image_2_SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production.tiff

<p>B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt’s lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK–Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.</p

image_3_SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production.tiff

<p>B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt’s lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK–Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.</p

image_1_SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production.tiff

<p>B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt’s lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK–Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.</p

image_1.tif

<p>Antibody-mediated rejection is currently the leading cause of transplant failure. Prevailing dogma predicts that B cells differentiate into anti-donor-specific antibody (DSA)-producing plasma cells only with the help of CD4+ T cells. Yet, previous studies have shown that dependence on helper T cells decreases when high amounts of protein antigen are recruited to the spleen, two conditions potentially met by organ transplantation. This could explain why a significant proportion of transplant recipients develop DSA despite therapeutic immunosuppression. Using murine models, we confirmed that heart transplantation, but not skin grafting, is associated with accumulation of a high quantity of alloantigens in recipients’ spleen. Nevertheless, neither naive nor memory DSA responses could be observed after transplantation of an allogeneic heart into recipients genetically deficient for CD4+ T cells. These findings suggest that DSA generation rather result from insufficient blockade of the helper function of CD4+ T cells by therapeutic immunosuppression. To test this second theory, different subsets of circulating T cells: CD8+, CD4+, and T follicular helper [CD4+CXCDR5+, T follicular helper cells (Tfh)], were analyzed in 9 healthy controls and 22 renal recipients. In line with our hypothesis, we observed that triple maintenance immunosuppression (CNI + MMF + steroids) efficiently blocked activation-induced upregulation of CD25 on CD8+, but not on CD4+ T cells. Although the level of expression of CD40L and ICOS was lower on activated Tfh of immunosuppressed patients, the percentage of CD40L-expressing Tfh was the same than control patients, as was Tfh production of IL21. Induction therapy with antithymocyte globulin (ATG) resulted in prolonged depletion of Tfh and reduction of CD4+ T cells number with depleting monoclonal antibody in murine model resulted in exponential decrease in DSA titers. Furthermore, induction with ATG also had long-term beneficial influence on Tfh function after immune reconstitution. We conclude that CD4+ T cell help is mandatory for naive and memory DSA responses, making Tfh cells attractive targets for improving the prevention of DSA generation and to prolong allograft survival. Waiting for innovative treatments to be translated into the clinical field ATG induction seems to currently offer the best clinical prospect to achieve this goal.</p

Treatment with IVIg is associated with a transient decrease in levels of PFR-MCA hydrolyzing IgG.

<p>IgG was purified from the plasma of patients who received IVIg therapy prior to transplantation (full circles) and from patients who did not received IVIg (empty circles). Plasma had been collected prior to renal transplant (D0) and 3 (M3), 12 (M12) and 24 (M24) months after renal transplant. IgG (66.67 nM) was incubated with PFR-MCA (100 µM), a peptide chromogenic substrate, for 24 hr at 37°C. The amount of hydrolysis was quantified by measuring the fluorescence of the leaving MCA moiety, and is expressed in femtomoles of substrate hydrolyzed per minute per picomoles of IgG. Pooled normal human IgG was used as a control source of IgG. Panel A depicts the raw results as scatter dot plots. Panel B depicts the evolution of the mean ± SEM levels of PFR-MCA-hydrolyzing IgG in the two groups of patients with time (*: P = 0.004). The dotted line represents the hydrolysis of PFR-MCA by normal pooled human IgG (mean of 29 measurements; Coefficient of variation: 0.29). Panel C depicts the levels of PFR-MCA-hydrolyzing IgG in patients treated with anti-thymocyte globulins (ATG, full squares) or not (empty squares), as measured in plasma collected 3 months post-transplantation.</p

Characteristics of the study population.

*<p>Two-tailed Mann-Whitney test; † Mean±SEM (range); ND: Not Documented; ^#Fisher's exact test.</p>‡<p>All patients received Steroids, Cyclosporin, Tacrolimus and/or Mycophenolate Mofetil; one patient received both Basiliximab and anti-thymocyte globulins.</p

Profiling Sirolimus-Induced Inflammatory Syndrome: A Prospective Tricentric Observational Study

<div><h3>Background</h3><p>The use of the immunosuppressant sirolimus in kidney transplantation has been made problematic by the frequent occurrence of various side effects, including paradoxical inflammatory manifestations, the pathophysiology of which has remained elusive.</p> <h3>Methods</h3><p>30 kidney transplant recipients that required a switch from calcineurin inhibitor to sirolimus-based immunosuppression, were prospectively followed for 3 months. Inflammatory symptoms were quantified by the patients using visual analogue scales and serum samples were collected before, 15, 30, and 90 days after the switch.</p> <h3>Results</h3><p>66% of patients reported at least 1 inflammatory symptom, cutaneo-mucosal manifestations being the most frequent. Inflammatory symptoms were characterized by their lability and stochastic nature, each patient exhibiting a unique clinical presentation. The biochemical profile was more uniform with a drop of hemoglobin and a concomitant rise of inflammatory acute phase proteins, which peaked in the serum 1 month after the switch. Analyzing the impact of sirolimus introduction on cytokine microenvironment, we observed an increase of IL6 and TNFα without compensation of the negative feedback loops dependent on IL10 and soluble TNF receptors. IL6 and TNFα changes correlated with the intensity of biochemical and clinical inflammatory manifestations in a linear regression model.</p> <h3>Conclusions</h3><p>Sirolimus triggers a destabilization of the inflammatory cytokine balance in transplanted patients that promotes a paradoxical inflammatory response with mild stochastic clinical symptoms in the weeks following drug introduction. This pathophysiologic mechanism unifies the various individual inflammatory side effects recurrently reported with sirolimus suggesting that they should be considered as a single syndromic entity.</p> </div

Clues for a defective immune regulation in TLT.

<p><b>A.</b> The percentage of CD86-expressing mature dendritic cells (OX62+ MHC II+) was measured in the three lymphoid organs (left panel). The ratio: number of CD4+ T cells/number of mature dendritic cells was also calculated (right panel). Adventitia <i>vs</i> spleen: ‡ p<0.05; adventitia <i>vs</i> draining lymph node: ** p<0.01. <b>B.</b> The percentage of IL10-producing CD4+ T cells (Tr1) dropped in TLT between 10 days and 1 month post-transplantation, an evolution inverted as compared with spleen and draining lymph node (left panel). The ratio: number of activated (CD25+ Foxp3−) CD4+ T cells/number of Tr1 cells was increased in TLT at each time points (right panel). Adventitia <i>vs</i> spleen: ‡ p<0.05; adventitia <i>vs</i> draining lymph node: * p<0.05. <b>C.</b> The percentage of regulatory T cells (CD4+ CD25+ Foxp3+) tended to decrease in TLT between 10 days and 1 month post-transplantation (left panel). The ratio: number of activated (CD25+ Foxp3−) CD4+ T cells/number of T reg was increased in TLT at each time points (right panel). Adventitia <i>vs</i> spleen: ‡ p<0.05; adventitia <i>vs</i> draining lymph node: * p<0.05.</p

Characteristics of the T helper response in the various lymphoid organs participating in chronic rejection.

<p><b>A. B.</b> The percentage of activated T lymphocytes was evaluated by: <b>A.</b> the expression of the α chain of the IL2 receptor (CD25), and <b>B.</b> the costimulatory molecule OX40 (CD134). Adventitia <i>vs</i> spleen: ‡ p<0.05, ‡‡ p<0.01; adventitia <i>vs</i> draining lymph node: * p<0.05, ** p<0.01. <b>C. D.</b> The TCR repertoire perturbations induced by chronic rejection in the spleen, the draining lymph nodes and the adventitial TLT were analyzed using the immunoscope method. Immunoscope divides T cell population into 180 “groups” defined upon the rearranged variable ß (Vß) gene segment used, and the length of the TCR CDR-3. In baseline conditions, the distribution profile of CDR3 lengths for each Vß family displays a Gaussian distribution. An increase in the height of a size peak signals an oligoclonal or a monoclonal expansion of this group that can be reliably quantified by the percentage of difference between the measured value the expected normal value. <b>C.</b> The mean percentage of perturbation for the 180 groups is shown for each lymphoid organ. Adventitia <i>vs</i> spleen: ‡‡‡ p<0.001; adventitia <i>vs</i> draining lymph node: *** p<0.001. <b>D.</b> The set of data was computed to group samples according to their pattern of TCR repertoire perturbation (Ward hierarchical clustering). Individual samples are listed in raw (ADV: adventitia; LN: draining lymph node; SPL: spleen), the 180 groups constituting the T cell population are in column. Perturbations are encoded from light green to bright red. On the right of the color map, a dendrogram list each observation, and shows which cluster it is in and when it entered its cluster.</p

Changes in concomitant immunosuppressive drugs after sirolimus introduction.

<p>Results are expressed in number of patients (%).</p