Biology subjects

Eiz-Vesper, B.

Publications and source records attributed to Eiz-Vesper, B..

7 recordsLinked to original sources

bioRxiv2026

Mutanome-guided immunopeptidomics of blood plasma for neoepitope detection in solid tumors is constrained by cfDNA variant calling sensitivity and MS detection limits

Introduction: Neoepitopes form the basis of tumor-specific immune responses. Tissue biopsy, the primary source for neoepitope detection, is limited and invasive. Therefore, we aimed to identify neoepitopes by mutanome-guided immunopeptidomics from plasma of cancer patients. Methods: Mass spectrometry (MS) data analysis of HLA ligands from plasma (n = 4) was guided by patient-specific mutanomes of cell-free DNA (cfDNA) from plasma or tumor genomic DNA (tgDNA) from tissue. Matched tumor tissue and healthy donor plasma served as controls. Neoepitopes were validated with synthetic peptides, and immunogenicity was assessed using IFN-gamma ELISpot and intracellular cytokine staining. Results: Wild-type immunopeptidomes from tissue and plasma overlapped by 58%, with 91% of plasma HLA ligands rediscovered in tissue. 13 out of 15 tumor-associated HLA ligands detected in plasma were rediscovered in the matching tissue. However, no neoepitopes in plasma were identified by immunopeptidomics guided by cfDNA mutanomes, likely reflecting the limited overlap between cfDNA and tgDNA mutanomes (15%). Using the tgDNA mutanome as a complementary reference, two neoepitopes were detected in one patient's plasma, albeit at the MS detection limit. Both neoepitopes were also discovered in tissue, along with three tissue-exclusive neoepitopes. Two tissue-exclusive neoepitopes induced antigen-specific T cell responses in healthy donor PBMCs. Conclusion: In summary, plasma immunopeptidomics enables profiling of HLA ligands from wild-type proteins, including TAAs. In principle, neoepitope detection from plasma at the peptide level is feasible, but tissue remains the gold standard for variant calling and neoepitope identification. Improved detection methods may enable minimally invasive approaches in the future.

immunology↗

bioRxiv2026

Memory T Cells in MHC-Deficient Humanized Mice

Major histocompatibility complexes (MHC) govern antigen presentation and T cell receptor (TCR) selection. Accurate in vivo modeling of human immunity therefore requires physiological human MHC TCR interactions. Humanized NOD scid IL2null (NSG) mice engrafted with human CD34+ hematopoietic stem cells are widely used to provide preclinical platforms for the development of advanced therapies; however, interactions between murine MHC and human TCR can promote xenoreactivity and alter T cell development. Here, we investigated how elimination of murine MHC together with different conditioning regimens shapes human T cell maturation in vivo. CD34+ cells from ten cord blood donors were transplanted into conventional NSG mice or murine MHC deficient NSG derivatives (DKO) following either sublethal irradiation or myeloablative busulfan conditioning. Integrated analyses combining flow cytometry, plasma cytokine profiling, and bulk and single cell TCR sequencing revealed marked differences in T cell differentiation across models. Busulfan conditioned DKO mice developed highly proliferative, activated, and cytotoxic T cells together with clonally expanded TCR repertoires. In contrast, irradiated NSG mice preferentially accumulated naive, NKT, and regulatory T cell populations. Busulfan-conditioned DKO mice showed no evidence of xenogeneic graft versus host disease and represent a refined enabling platform for human T cell development and provide a foundation for future preclinical evaluation of advanced gene and cell therapies.

immunology↗

bioRxiv2026

Next-generation all-in-one CRISPR/Cas9 multiply-edited CD30CAR-T cells:Potency despite risk of translocations

BackgroundChimeric antigen receptor (CAR)-T cells are therapeutic breakthroughs against advanced non-Hodgkin lymphomas and myelomas. On the other hand, no CAR-T cell product has been so far clinically approved for therapy of Hodgkin Lymphoma (HL), T cell lymphoma (TCL), or Epstein-Barr-Virus (EBV)-associated lymphoproliferative diseases (EBV-LPDs). CD30 (TNFRSF8) is commonly expressed on HL and on subsets of TCL and EBV-LPDs. CD30CAR-T cells generated via transduction with viral vectors have been tested in clinical trials, showing overall good responses against HL. CAR-T cells produced entirely with locus-specific gene editing methods are emerging as attractive next-generation engineered cell products for ease of multiple seamless cell modifications. MethodsUsing CRISPR/Cas9-mediated techniques, we optimized homology-directed repair templates (HDRTs) and performed all-in-one multiplex editing to knock-in (KI) CD30CAR within the TCR constant (TRAC) locus and to simultaneously knock-out (KO) PD-1 or/and {beta}2M. CD30CAR-T cells were tested in CD30+ cell models of HL, TCL, and EBV-LPDs. ResultsWe compared mouse versus human anti-CD30 scFv designs in HDRTs incorporating TRAC homology arms, FcIg spacer/detection domain, and CD28 / CD3{zeta} signaling domains. We obtained an average of 30% TRACKICD30CAR-T cells and efficient in vitro cytotoxicity with CD30+ cell targets. CARs incorporating the high-affinity humanized 5F11 scFv showed the highest CAR expression, and the editing templates were further modified to incorporate a truncated CD34 (tCD34) spacer/detection domain. 5F11-CD30CAR-tCD34-T cells showed high CAR-KI rates (approx. 50-80% 12-14 days after editing) and potency in vitro and in vivo. Subsequently, we tested all-in-one CAR KI with additional KOs by co-electroporation of guide RNAs (gRNAs) targeting the genes encoding PD-1 or /and {beta}2M to improve function and allow for improved cell persistence in allogeneic recipients, respectively. Compared with CD30CAR-T cells, CD30CAR-{beta}2MKO-T cells were similarly viable and functional and showed low risk of translocations. PD1KO enabled CD30CAR-T cells to produce higher levels of cytotoxic features upon exposure to targets. However, simultaneous {beta}2MKO and PD-1KO compromised the expansion capacity of CD30CAR-T cells and resulted in detectable translocations. ConclusionsNon-virally engineered 5F11-CD30CAR-T cells represent a novel cell therapy modality against CD30+ lymphomas. Multiplex editing remains to be optimized to avoid unwanted genomic alterations and chromosomal translocations.

cancer biology↗

bioRxiv2024

ENTPD3-specific CAR Regulatory T cells for Local Immune Control in T1D

Despite advances in Type 1 Diabetes (T1D) management such as hybrid closed loop systems, patients still face significant morbidity, reduced life expectancy, and impaired glucose regulation compared to healthy individuals or those with pancreas transplants. Here we developed beta cell-specific Chimeric Antigen Receptors (CAR) targeting the antigen ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) using a novel cell-based phage display methodology. ENTPD3 is highly expressed on beta cells of both early and progressed T1D patients. ENTPD3 CAR regulatory T cells (Tregs) homed, expanded and persisted in pancreatic islets in a T1D mouse model (NOD) and completely prevented disease progression. Human ENTPD3 CAR Tregs displayed a stable regulatory phenotype, strong activation, and suppression. Importantly, ENTPD3 CAR T cells recognised and were fully activated by human islets. This approach holds great promise as a durable treatment option for patients with prediabetes, new-onset diabetes, or those undergoing beta cell replacement therapy.

immunology↗

bioRxiv2023

DNA methylation profiling identifies TBKBP1 as potent amplifier of cytotoxic activity in CMV-specific human CD8+ T cells

Epigenetic mechanisms stabilize gene expression patterns during CD8+ T cell differentiation. However, although adoptive transfer of virus-specific T cells is clinically applied to reduce the risk of virus infection or reactivation in immunocompromised individuals, the DNA methylation pattern of virus-specific CD8+ T cells is largely unknown. Hence, we here performed whole-genome bisulfite sequencing of cytomegalovirus-specific human CD8+ T cells and found that they display a unique DNA methylation pattern consisting of 79 differentially methylated regions when compared to bulk memory CD8+ T cells. Among them was TBKBP1, coding for TBK-binding protein 1 that can interact with TANK-binding kinase 1 (TBK1) and mediate pro-inflammatory responses in innate immune cells downstream of intracellular virus sensing. Since TBKBP1 has not yet been reported in T cells, we aimed to unravel its role in virus-specific CD8+ T cells. TBKBP1 demethylation in terminal effector CD8+ T cells correlated with TBKBP1 expression and was stable upon long-term in vitro culture. TBKBP1 overexpression resulted in enhanced TBK1 phosphorylation upon stimulation of CD8+ T cells and significantly improved their virus neutralization capacity. Collectively, our data demonstrate that TBKBP1 modulates virus-specific CD8+ T cell responses and could be exploited as therapeutic target to improve adoptive T cell therapies.

immunology↗

bioRxiv2023

Depletion of alloreactive B cells by chimeric alloantigen receptor T cells with drug resistance to prevent antibody-mediated rejection in solid organ transplantation

In the present study, we developed a novel cell therapy approach to selectively combat antibody-mediated rejection (AMR), a major and unresolved complication after solid organ transplantation (SOT) caused by donor-HLA-specific, alloreactive B cells. Current treatment options including B-cell depletion protocols are inefficient and result in complete loss of humoral immunity. To selectively eliminate alloreactive B cells characterized by corresponding anti-donor-HLA B-cell receptors (BCRs), we engineered T cells with a novel chimeric receptor comprising a truncated HLA molecule fused to intracellular 4-1BB/CD3{xi} signaling domains to generate T cells overcoming rejection by antibodies (CORA-Ts). As proof-of-concept, CORA receptors based on HLA-A*02 were shown to bind anti-HLA-A*02 antibodies from the serum of kidney transplant recipients, indicating their suitability to also target the respective membrane-bound anti-HLA-A*02 BCRs on alloreactive B cells. In co-cultures with B-cell lines expressing and releasing anti-HLA-A*02 antibodies, CORA-Ts were specifically activated, released pro-inflammatory cytokines (e.g. IFN-{gamma}, granzyme B), and exhibited strong cytotoxicity resulting in an effective reduction of anti-HLA-A*02 antibody release. A modification of the HLA-A*02 3-domain within the CORA receptor effectively abrogated T-cell sensitization. Additionally, using CRISPR/Cas9-mediated knockout of a selected binding protein, CORA-Ts were able to resist immunosuppressive treatment to ensure high efficiency in transplant patients. Our results demonstrate that CORA-Ts are able to specifically recognize and eliminate alloreactive B cells, and thus selectively prevent formation of anti-HLA antibodies even under immunosuppressive conditions. This suggests CORA-Ts as potent novel approach to specifically combat AMR and improve long-term graft survival in SOT patients while preserving their overall B-cell immunity.

immunology↗

bioRxiv2023

Dynamic monitoring of viral gene expression reveals rapid antiviral effects of CD8 T cells recognizing the HCMV-pp65 antigen

Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. Therefore, we developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. Therefore, ARMATA does not only allow same-day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection.

immunology↗