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P006 Assessing the functional significance of HLA antibodies in renal allograft
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P005
Abstracts / Human Immunology 77 (2016) 40–156
HIGH TITER OF ANGIOTENSIN II RECEPTOR I ANTIBODIES MAY CAUSE PERSISTENT WEAK POSITIVE B CELL REACTIVITY IN FLOW CYTOMETRY CROSSMATCH IN THE ABSENCE OF HLA ANTIBODIES Valia Bravo-Egana, Dimitri Monos. Children’s Hospital of Philadelphia, Philadelphia, PA, USA. Aim: Patients exhibiting reactivity in flow cytometry crossmatch (FC XM) in the absence of DSA pose a dilemma to the laboratories when interpreting/reporting results. We report two cases where the patients consistently exhibit weak B cell positive reactivity in FC XM with different donor cells in the absence of DSA, other HLA antibodies (Ab) and MICA Ab. Interestingly, both patients have high titers of anti-angiotensin II receptor 1 (AT1R) Ab in their sera. Methods: Patients and donors typing was performed by NGS for A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1 loci. DRB345 were typed using SSO. The presence of anti-HLA and anti-MICA Ab in the patient’s sera was assessed using Luminex Single Antigen Bead technology. The MFI cut off used was 1500. FC XM were performed using IgG antibodies against T and B-cells. The MESF cut off for positivity was 2000 MESF shift. AT1R Ab were detected using ELISA immunoassay according to OneLambda protocol. Results: In both cases no anti-HLA or anti-MICA Ab were detected on the sera. However, they consistently showed weak B cell reactivity when FC XM were performed using different donor cells. High titers of antiAT1R Ab were detected in the sera of both patients. Conclusions: The initial contact point between a recipient’s immune system and a transplanted graft is the vascular endothelium. Numerous studies have found association between the presence of Ab to AT1R and Antibody-mediated rejection (AMR) in transplant recipients whose sera contained no Ab to donor HLA or MICA. Other studies found the presence of anti-AT1R Ab in the sera of pre-transplant patients as an independent risk factor for long-term graft loss in association with a higher risk of early AMR episodes. Here we present two patients waiting for transplants with no anti-HLA or anti-MICA Ab in their sera. High titers of antiAT1R Ab were detected on both sera. These Ab were enough to cause reactivity in B cell FC XM. Interestingly, recent studies demonstrated that AT1R is expressed in immune cells, including B cells, although its expression is lower in B cells than in other immune cell types. Assessing the AT1R Ab status provides additional information that may be important determining the immunologic risk for recipients.
P006
ASSESSING THE FUNCTIONAL SIGNIFICANCE OF HLA ANTIBODIES IN RENAL ALLOGRAFT Michael Conciatori 1, Michael Prod 1, Maria Oppermann 1, Maribeth Flaws 1, Demetra C. Castillo 2, Siva Kanangat 1. 1Rush University Medical Center, Pathology, Chicago, IL, United States; 2Rush University Medical Center, Medical Lab Sciences, Chicago, IL, United States. Objective: To assess potential function of the anti-HLA antibodies in terms of complement binding and non-complement binding in highly sensitized patients and to correlate the functional properties with invitro cytotoxicity assays and biopsy. Methods: The Class I and II HLA antibody profiles of 78 patients were analyzed to determine antibody strength and specificity and to determine if any of the identified antibodies are complement binding. In vivo correlation was done in 37 patients who had a biopsy and donor-specific antibody (DSA) assessment. The HLA class I and II antibody specificities were determined by single antigen bead assay (SAB) and the C1q binding assay (C1q-B) using Luminex platform (One Lambda, Canoga Park CA). In vitro cytotoxicity assays were done by T-AHG and Amos CDC. Results: Of the 2941 antibodies identified by SAB in 78 patients, 1940 (66%) were Class I and 1001 (34%) were Class II specific. In all, 758 (39%) of the Class I antibodies with P5000 MFI and 484 (48%) of the Class II antibodies were with an MFI of P5000. Of the total 2941 HLA antibodies, 238 (8%) were C1q-B. Of the C1q-B, 77 (32%) were directed against Class I HLA and 161 (68%) were directed against Class II. Importantly, 92% of all C1q Class I antibodies identified were defined as strong among the total 2941 HLA specific antibodies. Biopsy results were available from 37 patients and 15 of them had no C1q Class I or Class II DSAs and no AMR while 6 (16%) patients were C1q Class I and Class II positive DSA and AMR. Fourteen percent (n = 5) of the total patients were C1q Class I negative and C1q Class II positive and per biopsy were classified as C4d negative AMR. Of the 49 patients with CDC and C1q data available 19 (39%) nine were C1q Class I and Class II DSA negative and CDC B-cell and T-cell negative while 5 (10%) were C1q Class I and Class II DSA positive and CDC Bcell positive T-cell negative. In all, 4 were C1q Class I negative and Class II positive with CDC T-cell negative and CDC B-cell positive. Conclusions: This limited retrospective analysis confers that assaying for C1q binding antibodies is very likely to have clinical relevance in terms of the functional effect of the multiple HLA antibodies on the allograft. The class II antibodies and antibodies with higher MFIs are are more likely to be C1q-B.
P005
Abstracts / Human Immunology 77 (2016) 40–156
HIGH TITER OF ANGIOTENSIN II RECEPTOR I ANTIBODIES MAY CAUSE PERSISTENT WEAK POSITIVE B CELL REACTIVITY IN FLOW CYTOMETRY CROSSMATCH IN THE ABSENCE OF HLA ANTIBODIES Valia Bravo-Egana, Dimitri Monos. Children’s Hospital of Philadelphia, Philadelphia, PA, USA. Aim: Patients exhibiting reactivity in flow cytometry crossmatch (FC XM) in the absence of DSA pose a dilemma to the laboratories when interpreting/reporting results. We report two cases where the patients consistently exhibit weak B cell positive reactivity in FC XM with different donor cells in the absence of DSA, other HLA antibodies (Ab) and MICA Ab. Interestingly, both patients have high titers of anti-angiotensin II receptor 1 (AT1R) Ab in their sera. Methods: Patients and donors typing was performed by NGS for A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1 loci. DRB345 were typed using SSO. The presence of anti-HLA and anti-MICA Ab in the patient’s sera was assessed using Luminex Single Antigen Bead technology. The MFI cut off used was 1500. FC XM were performed using IgG antibodies against T and B-cells. The MESF cut off for positivity was 2000 MESF shift. AT1R Ab were detected using ELISA immunoassay according to OneLambda protocol. Results: In both cases no anti-HLA or anti-MICA Ab were detected on the sera. However, they consistently showed weak B cell reactivity when FC XM were performed using different donor cells. High titers of antiAT1R Ab were detected in the sera of both patients. Conclusions: The initial contact point between a recipient’s immune system and a transplanted graft is the vascular endothelium. Numerous studies have found association between the presence of Ab to AT1R and Antibody-mediated rejection (AMR) in transplant recipients whose sera contained no Ab to donor HLA or MICA. Other studies found the presence of anti-AT1R Ab in the sera of pre-transplant patients as an independent risk factor for long-term graft loss in association with a higher risk of early AMR episodes. Here we present two patients waiting for transplants with no anti-HLA or anti-MICA Ab in their sera. High titers of antiAT1R Ab were detected on both sera. These Ab were enough to cause reactivity in B cell FC XM. Interestingly, recent studies demonstrated that AT1R is expressed in immune cells, including B cells, although its expression is lower in B cells than in other immune cell types. Assessing the AT1R Ab status provides additional information that may be important determining the immunologic risk for recipients.
P006
ASSESSING THE FUNCTIONAL SIGNIFICANCE OF HLA ANTIBODIES IN RENAL ALLOGRAFT Michael Conciatori 1, Michael Prod 1, Maria Oppermann 1, Maribeth Flaws 1, Demetra C. Castillo 2, Siva Kanangat 1. 1Rush University Medical Center, Pathology, Chicago, IL, United States; 2Rush University Medical Center, Medical Lab Sciences, Chicago, IL, United States. Objective: To assess potential function of the anti-HLA antibodies in terms of complement binding and non-complement binding in highly sensitized patients and to correlate the functional properties with invitro cytotoxicity assays and biopsy. Methods: The Class I and II HLA antibody profiles of 78 patients were analyzed to determine antibody strength and specificity and to determine if any of the identified antibodies are complement binding. In vivo correlation was done in 37 patients who had a biopsy and donor-specific antibody (DSA) assessment. The HLA class I and II antibody specificities were determined by single antigen bead assay (SAB) and the C1q binding assay (C1q-B) using Luminex platform (One Lambda, Canoga Park CA). In vitro cytotoxicity assays were done by T-AHG and Amos CDC. Results: Of the 2941 antibodies identified by SAB in 78 patients, 1940 (66%) were Class I and 1001 (34%) were Class II specific. In all, 758 (39%) of the Class I antibodies with P5000 MFI and 484 (48%) of the Class II antibodies were with an MFI of P5000. Of the total 2941 HLA antibodies, 238 (8%) were C1q-B. Of the C1q-B, 77 (32%) were directed against Class I HLA and 161 (68%) were directed against Class II. Importantly, 92% of all C1q Class I antibodies identified were defined as strong among the total 2941 HLA specific antibodies. Biopsy results were available from 37 patients and 15 of them had no C1q Class I or Class II DSAs and no AMR while 6 (16%) patients were C1q Class I and Class II positive DSA and AMR. Fourteen percent (n = 5) of the total patients were C1q Class I negative and C1q Class II positive and per biopsy were classified as C4d negative AMR. Of the 49 patients with CDC and C1q data available 19 (39%) nine were C1q Class I and Class II DSA negative and CDC B-cell and T-cell negative while 5 (10%) were C1q Class I and Class II DSA positive and CDC Bcell positive T-cell negative. In all, 4 were C1q Class I negative and Class II positive with CDC T-cell negative and CDC B-cell positive. Conclusions: This limited retrospective analysis confers that assaying for C1q binding antibodies is very likely to have clinical relevance in terms of the functional effect of the multiple HLA antibodies on the allograft. The class II antibodies and antibodies with higher MFIs are are more likely to be C1q-B.