The Halifax flow Crossmatch Protocol Results According to the Class and MFI of the DSA

Introduction: The main strategy to reduce the immunogenicity of transplanted grafts is to seek maximum compatibility betweenalloantigens in the donor-recipient pair. It is understood that the virtual crossmatch (VXM) can be a good tool in the evaluation of the donor / recipient pair and that a broader application of these protocols will improve the pre-transplant immunological risk assessment. The aim of this study was to correlate the physical flow crossmatches results, looking at different mean fluorescence intensity (MFI) values of the donor specific antibodies (DSA), and the probability of a positive crossmatch. We also aimed to validate this tool using a well standardized flow crossmatch protocol. Methods: We performed a total of 15,217 FCXM between 2015 and 2019. All were tested by the Halifax Flow Cytometer Crossmatch (FCXM) protocol, with cells from deceased donors and serum from renal recipients. For this analysis we selected only samples that had one or two DSA per locus (N = 1,081), when the MFI was above 1,000, and they were divided according to the allelic group recognized by the antibody (anti-HLA-A, B, C, DR or DQ) looking at the probability of a positive crossmatch with different MFI values of the DSAs. Combinations among them were alsoanalyzed in the same way (N=175). Results: In the presence of an exclusive DSA against the allelic groups A, B and DR, with an index MFI above 5,000, all the FCXM were positives. With exclusively antibodies against groups C or DQ, all cases with DSAsabove 15,000 MFIs were positives. With two or more DSAs anti A and/or B, and/or DR, when their MFIs sums exceeded 5,000,all FCXM results against B cells were positive. The presence of anti-Class I DSAs (A, B and A+B), regardless of the MFI value, was responsible for 71% (N=424 of 601) of the T cell positivity and 77% (N=460 of 661) in the B cell crossmatches. The presence of only anti-Class II DSAs (DR and DQ) accounted for 55% (N=168 of 303) of FCXM positivity in B cells. The overall mean of the MFI of the DSAs was higher in the FCXM positive group when compared to negative crossmatch. The group with sum of DSAs A (N=217) showed that when the sum was 4,000 MFI or higher, there was a 24 times higher probability for a positive B cell crossmatch when compared with lowers MFIs. Conclusions: Our results show a strong association between the DSA MFI and the FCXM result. The data here presented confirm the results of our previous studies, justifying the VXMstandard used by our center, proving to be a good tool to streamline the selection of transplant recipients and facilitate the sharing of organs from national donors

Low Hydrophobic Mismatch Scores Calculated for HLA-A/B/DR/DQ Loci Improve Kidney Allograft Survival

We evaluated the impact of human leukocyte antigen (HLA) disparity (immunogenicity; IM) on long-term kidney allograft survival. The IM was quantified based on physicochemical properties of the polymorphic linear donor/recipient HLA amino acids (the Cambridge algorithm) as a hydrophobic, electrostatic, amino acid mismatch scores (HMS\AMS\EMS) or eplet mismatch (EpMM) load. High-resolution HLA-A/B/DRB1/DQB1 types were imputed to calculate HMS for primary/re-transplant recipients of deceased donor transplants. The multiple Cox regression showed the association of HMS with graft survival and other confounders. The HMS integer 0-10 scale showed the most survival benefit between HMS 0 and 3. The Kaplan-Meier analysis showed that: the HMS=0 group had 18.1-year median graft survival, a 5-year benefit over HMS\u3e0 group; HMS ≤ 3.0 had 16.7-year graft survival, a 3.8-year better than HMS\u3e3.0 group; and, HMS ≤ 7.8 had 14.3-year grafts survival, a 1.8-year improvement over HMS\u3e7.8 group. Stratification based on EMS, AMS or EpMM produced similar results. Additionally, the importance of HLA-DR with/without -DQ IM for graft survival was shown. In our simulation of 1,000 random donor/recipient pairs, 75% with HMS\u3e3.0 were re-matched into HMS ≤ 3.0 and the remaining 25% into HMS≥7.8: after re-matching, the 13.5 years graft survival would increase to 16.3 years. This approach matches donors to recipients with low/medium IM donors thus preventing transplants with high IM donors

Treatment with activated protein C (aPC) is protective during the development of myocardial fibrosis: an angiotensin II infusion model in mice.

Myocardial fibrosis contributes to the development of heart failure. Activated Protein C (aPC) is a circulating anticoagulant with anti-inflammatory and cytoprotective properties. Using a model of myocardial fibrosis second to Angiotensin II (AngII) infusion, we investigated the novel therapeutic function aPC in the development of fibrosis.C57Bl/6 and Tie2-EPCR mice were infused with AngII (2.0 µg/kg/min), AngII and aPC (0.4 µg/kg/min) or saline for 3d. Hearts were harvested and processed for analysis or used for cellular isolation. Basic histology and collagen deposition were assessed using histologic stains. Transcript levels of molecular mediators were analyzed by quantitative RT-PCR. Mice infused with AngII exhibited multifocal areas of myocardial cellular infiltration associated with significant collagen deposition compared to saline control animals (p<0.01). AngII-aPC infusion inhibited this cellular infiltration and the corresponding collagen deposition. AngII-aPC infusion also inhibited significant expression of the pro-fibrotic cytokines TGF-β1, CTGF and PDGF found in AngII only infused animals (p<0.05). aPC signals through its receptor, EPCR. Using Tie2-EPCR animals, where endothelial cells over-express EPCR and exhibit enhanced aPC-EPCR signaling, no significant reduction in cellular infiltration or fibrosis was evident with AngII infusion suggesting aPC-mediate protection is endothelial cell independent. Isolated infiltrating cells expressed significant EPCR transcripts suggesting a direct effect on infiltrating cells.This data indicates that aPC treatment abrogates the fibrogenic response to AngII. aPC does not appear to confer protection by stimulating the endothelium but by acting directly on the infiltrating cells, potentially inhibiting migration or activation

Poly I:C and Pam3CSK4 enhance T-cell-dependent B cell activation.

<p>Purified B cells from C57BL/6 mice were stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL) or a combination of both, with T-cell-dependent co-stimulation provided by anti-CD40 and anti-Ig. Expression of surface receptor was determined after 24 hours by flow cytometry. (A) CD80 (n = 8), (B) CD86 (n = 5), (C) CD25 (n = 6), (D) MHC class II (n = 7) and (E) CD69 (n = 3). Data are shown as average ± SEM of individual B cell preparations as indicated, data is pooled from two to four independent experiments. Statistics performed by ANOVA with Tukey post-test: “#” indicates significance relative to untreated, “+” indicates significance relative to poly I:C and “*” indicates significance relative to Pam3CSK4.</p

Allogeneic T cell response is increased in B cells stimulated with poly I:C and Pam3CSK4.

<p>Purified B cells from C57BL6 mice and stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL) or a combination of both with T-cell-dependent co-stimulation provided by anti-CD40 and anti-Ig. After 24 hours, B cells were inactivated by mitomycin C, and resuspended at various concentrations. B cells were co-cultured with 100,000 CD4⁺ T cells isolated from BALB/c mice at ratios 1:10 (10,000 B cells), 1:25 (4,000 B cells) or 1:50 (2,000 B cells). (A) Proliferation was measured after 3 days by [³H]-TdR incorporation. Separate co-cultures were setup in parallel to detect IL-2 production (B) in supernatant as well as expression of CD25 (C) on CD4⁺ T cells after 3 days. Results are shown as the average ± SEM of 5 individual B cell preparations from 5 independent experiments. Statistics performed by ANOVA with Tukey post-test: “#” indicates significance relative to untreated.</p

Proliferative response of B cells stimulated with poly I:C and Pam3CSK.

<p>Purified B cells from C57BL/6 mice were stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL) or a combination of both, with T-cell-dependent co-stimulation provided by anti-CD40 and anti-Ig. Proliferation was measured after 3 days by [³H]-TdR uptake. Data are shown as average ± SEM of 5 individual B cell preparations, data is pooled from at two independent experiments. Statistics performed by ANOVA with Tukey post-test: “#” indicates significance relative to untreated, “+” indicates significance relative to poly I:C and “*” indicates significance relative to Pam3CSK4.</p

Poly I:C and Pam3CSK4 stimulate expression of surface receptors in B cells independent of T cell help.

<p>CD19⁺ B cells were purified from spleens of C57BL6 mice and stimulated with poly I:C (25 μg/mL), Pam3CSK4 (1 μg/mL), or the combination of both. Expression of surface receptor was determined after 24 hours by flow cytometry. (A) CD80 (n = 11), (B) CD86 (n = 7), (C) CD25 (n = 6), (D) MHC class II (n = 5), (E) CD69 (n = 4), (F) CD40 (n = 6). Data are shown as average ± SEM of individual B cell preparations as indicated, data is pooled from two to four independent experiments. Statistics performed by ANOVA with Tukey post-test: “#” indicates significance relative to untreated, “+” indicates significance relative to poly I:C and “*” indicates significance relative to Pam3CSK4.</p