The serum diluted 1:16 was assayed for binding of IgM (A) or IgG (B) to donor fibroblasts and expressed as absorbance at 405nm (Y-axis). The results show that renal transplant recipients had no detectable donor-specific antibodies two months after transplantation. The average ODs and standard deviations for each population were 0.04±0.003, IgM pre-TX; 0.04±0.004, IgM post-TX; 0.03±0.003, IgG pre-TX; 0.03±0.004, IgG post-TX.
The number and specificity of donor-specific antibody-secreting cells was determined using a novel ELISPOT in which cultured fibroblasts were used as targets and following incubation with 5 x 104 stimulated B cells in unfractionated PBMC for 24 hours. Antibody bound to the fibroblasts was detected by ELISPOT using murine monoclonal anti-human-IgM or –IgG. (A) Typical cellular ELISPOT wells. Cellular ELISPOT wells revealing anti-donor or anti-recipient (autologous) IgM or IgG antibodies. Numbers indicate the ELISPOT readings. (B) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgM increased significantly after renal transplantation (p=0.0001, paired T test). (C) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation in individual recipients. Averages of ASC before and after transplantation were compared by paired T test (p=0.0001). (D) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgG increased (indicated in red) in four and decreased in two (indicated in green) of the nine subjects after renal transplantation. Pre- and post transplant values did not significantly differ by paired T test analysis (p=0.0574). (E) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation in three typical patients. Individual experimental values and standard errors are shown. Averages of ASC before and after transplantation were compared by paired T test (p=0.0574). (F) Frequency of total IgM and IgG producing cells before and 2 months after transplantation for a typical recipient. Recipients had 1417 IgM and 411.7 IgG secreting B cells per 5x104 B cells pre-transplantation, and 1003 IgM and 265.7 IgG secreting B cells per 5x104 B cells post-transplantation, respectively. The number of IgM or IgG secreting B cells before and after transplantation did significantly differ by T test analysis (for IgM p=0.0256, for IgG p=0.0337).
The number and specificity of donor-specific antibody-secreting cells was determined using a novel ELISPOT in which cultured fibroblasts were used as targets and following incubation with 5 x 104 stimulated B cells in unfractionated PBMC for 24 hours. Antibody bound to the fibroblasts was detected by ELISPOT using murine monoclonal anti-human-IgM or –IgG. (A) Typical cellular ELISPOT wells. Cellular ELISPOT wells revealing anti-donor or anti-recipient (autologous) IgM or IgG antibodies. Numbers indicate the ELISPOT readings. (B) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgM increased significantly after renal transplantation (p=0.0001, paired T test). (C) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation in individual recipients. Averages of ASC before and after transplantation were compared by paired T test (p=0.0001). (D) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgG increased (indicated in red) in four and decreased in two (indicated in green) of the nine subjects after renal transplantation. Pre- and post transplant values did not significantly differ by paired T test analysis (p=0.0574). (E) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation in three typical patients. Individual experimental values and standard errors are shown. Averages of ASC before and after transplantation were compared by paired T test (p=0.0574). (F) Frequency of total IgM and IgG producing cells before and 2 months after transplantation for a typical recipient. Recipients had 1417 IgM and 411.7 IgG secreting B cells per 5x104 B cells pre-transplantation, and 1003 IgM and 265.7 IgG secreting B cells per 5x104 B cells post-transplantation, respectively. The number of IgM or IgG secreting B cells before and after transplantation did significantly differ by T test analysis (for IgM p=0.0256, for IgG p=0.0337).
The number and specificity of donor-specific antibody-secreting cells was determined using a novel ELISPOT in which cultured fibroblasts were used as targets and following incubation with 5 x 104 stimulated B cells in unfractionated PBMC for 24 hours. Antibody bound to the fibroblasts was detected by ELISPOT using murine monoclonal anti-human-IgM or –IgG. (A) Typical cellular ELISPOT wells. Cellular ELISPOT wells revealing anti-donor or anti-recipient (autologous) IgM or IgG antibodies. Numbers indicate the ELISPOT readings. (B) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgM increased significantly after renal transplantation (p=0.0001, paired T test). (C) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation in individual recipients. Averages of ASC before and after transplantation were compared by paired T test (p=0.0001). (D) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgG increased (indicated in red) in four and decreased in two (indicated in green) of the nine subjects after renal transplantation. Pre- and post transplant values did not significantly differ by paired T test analysis (p=0.0574). (E) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation in three typical patients. Individual experimental values and standard errors are shown. Averages of ASC before and after transplantation were compared by paired T test (p=0.0574). (F) Frequency of total IgM and IgG producing cells before and 2 months after transplantation for a typical recipient. Recipients had 1417 IgM and 411.7 IgG secreting B cells per 5x104 B cells pre-transplantation, and 1003 IgM and 265.7 IgG secreting B cells per 5x104 B cells post-transplantation, respectively. The number of IgM or IgG secreting B cells before and after transplantation did significantly differ by T test analysis (for IgM p=0.0256, for IgG p=0.0337).
The number and specificity of donor-specific antibody-secreting cells was determined using a novel ELISPOT in which cultured fibroblasts were used as targets and following incubation with 5 x 104 stimulated B cells in unfractionated PBMC for 24 hours. Antibody bound to the fibroblasts was detected by ELISPOT using murine monoclonal anti-human-IgM or –IgG. (A) Typical cellular ELISPOT wells. Cellular ELISPOT wells revealing anti-donor or anti-recipient (autologous) IgM or IgG antibodies. Numbers indicate the ELISPOT readings. (B) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgM increased significantly after renal transplantation (p=0.0001, paired T test). (C) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation in individual recipients. Averages of ASC before and after transplantation were compared by paired T test (p=0.0001). (D) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgG increased (indicated in red) in four and decreased in two (indicated in green) of the nine subjects after renal transplantation. Pre- and post transplant values did not significantly differ by paired T test analysis (p=0.0574). (E) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation in three typical patients. Individual experimental values and standard errors are shown. Averages of ASC before and after transplantation were compared by paired T test (p=0.0574). (F) Frequency of total IgM and IgG producing cells before and 2 months after transplantation for a typical recipient. Recipients had 1417 IgM and 411.7 IgG secreting B cells per 5x104 B cells pre-transplantation, and 1003 IgM and 265.7 IgG secreting B cells per 5x104 B cells post-transplantation, respectively. The number of IgM or IgG secreting B cells before and after transplantation did significantly differ by T test analysis (for IgM p=0.0256, for IgG p=0.0337).
The number and specificity of donor-specific antibody-secreting cells was determined using a novel ELISPOT in which cultured fibroblasts were used as targets and following incubation with 5 x 104 stimulated B cells in unfractionated PBMC for 24 hours. Antibody bound to the fibroblasts was detected by ELISPOT using murine monoclonal anti-human-IgM or –IgG. (A) Typical cellular ELISPOT wells. Cellular ELISPOT wells revealing anti-donor or anti-recipient (autologous) IgM or IgG antibodies. Numbers indicate the ELISPOT readings. (B) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgM increased significantly after renal transplantation (p=0.0001, paired T test). (C) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation in individual recipients. Averages of ASC before and after transplantation were compared by paired T test (p=0.0001). (D) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgG increased (indicated in red) in four and decreased in two (indicated in green) of the nine subjects after renal transplantation. Pre- and post transplant values did not significantly differ by paired T test analysis (p=0.0574). (E) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation in three typical patients. Individual experimental values and standard errors are shown. Averages of ASC before and after transplantation were compared by paired T test (p=0.0574). (F) Frequency of total IgM and IgG producing cells before and 2 months after transplantation for a typical recipient. Recipients had 1417 IgM and 411.7 IgG secreting B cells per 5x104 B cells pre-transplantation, and 1003 IgM and 265.7 IgG secreting B cells per 5x104 B cells post-transplantation, respectively. The number of IgM or IgG secreting B cells before and after transplantation did significantly differ by T test analysis (for IgM p=0.0256, for IgG p=0.0337).
The number and specificity of donor-specific antibody-secreting cells was determined using a novel ELISPOT in which cultured fibroblasts were used as targets and following incubation with 5 x 104 stimulated B cells in unfractionated PBMC for 24 hours. Antibody bound to the fibroblasts was detected by ELISPOT using murine monoclonal anti-human-IgM or –IgG. (A) Typical cellular ELISPOT wells. Cellular ELISPOT wells revealing anti-donor or anti-recipient (autologous) IgM or IgG antibodies. Numbers indicate the ELISPOT readings. (B) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgM increased significantly after renal transplantation (p=0.0001, paired T test). (C) Frequency of B cells producing donor-specific IgM detected by cellular ELISPOT before and 2 months after renal transplantation in individual recipients. Averages of ASC before and after transplantation were compared by paired T test (p=0.0001). (D) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation. The number of antibody-secreting cells producing donor-specific IgG increased (indicated in red) in four and decreased in two (indicated in green) of the nine subjects after renal transplantation. Pre- and post transplant values did not significantly differ by paired T test analysis (p=0.0574). (E) Frequency of B cells producing donor-specific IgG detected by cellular ELISPOT before and 2 months after renal transplantation in three typical patients. Individual experimental values and standard errors are shown. Averages of ASC before and after transplantation were compared by paired T test (p=0.0574). (F) Frequency of total IgM and IgG producing cells before and 2 months after transplantation for a typical recipient. Recipients had 1417 IgM and 411.7 IgG secreting B cells per 5x104 B cells pre-transplantation, and 1003 IgM and 265.7 IgG secreting B cells per 5x104 B cells post-transplantation, respectively. The number of IgM or IgG secreting B cells before and after transplantation did significantly differ by T test analysis (for IgM p=0.0256, for IgG p=0.0337).
Shown are results from cellular ELISPOTS of B cells obtained from two recipients with donor or third party fibroblasts. The number of antibody-secreting cells producing donor-specific IgM increased significantly (pair A, p=0.0015; pair B, p=0.0035, unpaired, T test) after renal transplantation while the number the number of antibody-secreting cells producing third party-specific IgM did not (pair A–B, p=0.6; pair B–A p=0.3). (C) and (D). Specificity of donor-specific B cells for HLA class I. To determine the extent to which donor-specific B cells recognize HLA class I versus other potential targets on fibroblast cell surfaces, cultured donor fibroblasts were blocked with murine anti-HLA class I antibodies and then the ELISPOT was performed. Figures 5C and 5D depict the number of anti-donor-specific IgM (C) or IgG (D)-secreting cells before and 2 months after transplantation with (+) or without (−) MHC class I blockade and shows that donor-specific antibody-secreting cells predominantly recognize HLA class I. HLA blockade was specific since incubating with an isotype control did not decrease anti-donor reactivity. Averages and standard errors of samples obtained from 4 distinct recipients were assayed in triplicate. Comparison of the means was by paired T test (IgM pre-transplantation p<0.0001, IgM post-transplantation, p=0.001, IgG pre-transplantation, p=0.0482; IgG post-transplantation, p=0.0468). * (P<0.05),** (P<0.01), *** (p<0.001).
Shown are results from cellular ELISPOTS of B cells obtained from two recipients with donor or third party fibroblasts. The number of antibody-secreting cells producing donor-specific IgM increased significantly (pair A, p=0.0015; pair B, p=0.0035, unpaired, T test) after renal transplantation while the number the number of antibody-secreting cells producing third party-specific IgM did not (pair A–B, p=0.6; pair B–A p=0.3). (C) and (D). Specificity of donor-specific B cells for HLA class I. To determine the extent to which donor-specific B cells recognize HLA class I versus other potential targets on fibroblast cell surfaces, cultured donor fibroblasts were blocked with murine anti-HLA class I antibodies and then the ELISPOT was performed. Figures 5C and 5D depict the number of anti-donor-specific IgM (C) or IgG (D)-secreting cells before and 2 months after transplantation with (+) or without (−) MHC class I blockade and shows that donor-specific antibody-secreting cells predominantly recognize HLA class I. HLA blockade was specific since incubating with an isotype control did not decrease anti-donor reactivity. Averages and standard errors of samples obtained from 4 distinct recipients were assayed in triplicate. Comparison of the means was by paired T test (IgM pre-transplantation p<0.0001, IgM post-transplantation, p=0.001, IgG pre-transplantation, p=0.0482; IgG post-transplantation, p=0.0468). * (P<0.05),** (P<0.01), *** (p<0.001).
Shown are results from cellular ELISPOTS of B cells obtained from two recipients with donor or third party fibroblasts. The number of antibody-secreting cells producing donor-specific IgM increased significantly (pair A, p=0.0015; pair B, p=0.0035, unpaired, T test) after renal transplantation while the number the number of antibody-secreting cells producing third party-specific IgM did not (pair A–B, p=0.6; pair B–A p=0.3). (C) and (D). Specificity of donor-specific B cells for HLA class I. To determine the extent to which donor-specific B cells recognize HLA class I versus other potential targets on fibroblast cell surfaces, cultured donor fibroblasts were blocked with murine anti-HLA class I antibodies and then the ELISPOT was performed. Figures 5C and 5D depict the number of anti-donor-specific IgM (C) or IgG (D)-secreting cells before and 2 months after transplantation with (+) or without (−) MHC class I blockade and shows that donor-specific antibody-secreting cells predominantly recognize HLA class I. HLA blockade was specific since incubating with an isotype control did not decrease anti-donor reactivity. Averages and standard errors of samples obtained from 4 distinct recipients were assayed in triplicate. Comparison of the means was by paired T test (IgM pre-transplantation p<0.0001, IgM post-transplantation, p=0.001, IgG pre-transplantation, p=0.0482; IgG post-transplantation, p=0.0468). * (P<0.05),** (P<0.01), *** (p<0.001).
Shown are results from cellular ELISPOTS of B cells obtained from two recipients with donor or third party fibroblasts. The number of antibody-secreting cells producing donor-specific IgM increased significantly (pair A, p=0.0015; pair B, p=0.0035, unpaired, T test) after renal transplantation while the number the number of antibody-secreting cells producing third party-specific IgM did not (pair A–B, p=0.6; pair B–A p=0.3). (C) and (D). Specificity of donor-specific B cells for HLA class I. To determine the extent to which donor-specific B cells recognize HLA class I versus other potential targets on fibroblast cell surfaces, cultured donor fibroblasts were blocked with murine anti-HLA class I antibodies and then the ELISPOT was performed. Figures 5C and 5D depict the number of anti-donor-specific IgM (C) or IgG (D)-secreting cells before and 2 months after transplantation with (+) or without (−) MHC class I blockade and shows that donor-specific antibody-secreting cells predominantly recognize HLA class I. HLA blockade was specific since incubating with an isotype control did not decrease anti-donor reactivity. Averages and standard errors of samples obtained from 4 distinct recipients were assayed in triplicate. Comparison of the means was by paired T test (IgM pre-transplantation p<0.0001, IgM post-transplantation, p=0.001, IgG pre-transplantation, p=0.0482; IgG post-transplantation, p=0.0468). * (P<0.05),** (P<0.01), *** (p<0.001).
Source: PubMed