Unwanted alloimmune responses are a central driver of solid organ transplant rejection and currently managed with life-long immunosuppression, which imposes substantial risks and burdens on patients. Adoptive transfer of regulatory T cells (Tregs) offers a strategy to restore immunological balance and reduce long-term adverse effects of generalized immunosuppression. However, although Tregs can potently inhibit incipient immune activation, they struggle to suppress established memory effector T cells, necessitating the continued use of immunosuppression. Calcineurin inhibitors such as Tacrolimus effectively control both, newly activated and pre-existing effector T cells, but unfortunately, they also impair Treg function. Therefore, we hypothesized that gene-editing of Tregs inducing tacrolimus resistance (FKBP12KO) would enable combined therapy that curbs effector T cell responses without compromising Treg efficacy. Here, we developed FKBP12KO-Tregs using a ribonucleoprotein-based CRISPR-Cas9 approach and characterized them extensively in vitro. FKBP12KO-Tregs retained phenotype, high viability, and suppressive function comparable to unedited TregWT, and they remained functionally impervious to Tacrolimus, while preserving sensitivity to alternative CNIs. We additionally established a good-manufacturing practice process for FKBP12KO-Tregs. Comprehensive in vitro phenotypic, functional, and molecular characterization, together with the established manufacturing, provide the rationale for a proof-of-concept clinical trial assessing the feasibility and safety of co-administration of FKBP12KO-Tregs with Tacrolimus in living-donor kidney transplant recipients.

Class II histone deacetylases (HDAC) orchestrate T cell-dependent immune responses via the epigenetic control of genes and via the post-translational modification of cytoplasmic and nuclear proteins. However, the contribution of single HDAC family members to the differentiation and function of peripheral CD8+ T cells remains elusive. We here demonstrate that HDAC7-deficiency leads to the upregulation of immune checkpoint molecules, increased apoptosis and disturbed glutamine homeostasis of peripheral murine CD8+ T cells, which we could link to a MEF2D-dependent induction of FasL expression ultimately deterring the survival of HDAC7-deficient CD8+ T cells. Likewise, we observed in mouse models of lymphoma, that mice with a T cell-specific deletion of Hdac7 harbor impaired anti-tumor immune responses in syngeneic transfer models of lymphoma and we found that HDAC7 is required for CD8+ T cell-dependent memory recall responses in models of lymphocytic choriomeningitis virus infection. Taken together, we identify HDAC7 as a central regulator of cellular exhaustion and apoptosis of peripheral CD8+ T cells, controlling CD8+ T cell dependent anti-tumor and anti-viral immunity in mice.

Although CD8+ regulatory T cells (Tregs) were described in the 1970's, they remain poorly defined compared to CD4+ Tregs. Their phenotypic heterogeneity and lack of consensus markers have hindered mechanistic studies and slowed clinical development despite their therapeutic potential. In this study, we performed single-cell RNA sequencing coupled with CITE-seq and TCR-seq on peripheral blood CD8+ T cells from four healthy donors, including the CD45RC marker to distinguish pro-inflammatory from pro-regulatory subsets. We analyzed ∼7000 freshly isolated, non-stimulated CD8+ T cells and identified two distincts CD8+ Tregs subsets, defined by HELIOS or TNFR2 expression, with unique transcriptional and surface marker profiles. Functional assays revealed potent suppressive capacity of the TNFR2+CD29lowCD45RClow/- subset. These findings were independently validated using a publicly available single-cell dataset from four additional individuals. This work provides the most comprehensive profiling of human CD8+ Tregs to date and supports their translation to clinical application.

Background: Unwanted immune responses play a central role in the pathogenesis of solid organ allograft rejection. These are managed by life-long immunosuppression with considerable burden for the patient and society. Adoptive therapy with regulatory T-cells (Treg) is a promising approach to restore sustainable immune balance and avoid long-term adverse effects of immunosuppression. While Treg effectively inhibit activation of unwanted immune responses, they are less effective in controlling pre-existing/activated memory effector T-cells (Teff). Thus, co-administration of Treg with immunosuppressants is required to achieve a sustainable organ acceptance. Calcineurin inhibitors (CNI) are powerful in controlling de novo generated and preformed Teff. However, CNI also dampen Treg immunoregulatory function. Thus, we hypothesize improved results of adoptive Treg therapy in immunosuppressed patients applying tacrolimus-resistant Treg. Methods: While retaining CNI modulation of Teff with tacrolimus, we knocked-out FKBP12 in Treg (FKBP12KO-Treg) by gene-editing using ribonucleoprotein-based CRISPR/Cas9 technology to generate tacrolimus-resistant Treg and characterised them using flow cytometry, functional assays and in-depth phenotyping. Results: This detailed in vitro analysis showed FKBP12KO-Treg were comparable to non-gene edited Treg and impervious to tacrolimus while maintaining immunoregulatory function and sensitivity to alternative CNIs raising no safety concerns. Furthermore, we aligned our methodology to achieve GMP compliance laying the basis for a manufacturing license in preparation of a clinical trial. Conclusion: Based on the presented preclinical dataset implying safety and efficacy of FKBP12KO-Treg, we are now seeking to undertake a proof-of-concept clinical trial to evaluate the co-administration of FKBP12KO-Treg and tacrolimus to enhance the management of living donor kidney transplant recipients. ### Competing Interest Statement The authors declare that the research was conducted as part of a collaboration agreement between Charite-Universitaetsmedizin Berlin and Integrated DNA Technologies (IDT), IDT provided certain reagents and performed experiments. R.T., B.T., M.L.S., G.L.K., and A.M.J. are employees of IDT, which offers reagents for sale similar to some of the compounds described in the manuscript. Products and tools supplied by IDT are for research use only. Purchaser and/or user are solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. PR, HDV, DLW, MSH and LA hold a patent for immunosuppressant-resistant T-cells for adoptive immunotherapy (PCT/EP2021/072651), T.C. is an inventor of CAST-Seq (patent US11319580B2). PR, HDV and DLW founded the startup company TCbalance, which licensed the Treg part of PCT/EP2021/072651 from Charite-Universitaetsmedizin Berlin. European Union, https://ror.org/019w4f821, 825392, 101057438

Although CD8+ regulatory T cells (Tregs) were described in the 1970's, they remain poorly defined compared to CD4+ Tregs. Their phenotypic heterogeneity and lack of consensus markers have hindered mechanistic studies and slowed clinical development despite their therapeutic potential. In this study, we performed single-cell RNA sequencing coupled with CITE-seq and TCR-seq on peripheral blood CD8+ T cells from four healthy donors, including the CD45RC marker to distinguish pro-inflammatory from pro-regulatory subsets. We analyzed similar to 7000 freshly isolated, non-stimulated CD8+ T cells and identified two distincts CD8+ Tregs subsets, defined by HELIOS or TNFR2 expression, with unique transcriptional and surface marker profiles. Functional assays revealed potent suppressive capacity of the TNFR2+CD29lowCD45RClow/- subset. These findings were independently validated using a publicly available single-cell dataset from four additional individuals. This work provides the most comprehensive profiling of human CD8+ Tregs to date and supports their translation to clinical application.

Stage 5 chronic kidney disease (CKD) patients are at higher risk of various infections and malignancies due to uremia-associated alterations of the immune system. Little is known about other factors affecting peripheral blood immune cell compartment (PBICC) composition in this patient population. This cross-sectional study included 107 kidney transplantation candidates (CKD5 patients) and 29 healthy controls. B cell, T cell, and dendritic cell populations were measured using a standardized and validated flow cytometry panel of The ONE study. CKD patients exhibited B and T cell lymphopenia with an immunosenescent phenotype predominantly in the CD4+ compartment and significantly higher counts of APCs. Multivariable PERMANOVAs identified smoking (R2 = 0.041, P < 0.001), CMV seropositivity (R2 = 0.05, P < 0001), age (R2 = 0.02, P = 0.03), past dialysis treatment (R = 0.035, P = 0.019), and presence of atherosclerotic cardiovascular disease (R2 = 0.02, P = 0.03) as factors associated with the changes in PBICC composition. Poisson regression models revealed that smoking was associated mainly with increase in switched memory B cells (RR = 2,4, adj.P = 0.18), CMV seropositivity with increase in CD4+ (RR = 5.8, adj.P < 0.001) and CD8+ (RR = 3, adj.P < 0.001) TEMRA cells, age with decrease in naive CD8+ T-cells (RR = 0.34, adj.P < 0.001), and dialysis with increase in marginal-zone B cells (RR = 2.3, adj.P = 0.007). Factors associated with systemic inflammatory response significantly modify the composition of PBICC in patients with stage 5 CKD. Whether such alterations of immune compartments represent a risk for adverse outcomes needs to be explored further.

CAR T cell (CART) therapy holds promise for cancer treatment, but heterogeneity among products limits clinical effectiveness, making systematic profiling essential to identify predictors of success. Recently, a phase 1 clinical trial investigated whether a constitutively active IL-7 receptor (C7R) could safely improve the function and persistence of GD2-directed CARTs (GD2.CARTs) in pediatric patients with high-grade CNS tumors. We analyzed infusion products from trial participants using a custom-designed 33-color full spectrum flow cytometry (FSFC) panel combined with an image-based tumor killing assay to characterize CART products and evaluate the impact of C7R on GD2.CART performance. Patient-specific variations in T cell composition were linked to therapeutic success, with C7R co-expression enhancing the functional phenotype of GD2.CARTs compared to CAR-only products. Unsupervised clustering identified CD8+ T cells associated with clinical responses, marked by activation, infiltration, resilience, and cytotoxicity. Our FSFC-based profiling approach reveals determinants of CART efficacy and supports strategies to optimize adoptive immunotherapy.

T cells and their effector functions, in particular the canonical cytotoxicity of CD8+ T cells involving perforin, granzymes, Fas ligand (FasL), and tumor necrosis factor related apoptosis inducing ligand (TRAIL), are crucial for tumor immunity. Here, we reveal a previously unidentified mechanism by which CD40L-expressing CD8+ T cells induce cytotoxicity in cancer cells. In murine models, up to 50% of tumor-specific CD8+ T cells expressed CD40L, and conditional CD40L ablation in CD8+ T cells alone led to tumor formation. Mechanistically, CD40L+CD8+ T cells can induce cell death in CD40-expressing cancer cells by triggering caspase-8 activation. We demonstrate that a gene signature for resistance to CD40 signaling-induced cell death strongly correlates with worse survival in different human cancer cohorts. Our results introduce CD40L as a rather counterintuitive, noncanonical cytotoxic factor that complements the capabilities of CD8+ T cells to combat cancers and has the potential to enhance the efficacy of immunotherapies.

BackgroundStage 5 chronic kidney disease (CKD5) is linked to complex yet not fully understood disturbances in immune system. This study aimed to investigate these disturbances by exploring the detailed composition of peripheral blood immune cell compartments in CKD5 patients and to provide integrative, multivariable dissection of how common inflammatory risk factors shape the immune landscape.MethodsThis cross-sectional study included 107 patients with chronic kidney disease stage 5 (CKD5) and 29 healthy blood donors as controls. Peripheral blood B cells, T cells, and dendritic cells were measured using a standardized and validated flow cytometry panel. The impact of selected clinical factors on immune cell composition was initially evaluated using a robust multivariate method (PERMANOVA). Variables that significantly affected immune cell composition were then included in a subsequent series of Poisson regression models, assessing predictors influence on the counts of individual immune cell subpopulations.ResultsCompared to healthy controls, CKD5 patients presented with B cell lymphopenia across all measured subsets except for plasmablasts, T cell lymphopenia with an immunosenescent phenotype predominantly in the CD4+ compartment, and significantly higher counts of LIN-HLA-DR+ antigen-presenting cells, mainly due to an increase in myeloid dendritic cell subpopulations. PERMANOVA identified smoking, CMV seropositivity, age, dialysis treatment, and atherosclerotic cardiovascular disease as factors significantly influencing peripheral blood immune composition. Subsequent Poisson regression models revealed that smoking was associated mainly with an increase in switched memory B cells, CMV seropositivity with an increase in CD4+ and CD8+ TEMRA cells, age with a decrease in naive CD8+ T cells, and dialysis treatment with an increase in marginal-zone B cells.ConclusionsPatients with CKD5 exhibit distinct composition of peripheral blood immune cells, further modified by other factors associated with systemic inflammatory response. These factors should be considered in immunomonitoring protocols and may enhance prediction of clinical outcomes such as vaccine responses.

IntroductionWomen with a history of gestational diabetes mellitus (GDM) are at high risk of developing prediabetes or type 2 diabetes later in life. Recent studies have highlighted the regulation and function of innate lymphoid cells (ILCs) in metabolic homeostasis. However, the multifactorial impact of both overweight/obesity and GDM on the immunological profile of circulating ILCs and the progression to prediabetes are not yet fully elucidated.MethodsBlood samples from 42 women with a history of insulin-treated GDM (GDMi), 33 women with a history of GDM without insulin treatment during pregnancy (GDM), and 45 women after a normoglycemic pregnancy (Ctrl) participating in the ongoing observational PPSDiab study were analyzed by flow cytometry for markers of ILC subsets at the baseline visit (3-16 months postpartum; Visit 1) and 5 years postpartum (58-66 months postpartum; Visit 2).ResultsDuring the first 5 years postpartum, 18 women of the GDMi group (42.8%), 10 women of the GDM group (30.3%), and 8 participants of the Ctrl group (17.8%) developed prediabetes, respectively. Total circulating type 1 innate lymphoid cells (ILC1s) and NK cell numbers as well as percent HLA-DR+ ILC1s were increased in GDMi versus GDM and Ctrl women both at the baseline visit and the 5-year follow-up. Although ILC subsets at Visit 1 could not predict the progression from GDM to prediabetes, ILC2 frequency was associated with insulin sensitivity index (ISI), whereas percent HLA-DR+ ILC1s were inversely correlated. Moreover, circulating leukocytes and total NK cells were associated with waist circumference and fat mass both at Visit 1 and Visit 2.DiscussionOur findings introduce human ILCs as a potential therapeutic target deserving further exploration.Trial registrationStudy ID 300-11.

Regulatory T cells (T reg cells) hold promise for sustainable therapy of immune disorders. Recent advancements in chimeric antigen receptor development and genome editing aim to enhance the specificity and function of T reg cells. However, impurities and functional instability pose challenges for the development of safe gene-edited T reg cell products. Here, we examined different T reg cell subsets regarding their fate, epigenomic stability, transcriptomes, T cell receptor repertoires, and function ex vivo and after manufacturing. Each T reg cell subset displayed distinct features, including lineage stability, epigenomics, surface markers, T cell receptor diversity, and transcriptomics. Earlier-differentiated memory T reg cell populations, including a hitherto unidentified naïve-like memory T reg cell subset, outperformed late-differentiated effector memory–like T reg cells in regulatory function, proliferative capacity, and epigenomic stability. High yields of stable, functional T reg cell products could be achieved by depleting the small effector memory–like T reg cell subset before manufacturing. Considering T reg cell subset composition appears critical to maintain lineage stability in the final cell product.

Plasma cells (PCs) in bone marrow (BM) play an important role in both protective and pathogenic humoral immune responses, e.g. in various malignant and non-malignant diseases such as multiple myeloma, primary and secondary immunodeficiencies and autoimmune diseases. Dedicated microenvironmental niches in the BM provide PCs with biomechanical and soluble factors that support their long-term survival. There is a high need for appropriate and robust model systems to better understand PCs biology, to develop new therapeutic strategies for PCs-related diseases and perform targeted preclinical studies with high predictive value. Most preclinical data have been derived from in vivo studies in mice, as in vitro studies of human PCs are limited due to restricted survival and functionality in conventional 2D cultures that do not reflect the unique niche architecture of the BM. We have developed a microphysiological, dynamic 3D BM culture system (BM-MPS) based on human primary tissue (femoral biopsies), mechanically supported by a hydrogel scaffold casing. While a bioinert agarose casing did not support PCs survival, a photo-crosslinked collagen-hyaluronic acid (Col-HA) hydrogel preserved the native BM niche architecture and allowed PCs survival in vitro for up to 2 weeks. Further, the Col-HA hydrogel was permissive to lymphocyte migration into the microphysiological system´s circulation. Long-term PCs survival was related to the stable presence in the culture of soluble factors, as APRIL, BAFF, and IL-6. Increasing immunoglobulins concentrations in the medium confirm their functionality over culture time. To the best of our knowledge, this study is the first report of successful long-term maintenance of primary-derived non-malignant PCs in vitro . Our innovative model system is suitable for in-depth in vitro studies of human PCs regulation and exploration of targeted therapeutic approaches such as CAR-T cell therapy or biologics.

Phenotypic and compositional changes of immune cells in cerebrospinal fluid (CSF) can be used as biomarkers to help diagnose and track disease activity for neuroinflammatory and neurodegenerative diseases. Here, we describe an end-to-end workflow to perform high-dimensional immune profiling at single-cell resolution using Cytometry by Time-of-Flight (CyTOF) on cells isolated from the CSF of patients with neuroinflammation. We include protocols for sample collection and preparation, barcoding to allow for multiplexing, and downstream data analysis using R. For complete details on the use and execution of this protocol, please refer to Fernández-Zapata, C. et al1.

Background Innate lymphoid cells (ILCs) are key organizers of tissue immune responses and regulate tissue development, repair, and pathology. Persistent clinical sequelae beyond 12 weeks following acute COVID-19 disease, named post-COVID syndrome (PCS), are increasingly recognized in convalescent individuals. ILCs have been associated with the severity of COVID-19 symptoms but their role in the development of PCS remains poorly defined. Methods and results Here, we used multiparametric immune phenotyping, finding expanded circulating ILC precursors (ILCPs) and concurrent decreased group 2 innate lymphoid cells (ILC2s) in PCS patients compared to well-matched convalescent control groups at > 3 months after infection or healthy controls. Patients with PCS showed elevated expression of chemokines and cytokines associated with trafficking of immune cells (CCL19/MIP-3b, FLT3-ligand), endothelial inflammation and repair (CXCL1, EGF, RANTES, IL-1RA, PDGF-AA). Conclusion These results define immunological parameters associated with PCS and might help find biomarkers and disease-relevant therapeutic strategies.

Phenotypic and compositional changes of immune cells in cerebrospinal fluid (CSF) can be used as biomarkers to help diagnose and track disease activity for neuroinflammatory and neurodegenerative diseases. Here, we present a workflow to perform high-dimensional immune profiling at single-cell resolution using cytometry by time-of-flight (CyTOF) on cells isolated from the CSF of patients with neuroinflammation. We describe steps for sample collection and preparation, barcoding to allow for multiplexing, and downstream data analysis using R.For complete details on the use and execution of this protocol, please refer to Fernández-Zapata et al.1

Aims/hypothesis: Innate lymphoid cells (ILCs) represent a growing family of immune cells with diverse roles in tissue homeostasis and the initiation of inflammatory responses. Recent studies have highlighted the regulation and function of group 2 innate lymphoid cells (ILC2s) in metabolic homeostasis. Women with a history of gestational diabetes mellitus (GDM) are at high risk of developing and type 2 diabetes. However, it is still unclear whether circulating ILCs are dysregulated in patients with GDM and, thus, might contribute to insulin resistance.

Beyond SARS-CoV2 vaccines, mRNA drugs are being explored to overcome today's greatest healthcare burdens, including cancer and cardiovascular disease. Synthetic mRNA triggers immune responses in transfected cells, which can be reduced by chemically modified nucleotides. However, the side effects of mRNA-triggered immune activation on cell function and how different nucleotides, such as the N1-methylpseudouridine (m1Ψ) used in SARS-CoV2 vaccines, can modulate cellular responses is not fully understood. Here, cellular responses toward a library of uridine-modified mRNAs are investigated in primary human cells. Targeted proteomics analyses reveal that unmodified mRNA induces a pro-inflammatory paracrine pattern marked by the secretion of chemokines, which recruit T and B lymphocytes toward transfected cells. Importantly, the magnitude of mRNA-induced changes in cell function varies quantitatively between unmodified, Ψ-, m1Ψ-, and 5moU-modified mRNA and can be gradually tailored, with implications for deliberately exploiting this effect in mRNA drug design. Indeed, both the immunosuppressive effect of stromal cells on T-cell proliferation, and the anti-inflammatory effect of IL-10 mRNA are enhanced by appropriate uridine modification. The results provide new insights into the effects of mRNA drugs on cell function and cell-cell communication and open new possibilities to tailor mRNA-triggered immune activation to the desired pro- or anti-inflammatory application.