Maria Hernandez Fuentes

A High Throughput Molecular Assay for Detection of Renal Graft Acute Cellular Rejection Using Peripheral Blood

1 A Phase 2 Clinical Trial of Donor Specifi c Tolerance Induction in Recipients of HLA Disparate Living Donor Kidney Allografts by Donor Stem Cell Infusion. J. Leventhal,1 M. Abecassis,1 J. Miller,1 L. Gallon,1 R. Herzig,2 D. Tollerud,2 B. King,2 S. Ildstad.2 1Transplant Center, Northwestern Univ., Chicago, IL; 2Institute for Cellular Therapeutics, U of Louisville, Louisville, KY. Background: We have demonstrated that durable chimerism without GVHD can be established with minimal toxicity in highly-mismatched kidney Tx recipients through nonmyeloablative conditioning followed by infusion of a bioengineered stem cell graft. We herein provide intermediate-term F/U on our phase 2 trial. Methods: 14 HLA mismatched living donor renal Tx recipients entered into a tolerogenic protocol involving low intensity conditioning (fl udarabine, cyclophosphamide, 200cGy TBI days -4 to -1). Pts received a kidney Tx on day 0, then infusion of cryopreserved facilitating cell (FC)-enriched donor-derived CD34+ HSCT on Day +1. Subjects were discharged by POD 2 and managed as outpts. Maintenance IS consisted of Prograf and MMF w.o steroids. Weaning of IS occurred over a one year period. Results: Pt data is summarized below. All but one pt demonstrated peripheral blood macrochimerism post-Tx. Engraftment failure occurred in a highly sensitized (PRA > 40%) completely mismatched Tx recipient (NW9). Chimerism was lost in 4 pts at 2 (NW11), 3 (NW4), 6 (NW1) and 9 (NW12) months post-Tx. All pts demonstrated donor-specifi c hyporesponsiveness in the year post-Tx and were weaned from full dose IS. Complete IS withdrawal was successful in 6 of 10 pts with durable chimerism who are ≥ one year post-Tx; pts have been off IS for 2, 4, 7, 8, 9, & 19 months. There has been no GVHD or “engraftment syndrome”. Adverse events include herpes zoster reactivation in 3 pts; no CMV infection has occurred. Tx loss occurred in one pt (NW2) who developed aplastic anemia and sepsis following an atypical viral infection; successful rescue with banked autologous HSCT and subsequent re-Tx with a living donor kidney was performed. Conclusions: Low intensity conditioning + FC enriched HSCT can safely achieve durable chimerism in mismatched kidney Tx recipients, allowing for IS withdrawal. Prior sensitization represents an obstacle to successful induction of chimerism and tolerance induction. DISCLOSURE: King, B.: Employee, regenerex llc. Ildstad, S.: Employee, regenerex llc. Abstract# 2 B Cell Derived IL-10 Is Required for Co-Stimulatory Blockade Induced Tolerance. G. Lal,1 Y. Nakayama,2 B. E. Burrell,2 A. Sethi,1 Y. Ding,2 J. S. Bromberg.2 1National Centre for Cell Science, Pune, MH, India; 2University of Maryland, Baltimore, MD. Background: The anti-infl ammatory cytokine IL-10 plays a very important role in the maintenance of tolerance. Whether specifi c subsets of B cells and B cell derived IL-10 contribute to the generation of transplantation tolerance are not known. We hypothesized that specifi c subsets of B cells produce IL-10 that is required for tolerization. Methods: C57BL/6 mice received vascularized BALB/c cardiac allografts along with donor-specifi c splenocyte transfusion (DST, on day -7 prior to transplant) and anti-CD40L mAb (on days -7, -4, 0 and +4). One dose of anti-mouse CD20 mAb was administered on day +1 to deplete B cells. We created a novel mouse strain, CD19-cre+/+::IL-10fl /fl transgenic mice, in which IL-10 is specifi cally depleted in B cells, and they were used to investigate the function of B cell derived IL-10. Results: B cell depletion prevented co-stimulatory blockade induced allogeneic tolerance. Phenotypic analysis of different B cell subsets showed that tolerization with DST and anti-CD40L mAb did not significantly change the number of CD19+CD5+CD1d+ regulatory B cells (Breg) in spleen and lymph nodes. However, anti-CD20 mAb treatment significantly changed the number and percentage of the CD19+CD93-CD23+sIgMhisIgDhiCD21/CD35hi marginal zone precursor (MZP) subset in spleen and lymph nodes. DST plus anti-CD40L mAb treatment showed increased IL-10 mRNA expression in non-Breg cells, furthermore B cell depletion causes decreased IL-10 production in the residual MZP cells. Allogeneic transplantation under tolerogenic conditions in CD19-Cre+/+::IL-10fl /fl mice lead to acute rejection of the allograft, compared to in littermate controls, which resulted in tolerance. Immunohistochemical analysis of allografts showed signifi cantly increased perivascular and mononuclear infi ltration in the rejected grafts. CD19-Cre+/+::IL10fl /fl mice did not have any signifi cant change in the CD19+, CD4+, CD8+, Foxp3+ Treg, or Breg cell numbers or percentages in spleen and lymph nodes. However, MZP B cell number was reduced in the rejecting IL-10 defi cient mice compared to littermates in secondary lymphoid organs. Conclusion: IL-10 expression in B cell is required for the generation of transplantation tolerance. MZP subsets of B cell express IL-10 and MZP subset localization in the secondary lymphoid organs plays an important role in tolerance. These results demonstrate a novel subset of B cells is required for tolerization. 2 B Cell Derived IL-10 Is Required for Co-Stimulatory Blockade Induced Tolerance. G. Lal,1 Y. Nakayama,2 B. E. Burrell,2 A. Sethi,1 Y. Ding,2 J. S. Bromberg.2 1National Centre for Cell Science, Pune, MH, India; 2University of Maryland, Baltimore, MD. Background: The anti-infl ammatory cytokine IL-10 plays a very important role in the maintenance of tolerance. Whether specifi c subsets of B cells and B cell derived IL-10 contribute to the generation of transplantation tolerance are not known. We hypothesized that specifi c subsets of B cells produce IL-10 that is required for tolerization. Methods: C57BL/6 mice received vascularized BALB/c cardiac allografts along with donor-specifi c splenocyte transfusion (DST, on day -7 prior to transplant) and anti-CD40L mAb (on days -7, -4, 0 and +4). One dose of anti-mouse CD20 mAb was administered on day +1 to deplete B cells. We created a novel mouse strain, CD19-cre+/+::IL-10fl /fl transgenic mice, in which IL-10 is specifi cally depleted in B cells, and they were used to investigate the function of B cell derived IL-10. Results: B cell depletion prevented co-stimulatory blockade induced allogeneic tolerance. Phenotypic analysis of different B cell subsets showed that tolerization with DST and anti-CD40L mAb did not significantly change the number of CD19+CD5+CD1d+ regulatory B cells (Breg) in spleen and lymph nodes. However, anti-CD20 mAb treatment significantly changed the number and percentage of the CD19+CD93-CD23+sIgMhisIgDhiCD21/CD35hi marginal zone precursor (MZP) subset in spleen and lymph nodes. DST plus anti-CD40L mAb treatment showed increased IL-10 mRNA expression in non-Breg cells, furthermore B cell depletion causes decreased IL-10 production in the residual MZP cells. Allogeneic transplantation under tolerogenic conditions in CD19-Cre+/+::IL-10fl /fl mice lead to acute rejection of the allograft, compared to in littermate controls, which resulted in tolerance. Immunohistochemical analysis of allografts showed signifi cantly increased perivascular and mononuclear infi ltration in the rejected grafts. CD19-Cre+/+::IL10fl /fl mice did not have any signifi cant change in the CD19+, CD4+, CD8+, Foxp3+ Treg, or Breg cell numbers or percentages in spleen and lymph nodes. However, MZP B cell number was reduced in the rejecting IL-10 defi cient mice compared to littermates in secondary lymphoid organs. Conclusion: IL-10 expression in B cell is required for the generation of transplantation tolerance. MZP subsets of B cell express IL-10 and MZP subset localization in the secondary lymphoid organs plays an important role in tolerance. These results demonstrate a novel subset of B cells is required for tolerization. Abstract# 3 Sotrastaurin + Reduced-Exposure Tacrolimus Prevents Acute Rejection and Preserves Renal Function after De Novo Kidney Transplantation – 6 Month Results. G. Russ,1 H. Tedesco-Silva,1 D. Kuypers,1 S. Cohney,1 R. Langer,1 E. S. Woodle,1 J. Gill,1 J. Ng,2 J. Klupp,2 L. Chodoff,2 K. Budde.1 1Sotrastaurin A2214 Study Group; 2Novartis Pharma AG. Purpose: Sotrastaurin (STN, AEB071) is a novel low molecular weight immunosuppressant which blocks early T-cell activation through protein kinase C inhibition. This study examined the effi cacy and safety of reduced-exposure tacrolimus (TAC) with STN in low-moderate risk renal transplant recipients. Methods: Recipients of a fi rst or second kidney with cold ischemia time (CIT) <30 hours were randomized to STN 100 or 200mg bid + standard TAC (sTAC; 5-12ng/ mL) or STN 300mg bid + reduced TAC (rTAC; 2-5ng/mL) vs. enteric-coated mycophenolic acid (MPA) + sTAC in a partially blinded study. All patients received basiliximab and steroids. Results: 298 patients were randomized (59% deceased donors). Key results at Month 6 are provided in the table. Most rejections were mild (Banff grade IA/IB 17, IIA 12, IIB 1, III 0); 3 were antibody-mediated (none on STN); 7 were antibody-treated. Frequency and pattern of infections were comparable between groups. A slight dose-proportional increase in heart rate (5-7 bpm) was observed for STN patients. No evidence of other ECG abnormalities or cardiac AEs and no between-group differences in blood pressure were observed. Leucopenia was signifi cantly less frequent in all STN treatment groups compared to the MPA group. Treatment with STN 300mg bid + TAC 2-5 ng/mL was associated with numerically higher eGFR compared to MPA + sTAC. In the STN 200 + sTAC group, longer median CIT and more DGF may have contributed to reduced eGFR. Conclusion: STN 200mg bid + sTAC and STN 300mg bid + rTAC provided effective rejection prophylaxis through Month 6. STN-TAC regimens were well tolerated; discontinuation due to AEs was dose-proportional. The rTAC regimen was associated with the highest GFR. 3 Sotrastaurin + Redu

Lyoplate-based multiparameter flow cytometry for the analysis of T cell subsets in human immuno-monitoring studies

In recent years immuno-monitoring studies are becoming increasingly popular due to the relevant role the immune system plays in many pathologies and in treatment responses. n nHuman translational research is hampered by limited amounts of samples, intrinsic human variability and practical issues involving multi-centre sample collection and analysis. Therefore, human immuno-monitoring studies need to be standardized. Multicolour flow cytometry (MFC) provides a powerful tool to unravel the complexity of the immune system. However standardization of this technique is still in progress, due to differences in sample quality, reagents, antibodies and fluorchromes used, as well as instrument settings. n nPart of this variability could be overcome by using lyophilized reagents in a 96 well plate format for cell stimulation and staining. n nIn this pilot study we assess how lyoplate based-MFC performs compared to traditional liquid reagent-based MFC, mirroring larger human immuno-monitoring cohorts. n nPeripheral blood mononuclear cells were collected from healthy volunteers at two time points. Frozen samples were thawed, stimulated and stained using either liquid or lyophilized reagents. The 10 colour flow cytometry antibody cocktail used allowed the analysis of different T cell subsets (CD8 T cells, Th cells and Treg cells) and their cytokine production (IFNγ, IL17A, IL10). Quantitative and qualitative differences between liquid and lyophilized reagents were evaluated, as well as intra- and inter-assay variability. n nData from this study will assess the feasibility of standardized and high-throughput immuno-monitoring studies to discover pathology associated signatures and biomarkers predictive of therapy response.

Differential effect of memory, naive and transitional B cells in autologous CD4(+) T cell proliferation, activation and cytokine production

CD200 is a cell-surface glycoprotein that is normally expressed in tissues of the immune system, where its role is to protect immune privileged sites. We previously established CD200 to be frequently over-expressed and associated with poor AML patient outcome. In nthis study, we investigated the possibility that CD200 expression may mediate suppression of T-cell function in this disease. Using multiparameter flow cytometry, we compared PMA/ionomycin stimulated CD8+ T-cell cytotoxic potential (CD107a expression) and the frequency nof intracellular TNFa, IL-2 and IFNc producing CD4+/CD8+ nmemory T-cells between CD200hi and CD200lo patients. We demonstrated that both the magnitude of the CD8+ memory cytotoxic T-cell response and the Th1 cytokine producing CD4+ memory helper T-cells was significantly inhibited in CD200hi AML patients (P < 0.05). Further, using ELISPOT assays to measure IFNg release we showed that the Th1 memory response to common viral antigens was significantly reduced by 75% in CD200hi versus CD200lo AML patients(P < 0.05). Recovery of IFNc release in response to recall antigens nwas observed in CD4+ memory T-cells incubated with a nblocking antibody to CD200R. In conclusion, this study shows a correlation between T-cell dysfunction and expression of CD200 which suggests targeting this axis could be therapeutically beneficial for AML CD200hi patients.

A rapid diagnostic test for human regulatory T cell function to enable regulatory T cell therapy

CD200 is a cell-surface glycoprotein that is normally expressed in tissues of the immune system, where its role is to protect immune privileged sites. We previously established CD200 to be frequently over-expressed and associated with poor AML patient outcome. In nthis study, we investigated the possibility that CD200 expression may mediate suppression of T-cell function in this disease. Using multiparameter flow cytometry, we compared PMA/ionomycin stimulated CD8+ T-cell cytotoxic potential (CD107a expression) and the frequency nof intracellular TNFa, IL-2 and IFNc producing CD4+/CD8+ nmemory T-cells between CD200hi and CD200lo patients. We demonstrated that both the magnitude of the CD8+ memory cytotoxic T-cell response and the Th1 cytokine producing CD4+ memory helper T-cells was significantly inhibited in CD200hi AML patients (P < 0.05). Further, using ELISPOT assays to measure IFNg release we showed that the Th1 memory response to common viral antigens was significantly reduced by 75% in CD200hi versus CD200lo AML patients(P < 0.05). Recovery of IFNc release in response to recall antigens nwas observed in CD4+ memory T-cells incubated with a nblocking antibody to CD200R. In conclusion, this study shows a correlation between T-cell dysfunction and expression of CD200 which suggests targeting this axis could be therapeutically beneficial for AML CD200hi patients.

Chapter Title: Chronic graft loss. Immunological and non-immunological factors.

AIMSnLate loss of kidney grafts is an ongoing problem in the field of transplantation. This is caused by immunological and non-immunological factors, the main immunological driver of rejection is the immune response against HLA molecules that differ between donor and recipient.nnnMETHODSnTo measure the anti-donor responses that a recipient can mount, we have been quantifying anti-donor T-cell frequencies in recipients of renal transplants for several years. Anti-donor direct and indirect pathway frequencies have been measured in vitro in kidney and heart transplant patients by Limiting dilution analysis and other methods. Further, to elucidate the role of CD4+CD25+ regulatory T-cells, these cells have been depleted in ex vivo assays of cellular function. Antigen specific CD4+CD25+ cell lines are being expanded in vitro with a view to using them in immunotherapeutic strategies.nnnRESULTSnFrequencies of T-cells with direct pathway anti-donor specificity decline in most patients, while those with indirect anti-donor specificity increase in frequency in patients with late graft failure. In keeping with results from experimental models of transplantation tolerance, evidence for allospecific regulatory cells was found in some patients with good, stable transplant function. Interestingly, the regulatory cells appeared to have indirect allospecificity, and no evidence of direct pathway regulation was observed.nnnCONCLUSIONSnThe indirect pathway anti-donor alloresponse poses the major threat to long-term transplant survival. Indirect pathway regulatory T-cells arise in some patients. These data are consistent with the hypothesis that tolerance strategies require shrinkage of the direct, and regulation of the indirect, anti-donor response.