CD19 CAR T-Cell Therapy in Autoimmune Disease - A Case Series with Follow-up.

BACKGROUND: Treatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission. METHODS: We evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology-European League against Rheumatism (ACR-EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded. RESULTS: The median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR-EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell-associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization. CONCLUSIONS: In this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.).

Reconstitution of EBV-directed T cell immunity by adoptive transfer of peptide-stimulated T cells in a patient after allogeneic stem cell transplantation for AITL.

Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRß) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.

Retzius sparing robot-assisted radical prostatectomy: optimizing functional results.

PURPOSE: The aim of this study is to critically evaluate the existing body of evidence regarding the efficacy of Retzius-sparing radical prostatectomy (RS-RARP) in achieving improved functional outcomes. Moreover, we explored possible strategies to further optimize functional outcomes. METHODS: Following PRISMA guidelines, a systematic review (PROSPERO ID CRD42024539915) was performed on 9th September 2023 on PubMed, Scopus, and Web of Science. Only original articles in the English language reporting functional outcomes after RS-RARP were included. RESULTS: Overall, the search string yielded 99 results on PubMed, 122 on Scopus, and 120 on Web Of Science. After duplicate exclusion, initial screening and eligibility evaluation, a total of 47 studies were included in the qualitative analysis, corresponding to a cohort of 13.196 patients. All studies reported continence recovery. RS-RARP appeared to achieve better and faster continence recovery compared to S-RARP. However, it should be noted that continence definition was heterogeneous and not based on validated condition-specific questionnaires. Seven (15%) studies provided for any sort of rehabilitation for urinary incontinence after RS-RARP. 22 studies analyzed potency recovery rates, showing no difference between RS-RARP and S-RARP. The evaluation of this outcome poses a great challenge due to the lack of standardized assessment tools and reporting methods. Only two studies reported on the consistent use of post-operative PDE5i as penile rehabilitation. CONCLUSIONS: The current review highlights the satisfactory functional results of Retzius-sparing robot assisted radical prostatectomy, which holds true irrespective of disease stage and prostate volume, with promising results even in patients previously treated for BPH or in the salvage setting. How can we optimize those results? The answer does not probably lie in further refinement of the surgical technique, but in giving greater attention to patient counselling and rehabilitation strategies in order to minimize regret and maximize satisfaction.

Upregulation of CCR4 in activated CD8+ T cells indicates enhanced lung homing in patients with severe acute SARS-CoV-2 infection.

COVID-19 is a life-threatening disease leading to bilateral pneumonia and respiratory failure. The underlying reasons why a smaller percentage of patients present with severe pulmonary symptoms whereas the majority is only mildly affected are to date not well understood. Comparing the immunological phenotype in healthy donors and patients with mild versus severe COVID-19 shows that in COVID-19 patients, NK-/B-cell activation and proliferation are enhanced independent of severity. As an important precondition for effective antibody responses, T-follicular helper cells and antibody secreting cells are increased both in patients with mild and severe SARS-CoV-2 infection. Beyond this, T cells in COVID-19 patients exhibit a stronger activation profile with differentiation toward effector cell phenotypes. Importantly, when looking at the rates of pulmonary complications in COVID-19 patients, the chemokine receptor CCR4 is higher expressed by both CD4 and CD8 T cells of patients with severe COVID-19. This raises the hypothesis that CCR4 upregulation on T cells in the pathogenesis of COVID-19 promotes stronger T-cell attraction to the lungs leading to increased immune activation with presumably higher pulmonary toxicity. Our study contributes significantly to the understanding of the immunological changes during COVID-19, as new therapeutic agents, preferentially targeting the immune system, are highly warranted.

Successful treatment of COVID-19 infection with convalescent plasma in B-cell-depleted patients may promote cellular immunity.

Treatment with convalescent plasma has been shown to be safe in coronavirus disease in 2019 (COVID-19) infection, although efficacy reported in immunocompetent patients varies. Nevertheless, neutralizing antibodies are a key requisite in the fight against viral infections. Patients depleted of antibody-producing B cells, such as those treated with rituximab (anti-CD20) for hematological malignancies, lack a fundamental part of their adaptive immunity. Treatment with convalescent plasma appears to be of general benefit in this particularly vulnerable cohort. We analyzed clinical course and inflammation markers of three B-cell-depleted patients suffering from COVID-19 who were treated with convalescent plasma. In addition, we measured serum antibody levels as well as peripheral blood CD38/HLA-DR-positive T-cells ex vivo and CD137-positive T-cells after in vitro stimulation with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived peptides in these patients. We observed that therapy with convalescent plasma was effective in all three patients and analysis of CD137-positive T-cells after stimulation with SARS-CoV-2 peptides showed an increase in peptide-specific T-cells after application of convalescent plasma. In conclusion, we here demonstrate efficacy of convalescent plasma therapy in three B-cell-depleted patients and present data that suggest that while application of convalescent plasma elevates systemic antibody levels only transiently, it may also boost specific T-cell responses.

Human CD4+ T cells specific for dominant epitopes of SARS-CoV-2 Spike and Nucleocapsid proteins with therapeutic potential.

Since December 2019, Coronavirus disease-19 (COVID-19) has spread rapidly throughout the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as cancer patients who recently underwent a haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both short-lived neutralizing antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Here, we identify T cells from COVID-19 patients with a potentially protective response to two major antigens of the SARS-CoV-2 virus, Spike and Nucleocapsid protein. By generating clones of highly virus-reactive CD4+ T cells, we were able to confirm a set of nine immunodominant epitopes and characterize T cell responses against these. Accordingly, the sensitivity of T cell clones for their specific epitope, as well as the extent and focus of their cytokine response was examined. Moreover, using an advanced T cell receptor (TCR) sequencing approach, we determined the paired TCR-αß sequences of clones of interest. While these data on a limited population require further expansion for universal application, the results presented here form a crucial first step towards TCR-transgenic CD4+ T cell therapy of COVID-19.

Visualizing Single-Cell Secretion Dynamics with Single-Protein Sensitivity.

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein-Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa-1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity.

Clinical-grade generation of peptide-stimulated CMV/EBV-specific T cells from G-CSF mobilized stem cell grafts.

BACKGROUND: A major complication after allogeneic hematopoietic stem cell transplantation (aSCT) is the reactivation of herpesviruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Both viruses cause significant mortality and compromise quality of life after aSCT. Preventive transfer of virus-specific T cells can suppress reactivation by re-establishing functional antiviral immune responses in immunocompromised hosts. METHODS: We have developed a good manufacturing practice protocol to generate CMV/EBV-peptide-stimulated T cells from leukapheresis products of G-CSF mobilized and non-mobilized donors. Our procedure selectively expands virus-specific CD8+ und CD4+ T cells over 9 days using a generic pool of 34 CMV and EBV peptides that represent well-defined dominant T-cell epitopes with various HLA restrictions. For HLA class I, this set of peptides covers at least 80% of the European population. RESULTS: CMV/EBV-specific T cells were successfully expanded from leukapheresis material of both G-CSF mobilized and non-mobilized donors. The protocol allows administration shortly after stem cell transplantation (d30+), storage over liquid nitrogen for iterated applications, and protection of the stem cell donor by avoiding a second leukapheresis. CONCLUSION: Our protocol allows for rapid and cost-efficient production of T cells for early transfusion after aSCT as a preventive approach. It is currently evaluated in a phase I/IIa clinical trial.

Impact of collection programs for the generation of monocyte apheresis products on product quality and composition as starting material for the generation of cellular therapeutics.

BACKGROUND: With the discontinuation of the last generation of apheresis machines, new options for monocyte apheresis became available. As apheresis products play a crucial role in the generation of new cellular therapeutics (e.g., generation of dendritic cells [DCs] or precursor for T-cell experiments) we sought to compare two different collection programs for potential benefits or disadvantages due to different composition of the cellular products. STUDY DESIGN AND METHODS: Composition of discontinuously and continuously collected monocyte products from the same 13 donors was analyzed. For further evaluation as starting material for manufacturing of cellular therapeutics typically used steps such as Ficoll Isopaque, cryoconservation and monocyte isolation, with subsequent generation of mature DCs (mDCs) and assessment of T-cell function, were performed on seven of these apheresis pairs. RESULTS: Yield of total cells, monocytes, and mDCs was equal with both methods. T-cell composition did not differ significantly in content of CD3+, CD4+, and CD8+ cells. Differentiation status and cytokine production of CD8+ T cells upon stimulation with cytomegalovirus pp65 antigen was not significantly different. CONCLUSION: Both methods seem comparably suited for the generation of cellular products. If the intended use is "fresh" (without cryoconservation), continuously harvested cells show better cell numbers, while discontinuously harvested cells show better recovery after cryoconservation.

Null mutation in PGAP1 impairing Gpi-anchor maturation in patients with intellectual disability and encephalopathy.

Many eukaryotic cell-surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). There are at least 26 genes involved in biosynthesis and remodeling of GPI anchors. Hypomorphic coding mutations in seven of these genes have been reported to cause decreased expression of GPI anchored proteins (GPI-APs) on the cell surface and to cause autosomal-recessive forms of intellectual disability (ARID). We performed homozygosity mapping and exome sequencing in a family with encephalopathy and non-specific ARID and identified a homozygous 3 bp deletion (p.Leu197del) in the GPI remodeling gene PGAP1. PGAP1 was not described in association with a human phenotype before. PGAP1 is a deacylase that removes an acyl-chain from the inositol of GPI anchors in the endoplasmic reticulum immediately after attachment of GPI to proteins. In silico prediction and molecular modeling strongly suggested a pathogenic effect of the identified deletion. The expression levels of GPI-APs on B lymphoblastoid cells derived from an affected person were normal. However, when those cells were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC), GPI-APs were cleaved and released from B lymphoblastoid cells from healthy individuals whereas GPI-APs on the cells from the affected person were totally resistant. Transfection with wild type PGAP1 cDNA restored the PI-PLC sensitivity. These results indicate that GPI-APs were expressed with abnormal GPI structure due to a null mutation in the remodeling gene PGAP1. Our results add PGAP1 to the growing list of GPI abnormalities and indicate that not only the cell surface expression levels of GPI-APs but also the fine structure of GPI-anchors is important for the normal neurological development.

Hypomorphic mutations in PGAP2, encoding a GPI-anchor-remodeling protein, cause autosomal-recessive intellectual disability.

PGAP2 encodes a protein involved in remodeling the glycosylphosphatidylinositol (GPI) anchor in the Golgi apparatus. After synthesis in the endoplasmic reticulum (ER), GPI anchors are transferred to the proteins and are remodeled while transported through the Golgi to the cell membrane. Germline mutations in six genes (PIGA, PIGL, PIGM, PIGV, PIGN, and PIGO) in the ER-located part of the GPI-anchor-biosynthesis pathway have been reported, and all are associated with phenotypes extending from malformation and lethality to severe intellectual disability, epilepsy, minor dysmorphisms, and elevated alkaline phosphatase (ALP). We performed autozygosity mapping and ultra-deep sequencing followed by stringent filtering and identified two homozygous PGAP2 alterations, p.Tyr99Cys and p.Arg177Pro, in seven offspring with nonspecific autosomal-recessive intellectual disability from two consanguineous families. Rescue experiments with the altered proteins in PGAP2-deficient Chinese hamster ovary cell lines showed less expression of cell-surface GPI-anchored proteins DAF and CD59 than of the wild-type protein, substantiating the pathogenicity of the identified alterations. Furthermore, we observed a full rescue when we used strong promoters before the mutant cDNAs, suggesting a hypomorphic effect of the mutations. We report on alterations in the Golgi-located part of the GPI-anchor-biosynthesis pathway and extend the phenotypic spectrum of the GPI-anchor deficiencies to isolated intellectual disability with elevated ALP. GPI-anchor deficiencies can be interpreted within the concept of a disease family, and we propose that the severity of the phenotype is dependent on the location of the altered protein in the biosynthesis chain.

IFN-γ-driven intratumoral microenvironment exhibits superior prognostic effect compared with an IFN-α-driven microenvironment in patients with colon carcinoma.

Interferon (IFN)-α and IFN-γ are cytokines with potent immunomodulating and anti-tumor activities. It is unknown which of the two IFNs may be more potent in the regulation of an anti-tumorigenic response in colorectal carcinoma or whether both cytokines cooperate. We, therefore, established human myxovirus resistance protein A and human guanylate-binding protein-1 as markers for the differential detection of IFN-α- and IFN-γ-driven tumor micromilieus, respectively. In vitro studies with different cultures of tumor cells from colorectal carcinoma and stroma cells showed that the expression of myxovirus resistance protein A was exclusively induced by IFN-α, whereas guanylate-binding protein-1 was strongly induced by IFN-γ and only weakly by IFN-α. This expression pattern was used to distinguish cell activation caused by the two cytokines in a clinical cohort of patients with colon carcinoma (n = 378). Patients with primary tumors expressing only guanylate-binding protein-1 exhibited the highest cancer-specific 5-year survival (94.0%, P = 0.006) compared with those expressing both factors (90.3%, P = 0.006), myxovirus resistance protein A alone (83.5%, P = 0.096), or none (72.8%). Our study describes a successful proof-of-principle approach that complex cytokine interaction networks can be dissected in human tissues and demonstrates that an IFN-γ-driven tumor microenvironment exhibits a superior prognostic effect compared with an IFN-α-driven tumor microenvironment in colon carcinoma.

CD19 Chimeric Antigen Receptor T Cell Treatment: Unraveling the Role of B Cells in Systemic Lupus Erythematosus.

B cell generation of autoantibodies is a crucial step in the pathogenesis of systemic lupus erythematosus (SLE). After their differentiation in the bone marrow, B cells populate the secondary lymphatic organs, where they undergo further maturation leading to the development of memory B cells as well as antibody-producing plasmablasts and plasma cells. Targeting B cells is an important strategy to treat autoimmune diseases such as SLE, in which B cell tolerance is disturbed and autoimmune B cells and autoantibodies emerge. This review discusses the functional aspects of antibody- and cell-based B cell-depleting therapy in SLE. It thereby particularly focuses on lessons learned from chimeric antigen receptor (CAR) T cell treatment on the role of B cells in SLE for understanding B cell pathology in SLE. CAR T cells model a deep B cell depletion and thereby allow understanding the role of aberrant B cell activation in the pathogenesis of SLE. Furthermore, the effects of B cell depletion on autoantibody production can be better described, ie, explaining the concept of different cellular sources of (auto-) antibodies in the form of short-lived plasmablasts and long-lived plasma cells, which differ in their susceptibility to B cell depletion and require different targeted therapeutic approaches. Finally, the safety of deep B cell depletion in autoimmune disease is discussed.