Chantal Gautreau

B Cell Survival in Intragraft Tertiary Lymphoid Organs After Rituximab Therapy

Background. Rituximab is emerging as a potent therapeutic option in chronic inflammatory diseases associated with a prominent humoral component. Recent studies have demonstrated that chronic inflammatory infiltrate organize progressively themselves into ectopic lymphoid tissues (tertiary lymphoid organs; TLOs) supporting a local humoral immune response. In the present study, we evaluated the impact of rituximab therapy on TLOs associated with chronic active antibody-mediated rejection, a prototypic humoral chronic inflammatory condition. Methods. Renal allografts removed for terminal chronic rejection were prospectively collected in four transplantation centers over 4 years. Among 38 grafts collected, two were explanted after rituximab therapy for chronic active antibody-mediated rejection. Clinical characteristics and circulating B cell count were recorded for these two patients. The composition and the microarchitecture of the inflammatory infiltrate were analyzed by flow cytometry and immunohistochemistry. Organotypic cultures were performed to evaluate the intragraft production of alloantibody. Levels of expression of BAFF (Blys, CD257) were evaluated by quantitative reverse transcriptase-polymerase chain reaction. Results. Despite the complete depletion of circulating B cells in peripheral blood, TLOs were evidenced in the interstitium of both explanted grafts. Their functionality was assessed by the demonstration of a persistent local production of alloantibody. BAFF, a potent survival factor for B cells, was found to be overexpressed (both at the gene and the protein levels) in chronically rejected grafts when compared with normal kidneys and lymph nodes. Conclusions. In certain patients, inflammatory microenvironment provides BAFF-dependent paracrine survival signal to B-cells in TLOs, allowing them to escape rituximab-induced apoptosis, thereby thwarting therapeutic efficiency.

Chronic Rejection Triggers the Development of an Aggressive Intragraft Immune Response through Recapitulation of Lymphoid Organogenesis

The unwarranted persistence of the immunoinflammatory process turns this critical component of the body’s natural defenses into a destructive mechanism, which is involved in a wide range of diseases, including chronic rejection. Performing a comprehensive analysis of human kidney grafts explanted because of terminal chronic rejection, we observed that the inflammatory infiltrate becomes organized into an ectopic lymphoid tissue, which harbors the maturation of a local humoral immune response. Interestingly, intragraft humoral immune response appeared uncoupled from the systemic response because the repertoires of locally produced and circulating alloantibodies only minimally overlapped. The organization of the immune effectors within adult human inflamed tissues recapitulates the biological program recently identified in murine embryos during the ontogeny of secondary lymphoid organs. When this recapitulation was incomplete, intragraft B cell maturation was impeded, limiting the aggressiveness of the local humoral response. Identification of the molecular checkpoints critical for completion of the lymphoid neogenesis program should help develop innovative therapeutic strategies to fight chronic inflammation.

Hyperacute xenograft rejection in the swine-to-human donor-recipient combination : in vitro analysis of complement activation

Complement activation is central to the rejection of discordant xenografts. In order to assess the respective roles of direct and alternative pathways, an in vitro model of hyperacute rejection in the swine-to-human donor-recipient combination was designed, using a complement-dependent cytotoxicity test with swine endothelial cells in culture as targets, and fresh human serum as the source of xenogeneic antibodies and complement. The cytotoxic activity of the sera was evaluated by a colorimetric assay using (3-[4,5-dimethyldiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). Pure human serum lysed 58 +/- 5% of swine endothelial cells. Selective inhibition of the direct pathway by adding EGTA to the serum reduced cytolysis to 51 +/- 2% (P < 0.01 versus normal serum). Similarly, when using C1q-deficient human sera, only 37 +/- 7% of swine endothelial cells were killed (P < 0.001 versus normal serum). When the alternative pathway was selectively inhibited by heating for 20 min at 50 degrees C, the lytic activity of human serum dropped to 42 +/- 5% (P < 0.001 versus normal serum). Factor B-deficient human serum could only lyse 42 +/- 10% of porcine endothelial cells (P < 0.001 versus normal serum). Syngeneic normal swine serum and heat-inactivated serum were not cytotoxic. Mixing serum with deficient direct pathway and serum with deficient alternative pathway restored the cytotoxicity to normal levels. Similarly, the cytotoxic activity of deficient serum supplemented with purified C1q or factor B at physiological concentrations reached that of normal human serum. In this model of in vitro hyperacute rejection, both pathways of complement activation are involved, suggesting that regimens designed to inhibit hyperacute rejection of swine xenografts into humans should take into account the dual activation of complement in this donor-recipient combination.

Lung transplantation in patients with pretransplantation donor-specific antibodies detected by Luminex assay.

Background New methods of solid-phase assays, such as Luminex assay, with high sensitivity in detecting anti–human leukocyte antigen (HLA) antibodies (Abs), have increased the proportion of sensitized candidates waiting for lung transplantation (LTx). However, how to apply these results clinically during graft allocation is debated: strict exclusion of candidates with Luminex-positive results can lead to lost opportunities for Tx. We retrospectively analyzed the clinical impact of pre-LTx Luminex-detected Abs on post-LTx outcomes for patients who underwent LTx before the availability of Luminex assay. Methods We analyzed data for 56 successive patients who underwent LTx before 2008 and were considered to not have anti-HLA Abs by then-available methods of detection at the date of their LTx. Pre-LTx sera from these patients were retested by Luminex assay. Using log-rank test, freedom from bronchiolitis obliterans syndrome (BOS) and graft survival were compared between patients with and without pre-LTx Luminex-detected anti-HLA Abs classes I and II and donor-specific Abs (DSA) classes I and II. Results Freedom from bronchiolitis obliterans syndrome was lower, and mortality was higher for patients with than those without pre-LTx Luminex-detected DSA class II (P=0.004 and P=0.007, respectively) but did not differ for patients with and without DSA class I or anti-HLA Abs class I or II. Conclusions It suggests to avoid attributing graft with forbidden antigens to sensitized candidates with Luminex-detected DSA class II and to evaluate the role of specific posttransplantation protocols for LTx candidates who require emergency LTx.

Use of intravenous immunoglobulin to delay xenogeneic hyperacute rejection. An in vivo and in vitro evaluation.

The delaying action of intravenous immunoglobulin (WIG) from human origin on hyperacute xenogeneic rejection was assessed in the guinea pig-to-rat combination. IVIG (1500 mg/kg) injected i.v. into Lewis rats 1 hr before grafting significantly prolonged the mean guinea pig heart survival time (167 min, P<0.005) compared with control injections using NaCl (12 min) or the IVIG glycine vehicle (11 min). The effect of IVIG was also assessed in vitro in the pig-to-human combination. A dose-dependent inhibition of the complement-mediated direct cytotoxicity of human serum on pig RBC was shown using IVIG. The weak direct cytotoxicity of IVIG to pig RBC, which was abolished after preincubating IVIG with pig RBC, was attributed to the anti-pig xenoreactive natural antibodies (XNA) contained in the IVIG preparation. In vitro, XNA-de-pleted IVIG exerted a significantly stronger inhibitory effect than non-XNA-depleted IVIG, suggesting the use XNA-depleted IVIG in the pig-to-human combination. Although the mechanism of the inhibitory effect of IVIG remains to be clarified, IVIG may represent a new and simple therapeutic modality against xenogeneic hyperacute rejection.

Predominant role of the Fab fragment in delaying hyperacute rejection in guinea pig-to-rat xenotransplantation

BACKGROUND Human intravenous immunoglobulin G delayed xenogeneic hyperacute rejection (HAR) in the guinea pig-to-rat combination. We investigated the respective roles of the Fc and Fab fragments of the IgG molecule in this inhibitory effect. METHODS By using a guinea pig-to-rat heart transplantation model, the efficiency of IgG, Fab, and Fc in prolonging the grafted hearts survival time (ST) was compared. RESULTS A dose-dependent increase in the ST was observed with Fab (r=0.74, P < 0.0001), IgG (r=0.57, P < 0.001), and Fc (r=0.51, P < 0.01). The linear regression slopes with Fab and with IgG were, respectively, sevenfold and fourfold steeper than with Fc. The ST was significantly longer than controls (23+/-7 min) after infusion of 2 g/kg IgG (147+/-42 min) or 1 g/kg Fab (176+/-38 min), whereas the highest dose of Fc (1.5 g/kg) did not induce significant prolongation of ST. In terms of equivalent functional doses, 1 g/kg Fab was significantly more potent in prolonging the ST than 1.5 g/kg IgG (87+/-25 min) or 0.5 g/kg Fc (33+/-14 min). Analysis of the rejected hearts evidenced edema, necrosis, and rat C3 deposits characteristic of HAR. CONCLUSION These results indicated that the delaying action of intravenous immunoglobulin G on HAR in the guinea pig-to-rat combination is mostly mediated through the Fab fragment.

Characteristics of Donor-Specific Antibodies Associated With Antibody-Mediated Rejection in Lung Transplantation

Although donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) are frequently found in recipients after lung transplantation (LT), the characteristics of DSA which influence antibody-mediated rejection (AMR) in LT are not fully defined. We retrospectively analyzed 206 consecutive LT patients of our center (2010–2013). DSAs were detected by using luminex single antigen beads assay and mean fluorescence intensity was assessed. Within the study population, 105 patients had positive DSA. Patients with and without AMR (AMRPos, n = 22, and AMRNeg, n = 83, respectively) were compared. AMRPos patients had significantly greater frequencies of anti-HLA DQ DSA (DQ DSA) than AMRNeg patients (95 vs 58%, respectively, p < 0.0001). Compared to AMRNeg patients, AMRPos patients had higher DQ DSA sum MFI [7,332 (2,067–10,213) vs 681 (0–1,887), p < 0.0001]. DQ DSA when associated with AMR, had more frequent graft loss and chronic lung allograft dysfunction (CLAD). These data suggest (i) that DSA characteristics clearly differ between AMRPos and AMRNeg patients and (ii) the deleterious impact of DQ DSA on clinical outcome.

Role of C1q-binding anti-HLA antibodies as a predictor of lung allograft outcome

Presence of anti-human leukocyte antigen donor-specific antibodies (DSAs) is associated with poor outcome after lung transplantation. Currently, DSAs are detected using the Luminex technique, which may be overly sensitive. The new C1q assay allows for the exclusive detection of complement (C1q)-binding antibodies, involved in antibody-mediated rejection. We investigated whether early detection of complement-binding DSAs is associated with chronic lung allograft dysfunction (CLAD) and survival. From 2009 to 2012, lung transplant recipients from three transplantation centres were screened for the presence of DSA and their complement-binding capacity during the 6–12 months post-transplantation in a stable condition. The analysis included 168 patients. The 3-year rates of freedom from CLAD and graft survival were lower for patients with complement-binding DSAs (33.6% and 53.7%, respectively), as compared with patients with non-complement-binding DSAs (61.9% and 77.4%, respectively) and patients without DSA (70% and 84.9%, respectively) (p<0.001 and p=0.001, respectively). Detection of complement-binding DSA was associated with a risk of graft loss that was nearly tripled after adjustment for clinical, functional, histological and immunological factors (hazard ratio 2.98, 95% CI 1.33–6.66; p=0.008). Assessment of the C1q-binding capacity of DSA appears to be useful in identifying stable lung transplant recipients at high risk of lung allograft loss. The C1q assay allows for a better identification of lung-transplant recipients with the highest risk of graft failure http://ow.ly/CkmY30kz7P8