Introduction: Chimeric antigen receptors CARs can be used to change the specificity of FOXP3+ regulatory T cells (Tregs) for the induction of tissue specific tolerance. We and others have shown that CAR-Tregs specific for transplant mismatched HLA-A*0201 (A2-CAR) can be used for tolerance induction in humanized mouse models. Methods: We now wanted to study the therapeutic potential of MHC-specific CAR Tregs in an immunogenic, non-lymphopenic mouse model. To this end, we generated H-2Kb-specific scFv from phage display libraries to create second generation CARs with a CD28/CD3z signaling domain. Results: The CARs showed good surface expression and strong activation of Tregs with an exclusive Kb-specificity. They were much more effective in suppression of CD4+ and CD8+ effector T cell response in allogeneic mixed lymphocyte reaction as compared to nTregs. Kb-CAR-Tregs prevented skin transplant rejection in BALB/c RAG1-/-after cotransfer with equal numbers of effector T cells. To our major surprise adoptive transfer of CAR-Tregs in an immunogenic C57BL/6 to BALB/c skin transplant did not result in any meaningful prolongation of transplant survival in a non-lymphopenic setting. We therefore cloned our anti-MHC*0201 scFv from the initial experiments in our humanized mouse models into a murine CAR vector. However, the rejection of A2-transgenic C57BL/6 skin grafts into C57BL/6 recipients (single MHC mismatch) was also not prolonged by adoptive transfer of A2-CAR Tregs. Although lymphodepletion and rapamycin prolonged graft survival, the addition of Kb-CAR Tregs had no added benefit, although we observed long-term persistence of CAR-Tregs and a strong accumulation of the transferred Tregs within the graft. In fact, the transferred CAR-Tregs made up to 40% of all intragraft Tregs ruling out too small local Treg numbers as the reason for the low in vivo efficiency. Conclusion: Weakly immunogenic humanized mouse models can be misleading to evaluate the effectivity of CAR-Tregs for clinical translation. Lymphodepleting strategies strongly increase the accumulation of graft-specific Tregs within the grafts. In order to develop clinically meaningful protocols for CAR-Treg therapies more immunogenic, non-lymphopenic models are required.
Autoimmune hepatitis (AIH) is detected at a late stage in the course of the disease. Therefore, induction and etiology are largely unclear. It is controversial if the induction of autoimmunity occurs in the liver or in the spleen. In our experimental murine AIH model, the induction of autoimmunity did not occur in the spleen. Instead, a protective role of the spleen could be more likely. Therefore, we splenectomized mice followed by induction of experimental murine AIH. Splenectomized mice presented more severe portal inflammation. Furthermore, these mice had more IL-17, IL-23 receptor (IL-23R) and caspase 3 (casp3) and a decreased amount of erythropoietin in serum, while intrahepatic T cell compartments were unaffected. These results indicate that the spleen is not necessary for induction of AIH, and splenectomy disrupts the ability to immune regulate the intensity of hepatic inflammation, production of IL-17 and apoptosis.
Abstract To evaluate the feasibility and potential value of 2D Parametric Parenchymal Blood Flow (2D-PPBF) for the assessment of perfusion changes following partial spleen embolization (PSE) in a retrospective observational study design. Overall, 12 PSE procedures in 12 patients were included in this study. The outcome of the study was the platelet response (PR), calculated as the percentage increase of platelet count (PLT), following PSE. To quantify perfusion changes using 2D-PPBF, the acquired digital subtraction angiography series were post-processed. A reference region-of-interest (ROI) was placed in the afferent splenic artery and a target ROI was positioned on the embolization territory of the spleen on digital subtraction angiography series pre- and post-embolization. The ratios of the target ROIs to the reference ROIs were calculated for the Wash-In-Rate (WIR), the Time-To-Peak (TTP) and the Area-Under-the-Curve (AUC). Comparisons between pre- and post-embolization data were made using Wilcoxon signed-rank test and Spearman's rank correlation coefficient (r). Afterwards, the study population was divided by the median of the TTP before PSE to analyze its value for the prediction of PR following PSE. Following PSE, PLT increased significantly from 43,000 ± 21,405 platelets/μL to 128,500 ± 66,083 platelets/μL with a PR of 255 ± 243% ( P = .003). In the embolized splenic territory, the pre-/post-embolization 2D-PPBF parameter changed significantly: WIR pre-PSE 1.23 ± 2.42/WIR post-PSE 0.09 ± 0.07; -64 ± 46% (p = 0.04), TTP pre-PSE 4.41 ± 0.99/TTP post-PSE 5.67 ± 1.52 ( P = .041); +34 ± 47% and AUC post-PSE 0.81 ± 0.85/AUC post-PSE 0.14 ± 0.08; -71 ± 18% ( P = .002). A significant correlation of a 2D-PPBF parameter with the PLT was found for TTP pre-PSE /PLT pre-PSE r = -0.66 ( P = .01). Subgroup analysis showed a significantly increased PR for the group with TTP pre-PSE >4.44 compared to the group with TTP pre-PSE ≤4.44 (404 ± 267% versus 107 ± 76%; P = .04). 2D-PPBF is an objective approach to analyze the perfusion reduction of embolized splenic tissue. TTP derived from 2D-PPBF has the potential to predict the extent of PR during PSE.
Following organ transplantation, lifelong immunosuppressive therapy is required to prevent the host immune system from destroying the allograft. This can cause severe side effects and increased recipient morbidity and mortality. Complete cessation of immunosuppressive drugs has been successfully accomplished in selected transplant recipients, providing proof of principle that operational allograft tolerance is attainable in clinical transplantation. The intra-graft molecular pathways associated with successful drug withdrawal, however, are not well defined. In this study, we analyzed sequential blood and liver tissue samples collected from liver transplant recipients enrolled in a prospective multicenter immunosuppressive drug withdrawal clinical trial. Before initiation of drug withdrawal, operationally tolerant and non-tolerant recipients differed in the intra-graft expression of genes involved in the regulation of iron homeostasis. Furthermore, as compared with non-tolerant recipients, operationally tolerant patients exhibited higher serum levels of hepcidin and ferritin and increased hepatocyte iron deposition. Finally, liver tissue gene expression measurements accurately predicted the outcome of immunosuppressive withdrawal in an independent set of patients. These results point to a critical role for iron metabolism in the regulation of intra-graft alloimmune responses in humans and provide a set of biomarkers to conduct drug-weaning trials in liver transplantation.
Objective: Assess the impact of having a living donor on waitlist outcomes and overall survival through an intention-to-treat analysis. Background: Living-donor liver transplantation (LDLT) offers an alternative to deceased donation in the face of organ shortage. An as-treated analysis revealed that undergoing LDLT, compared to staying on the waiting list, is associated with improved survival, even at Model for End-stage Liver Disease-sodium (MELD-Na) score of 11. Methods: Liver transplant candidates listed at the Ajmera Transplant Centre (2000-2021) were categorized as pLDLT (having a potential living donor) or pDDLT (without a living donor). Employing Cox proportional-hazard regression with time-dependent covariates, we evaluated pLDLT’s impact on waitlist dropout and overall survival through a risk-adjusted analysis. Results: Of 4,124 candidates, 984 (24%) had potential living donors. The pLDLT group experienced significantly lower overall waitlist dropouts (5.2%vs. 34.4%, P <0.001) and mortality (3.8%vs. 24.4%, P <0.001) compared to the pDDLT group. Possessing a living donor correlated with a 26% decline in the risk of waitlist dropout (adjusted hazard ratio 0.74, 95%CI 0.55-0.99, P =0.042). The pLDLT group also demonstrated superior survival outcomes at 1- (84.9%vs. 80.1%), 5- (77.6%vs. 61.7%), and 10-year (65.6%vs.52.9%) from listing (log-rank P <0.001) with a 35% reduced risk of death (adjusted hazard ratio 0.65, 95%CI 0.56-0.76, P <0.001). Moreover, the predicted hazard ratios consistently remained below 1 across the MELD-Na range 11-26. Conclusions: Having a potential living donor significantly improves survival in end-stage liver disease patients, even with MELD-Na scores as low as 11. This emphasizes the need to promote awareness and adoption of LDLT in liver transplant programs worldwide.
Hepatocellular carcinoma (HCC) recurs after liver transplantation (LT) in ~17% of patients. We aimed to retrospectively compare the outcomes of patients treated with different tyrosine kinase inhibitors (TKIs) for recurrent HCC post-LT.
Adoptive transfer of CD4+Foxp3+ regulatory T cells (Tregs) might be an alternative option to achieve tissue specific tolerance without perturbance of general immunocompetence. While polyspecific Tregs could control graft versus host diseases under lymphopenic conditions, they were largely ineffective under non-lymphopenic conditions as seen in patients after organ transplantation. Herein, we describe that the surface molecules latency associated peptide (LAP) and glycoprotein A repetitions predominant (GARP) can specifically identify human Tregs activated by their T cell receptor and not in bystander fashion. Using these markers we can show for the first time that the human natural Treg repertoire contains about 10% of alloreactive Tregs. In addition we show that CD154 is neither expressed on resting nor on activated Tregs and can therefore be used to increase the purity of isolated Tregs. The combination of CD154-LAP+ or CD154-GARP+ markers allowed the isolation of highly pure antigen-specific Tregs (purity>83%). The purity, assessed by epigentic methylation analysis, exceeds all other published Treg isolations and identifies furthermore just antigen-specific Tregs. Furthermore, we demonstrated that those LAP+ allospecific Tregs are highly capable in the prevention of potent allospecific DTH responses in humanized mice and in the prevention of rejection of allogeneic cells in immune reconstituted humanized mice. Finally we can show that the transfer of donor-specific Tregs prevents the rejection of allogeneic human skin trasplanted on immune reconstituted humaized NRG mice.These results represent a major step forward for the use of adoptively transferred Tregs in transplantation. DISCLOSURE:Jaeckel, E.: Speaker’s Bureau, Miltenyi Biotech.
In the past, endogenous retroviral sequences have been isolated from patients suffering from different kinds of autoimmune diseases. Recently, a full length retroviral genome, termed IDDMK(1,2)22, was isolated from patients with new-onset IDDM. This genome contains a major histocompatibility complex II-dependent superantigen within its envelope gene. The viral sequence was found in ten patients with new-onset IDDM, but not in age-matched control subjects (Conrad et al. [9]). We searched for the presence of this viral genome by nested reverse transcription-polymerase chain reaction (RT-PCR) in a cohort of six patients with new-onset IDDM and six control subjects of the same age. We found all samples to be positive without any differences between patients and control subjects. The same results were obtained with supernatants of activated peripheral blood mononuclear cells. We performed isopycnic ultracentrifugation in sucrose density gradients on all samples and were unable to detect particles of the new virus in any of our samples. However, positive signals were obtained from all pellet fractions. RNase, DNase treatment and nested PCRs without reverse transcription showed that the positive signals were probably derived from intracellular RNA and DNA. In summary, no correlation between a positive nested PCR signal for IDDMK(1,2)22 and diabetes was found indicating that the new sequence represents just an additional member of the human endogenous retrovirus (HERV) family with lack of an exogenous counterpart.
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