From stem cells to regulatory T cells: A tale of plasticity.

In this issue, Yano et al.1 present a method to obtain suppressive regulatory T (Treg) cells from human induced pluripotent stem cells (hiPSCs). This approach has the potential to address the low Treg cell yields of current ex vivo Treg cell expansion and induction protocols, an unmet challenge for autologous Treg cell treatments.

Harnessing Immune Cell Metabolism to Modulate Alloresponse in Transplantation.

Immune cell metabolism plays a pivotal role in shaping and modulating immune responses. The metabolic state of immune cells influences their development, activation, differentiation, and overall function, impacting both innate and adaptive immunity. While glycolysis is crucial for activation and effector function of CD8 T cells, regulatory T cells mainly use oxidative phosphorylation and fatty acid oxidation, highlighting how different metabolic programs shape immune cells. Modification of cell metabolism may provide new therapeutic approaches to prevent rejection and avoid immunosuppressive toxicities. In particular, the distinct metabolic patterns of effector and suppressive cell subsets offer promising opportunities to target metabolic pathways that influence immune responses and graft outcomes. Herein, we review the main metabolic pathways used by immune cells, the techniques available to assay immune metabolism, and evidence supporting the possibility of shifting the immune response towards a tolerogenic profile by modifying energetic metabolism.

T-cell receptor sequencing reveals selected donor-reactive CD8+ T cell clones resist antithymocyte globulin depletion after kidney transplantation.

High frequencies of donor-reactive memory T cells in the periphery of transplant candidates prior to transplantation are linked to the development of posttransplant acute rejection episodes and reduced allograft function. Rabbit antithymocyte globulin (rATG) effectively depletes naïve CD4+ and CD8+ T cells for >6 months posttransplant, but rATG's effects on human donor-reactive T cells have not been carefully determined. To address this, we performed T cell receptor ß-chain sequencing on peripheral blood mononuclear cells aliquots collected pretransplant and serially posttransplant in 7 kidney transplant recipients who received rATG as induction therapy. We tracked the evolution of the donor-reactive CD4+ and CD8+ T cell repertoires and identified stimulated pretransplant, CTV-(surface dye)-labeled, peripheral blood mononuclear cells from each patient with donor cells or third-party cells. Our analyses showed that while rATG depleted CD4+ T cells in all tested subjects, a subset of donor-reactive CD8+ T cells that were present at high frequencies pretransplant, consistent with expanded memory cells, resisted rATG depletion, underwent posttransplant expansion and were functional. Together, our data support the conclusion that a subset of human memory CD8+ T cells specifically reactive to donor antigens expand in vivo despite induction therapy with rATG and thus have the potential to mediate allograft damage.

Small-molecule TIP60 inhibitors enhance regulatory T cell induction through TIP60-P300 acetylation crosstalk.

Foxp3 acetylation is essential to regulatory T (Treg) cell stability and function, but pharmacologically increasing it remains an unmet challenge. Here, we report that small-molecule compounds that inhibit TIP60, an acetyltransferase known to acetylate Foxp3, unexpectedly increase Foxp3 acetylation and Treg induction. Utilizing a dual experimental/computational approach combined with a newly developed FRET-based methodology compatible with flow cytometry to measure Foxp3 acetylation, we unraveled the mechanism of action of these small-molecule compounds in murine and human Treg induction cell cultures. We demonstrate that at low-mid concentrations they activate TIP60 to acetylate P300, a different acetyltransferase, which in turn increases Foxp3 acetylation, thereby enhancing Treg cell induction. These results reveal a potential therapeutic target relevant to autoimmunity and transplant.

Understanding the heterogeneity of alloreactive natural killer cell function in kidney transplantation.

Human Natural Killer (NK) cells are heterogeneous lymphocytes regulated by variegated arrays of germline-encoded activating and inhibitory receptors. They acquire the ability to detect polymorphic self-antigen via NKG2A/HLA-E or KIR/HLA-I ligand interactions through an education process. Correlations among HLA/KIR genes, kidney transplantation pathology and outcomes suggest that NK cells participate in allograft injury, but mechanisms linking NK HLA/KIR education to antibody-independent pathological functions remain unclear. We used CyTOF to characterize pre- and post-transplant peripheral blood NK cell phenotypes/functions before and after stimulation with allogeneic donor cells. Unsupervised clustering identified unique NK cell subpopulations present in varying proportions across patients, each of which responded heterogeneously to donor cells based on donor ligand expression patterns. Analyses of pre-transplant blood showed that educated, NKG2A/KIR-expressing NK cells responded greater than non-educated subsets to donor stimulators, and this heightened alloreactivity persisted > 6 months post-transplant despite immunosuppression. In distinct test and validation sets of patients participating in two clinical trials, pre-transplant donor-induced release of NK cell Ksp37, a cytotoxicity mediator, correlated with 2-year and 5-year eGFR. The findings explain previously reported associations between NK cell genotypes and transplant outcomes and suggest that pre-transplant NK cell analysis could function as a risk-assessment biomarker for transplant outcomes.

TACI and endogenous APRIL in B cell maturation.

While many of the genes and molecular pathways in the germinal center B cell response which initiate protective antibody production are known, the contributions of individual molecular players in terminal B cell differentiation remain unclear. We have previously investigated how mutations in TACI gene, noted in about 10% of patients with common variable immunodeficiency, impair B cell differentiation and often, lead to lymphoid hyperplasia and autoimmunity. Unlike mouse B cells, human B cells express TACI-L (Long) and TACI-S (Short) isoforms, but only TACI-S promotes terminal B cell differentiation into plasma cells. Here we show that the expression of intracellular TACI-S increases with B cell activation, and colocalizes with BCMA and their ligand, APRIL. We show that the loss of APRIL impairs isotype class switch and leads to distinct metabolic and transcriptional changes. Our studies suggest that intracellular TACI-S and APRIL along with BCMA direct long-term PC differentiation and survival.

Persistent SARS-CoV-2-specific immune defects in kidney transplant recipients following third mRNA vaccine dose.

Kidney transplant recipients (KTRs) show poorer response to SARS-CoV-2 mRNA vaccination, yet response patterns and mechanistic drivers following third doses are ill-defined. We administered third monovalent mRNA vaccines to n = 81 KTRs with negative or low-titer anti-receptor binding domain (RBD) antibody (n = 39 anti-RBDNEG; n = 42 anti-RBDLO), compared with healthy controls (HCs, n = 19), measuring anti-RBD, Omicron neutralization, spike-specific CD8+%, and SARS-CoV-2-reactive T cell receptor (TCR) repertoires. By day 30, 44% anti-RBDNEG remained seronegative; 5% KTRs developed BA.5 neutralization (vs 68% HCs, P < .001). Day 30 spike-specific CD8+% was negative in 91% KTRs (vs 20% HCs; P = .07), without correlation to anti-RBD (rs = 0.17). Day 30 SARS-CoV-2-reactive TCR repertoires were detected in 52% KTRs vs 74% HCs (P = .11). Spike-specific CD4+ TCR expansion was similar between KTRs and HCs, yet KTR CD8+ TCR depth was 7.6-fold lower (P = .001). Global negative response was seen in 7% KTRs, associated with high-dose MMF (P = .037); 44% showed global positive response. Of the KTRs, 16% experienced breakthrough infections, with 2 hospitalizations; prebreakthrough variant neutralization was poor. Absent neutralizing and CD8+ responses in KTRs indicate vulnerability to COVID-19 despite 3-dose mRNA vaccination. Lack of neutralization despite CD4+ expansion suggests B cell dysfunction and/or ineffective T cell help. Development of more effective KTR vaccine strategies is critical. (NCT04969263).

Interferon-ß acts directly on T cells to prolong allograft survival by enhancing regulatory T cell induction through Foxp3 acetylation.

Type I interferons (IFNs) are pleiotropic cytokines with potent antiviral properties that also promote protective T cell and humoral immunity. Paradoxically, type I IFNs, including the widely expressed IFNß, also have immunosuppressive properties, including promoting persistent viral infections and treating T-cell-driven, remitting-relapsing multiple sclerosis. Although associative evidence suggests that IFNß mediates these immunosuppressive effects by impacting regulatory T (Treg) cells, mechanistic links remain elusive. Here, we found that IFNß enhanced graft survival in a Treg-cell-dependent murine transplant model. Genetic conditional deletion models revealed that the extended allograft survival was Treg cell-mediated and required IFNß signaling on T cells. Using an in silico computational model and analysis of human immune cells, we found that IFNß directly promoted Treg cell induction via STAT1- and P300-dependent Foxp3 acetylation. These findings identify a mechanistic connection between the immunosuppressive effects of IFNß and Treg cells, with therapeutic implications for transplantation, autoimmunity, and malignancy.

Self-Assembled Lanthanide Antenna Glutathione Sensor for the Study of Immune Cells.

The small molecule 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-carboxylic acid (2b) behaves as a reactive non-fluorescent Michael acceptor, which after reaction with thiols becomes fluorescent, and an efficient Eu3+ antenna, after self-assembling with this cation in water. This behavior makes 2b a highly selective GSH biosensor, which has demonstrated high potential for studies in murine and human cells of the immune system (CD4+ T, CD8+ T, and B cells) using flow cytometry. GSH can be monitored by the fluorescence of the product of addition to 2b (445 nm) or by the luminescence of Eu3+ (592 nm). 2b was able to capture baseline differences in GSH intracellular levels among murine and human CD4+ T, CD8+ T, and B cells. We also successfully used 2b to monitor intracellular changes in GSH associated with the metabolic variations governing the induction of CD4+ naïve T cells into regulatory T cells (TREG).

The spatially resolved transcriptional profile of acute T cell-mediated rejection in a kidney allograft.

Analysis of the transcriptional profile of graft biopsies represents a promising strategy to study T cell-mediated-rejection (TCMR), also known as acute cellular rejection. However, bulk RNA sequencing of graft biopsies may not capture the focal nature of acute rejection. Herein, we used the whole exome GeoMX Digital Space Profiling platform to study five tubular and three glomerular regions of interest in the kidney graft biopsy from a patient with a chronic-active TCMR episode and in analogous areas from two different normal kidney control biopsies. All kidney sections were from paraffin blocks. Overall, inflammatory genes were significantly upregulated in the tubular areas of the TCMR biopsy and showed an enrichment for gene-ontology terms associated with T-cell activation, differentiation, and proliferation. Enrichment analysis of the 100 genes with the highest coefficient of variation across the TCMR tubular regions of interest revealed that these highly variable genes are involved in kidney development and injury and interestingly do not associate with the 2019 Banff classification pathology scores within the individual regions of interest. Spatial transcriptomics allowed us to unravel a previously unappreciated variability across different areas of the TCMR biopsy related to the graft response to the alloimmune attack, rather than to the immune cells. Thus, our approach has the potential to decipher clinically relevant, new pathogenic mechanisms, and therapeutic targets in acute cellular rejection and other kidney diseases with a focal nature.

T-cell Exhaustion in Organ Transplantation.

Exhaustion of T cells occurs in response to long-term exposure to self and foreign antigens. It limits T cell capacity to proliferate and produce cytokines, leading to an impaired ability to clear chronic infections or eradicate tumors. T-cell exhaustion is associated with a specific transcriptional, epigenetic, and metabolic program and characteristic cell surface markers' expression. Recent studies have begun to elucidate the role of T-cell exhaustion in transplant. Higher levels of exhausted T cells have been associated with better graft function in kidney transplant recipients. In contrast, reinvigorating exhausted T cells by immune checkpoint blockade therapies, while promoting tumor clearance, increases the risk of acute rejection. Lymphocyte depletion and high alloantigen load have been identified as major drivers of T-cell exhaustion. This could account, at least in part, for the reduced rates of acute rejection in organ transplant recipients induced with thymoglobulin and for the pro-tolerogenic effects of a large organ such as the liver. Among the drugs that are widely used for maintenance immunosuppression, calcineurin inhibitors have a contrasting inhibitory effect on exhaustion of T cells, while the influence of mTOR inhibitors is still unclear. Harnessing or encouraging the natural processes of exhaustion may provide a novel strategy to promote graft survival and transplantation tolerance.

CyTOF-Enabled Analysis Identifies Class-Switched B Cells as the Main Lymphocyte Subset Associated With Disease Relapse in Children With Idiopathic Nephrotic Syndrome.

B cell depleting therapies permit immunosuppressive drug withdrawal and maintain remission in patients with frequently relapsing nephrotic syndrome (FRNS) or steroid-dependent nephrotic syndrome (SDNS), but lack of biomarkers for treatment failure. Post-depletion immune cell reconstitution may identify relapsing patients, but previous characterizations suffered from methodological limitations of flow cytometry. Time-of-flight mass cytometry (CyTOF) is a comprehensive analytic modality that simultaneously quantifies over 40 cellular markers. Herein, we report CyTOF-enabled immune cell comparisons over a 12-month period from 30 children with SDNS receiving B cell depleting therapy who either relapsed (n = 17) or remained stable (n = 13). Anti-CD20 treatment depleted all B cells subsets and CD20 depleting agent choice (rituximab vs ofatumumab) did not affect B cell subset recovery. Despite equal total numbers of B cells, 5 subsets of B cells were significantly higher in relapsing individuals; all identified subsets of B cells were class-switched. T cell subsets (including T follicular helper cells and regulatory T cells) and other major immune compartments were largely unaffected by B cell depletion, and similar between relapsing and stable children. In conclusion, CyTOF analysis of immune cells from anti-CD20 antibody treated patients identifies class-switched B cells as the main subset whose expansion associates with disease relapse. Our findings set the basis for future studies exploring how identified subsets can be used to monitor treatment response and improve our understanding of the pathogenesis of the disease.

Mitochondrial localization and moderated activity are key to murine erythroid enucleation.

Mammalian red blood cells (RBCs), which primarily contain hemoglobin, exemplify an elaborate maturation process, with the terminal steps of RBC generation involving extensive cellular remodeling. This encompasses alterations of cellular content through distinct stages of erythroblast maturation that result in the expulsion of the nucleus (enucleation) followed by the loss of mitochondria and all other organelles and a transition to anaerobic glycolysis. Whether there is any link between erythroid removal of the nucleus and the function of any other organelle, including mitochondria, remains unknown. Here we demonstrate that mitochondria are key to nuclear clearance. Using live and confocal microscopy and high-throughput single-cell imaging, we show that before nuclear polarization, mitochondria progressively move toward one side of maturing erythroblasts and aggregate near the nucleus as it extrudes from the cell, a prerequisite for enucleation to proceed. Although we found active mitochondrial respiration is required for nuclear expulsion, levels of mitochondrial activity identify distinct functional subpopulations, because terminally maturing erythroblasts with low relative to high mitochondrial membrane potential are at a later stage of maturation, contain greatly condensed nuclei with reduced open chromatin-associated acetylation histone marks, and exhibit higher enucleation rates. Lastly, to our surprise, we found that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation, primarily through pyruvate but independent of in situ glycolysis. These findings demonstrate the critical but unanticipated functions of mitochondria during the erythroblast enucleation process. They are also relevant to the in vitro production of RBCs as well as to disorders of the erythroid lineage.

Erythropoietin administration expands regulatory T cells in patients with autoimmune hepatitis.

OBJECTIVE: Autoimmune hepatitis (AIH) is a chronic liver disease associated with impaired regulatory T cell (Treg) number and function. Erythropoietin (EPO) is a kidney-produced erythropoietic hormone that has known immune-modulating effects, including Treg induction. Whether EPO administration increases Treg in patients with AIH is unknown. METHODS: We treated six stable AIH patients with a single 1000 IU dose of EPO and comprehensively characterized changes in Treg overall and in Treg subsets before and at 4 and 12 weeks after treatment using mass cytometry (CyTOF) combined with an unbiased clustering approach (Phenograph) based on 22 Treg-relevant cell-surface markers. RESULTS: EPO was well-tolerated and no patients showed significant changes in hematological parameters, liver enzymes, or IgG levels from baseline to 12 weeks following EPO administration. Total Treg and Treg/CD8+ T cell ratios significantly increased at 4 weeks and returned to baseline levels at 12 weeks after EPO injection. We identified 17 Treg subsets of which CD4+CD25HICD127NEG HLADR+ Treg had the highest increase and the most favorable Treg/CD8+ ratio upon EPO treatment. At 12 weeks after EPO administration, the HLADR+ Treg subset also returned to values comparable to those at baseline. Ex vivo assays documented that Treg were functional and the ones isolated at 12 weeks after EPO injection were significantly more suppressive than the ones isolated at baseline. In Treg-depleted assays, EPO did not show a significant effect on IFN-γ+, IL-2+, and IL-17+ CD4+ T cells. CONCLUSION: In stable AIH patients, EPO increases overall Treg and particularly those expressing the high function marker HLA-DR. These results provide the rationale for future studies testing the hypothesis that EPO or EPO analogues improve outcomes of AIH patients by increasing Treg.

Interclass GPCR heteromerization affects localization and trafficking.

Membrane trafficking processes regulate G protein-coupled receptor (GPCR) activity. Although class A GPCRs are capable of activating G proteins in a monomeric form, they can also potentially assemble into functional GPCR heteromers. Here, we showed that the class A serotonin 5-HT2A receptors (5-HT2ARs) affected the localization and trafficking of class C metabotropic glutamate receptor 2 (mGluR2) through a mechanism that required their assembly as heteromers in mammalian cells. In the absence of agonists, 5-HT2AR was primarily localized within intracellular compartments, and coexpression of 5-HT2AR with mGluR2 increased the intracellular distribution of the otherwise plasma membrane-localized mGluR2. Agonists for either 5-HT2AR or mGluR2 differentially affected trafficking through Rab5-positive endosomes in cells expressing each component of the 5-HT2AR-mGluR2 heterocomplex alone, or together. In addition, overnight pharmacological 5-HT2AR blockade with clozapine, but not with M100907, decreased mGluR2 density through a mechanism that involved heteromerization between 5-HT2AR and mGluR2. Using TAT-tagged peptides and chimeric constructs that are unable to form the interclass 5-HT2AR-mGluR2 complex, we demonstrated that heteromerization was necessary for the 5-HT2AR-dependent effects on mGluR2 subcellular distribution. The expression of 5-HT2AR also augmented intracellular localization of mGluR2 in mouse frontal cortex pyramidal neurons. Together, our data suggest that GPCR heteromerization may itself represent a mechanism of receptor trafficking and sorting.

Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis.

Kidney glomerulosclerosis commonly progresses to end-stage kidney failure, but pathogenic mechanisms are still poorly understood. Here, we show that podocyte expression of decay-accelerating factor (DAF/CD55), a complement C3 convertase regulator, crucially controls disease in murine models of adriamycin (ADR)-induced focal and segmental glomerulosclerosis (FSGS) and streptozotocin (STZ)-induced diabetic glomerulosclerosis. ADR induces enzymatic cleavage of DAF from podocyte surfaces, leading to complement activation. C3 deficiency or prevention of C3a receptor (C3aR) signaling abrogates disease despite DAF deficiency, confirming complement dependence. Mechanistic studies show that C3a/C3aR ligations on podocytes initiate an autocrine IL-1ß/IL-1R1 signaling loop that reduces nephrin expression, causing actin cytoskeleton rearrangement. Uncoupling IL-1ß/IL-1R1 signaling prevents disease, providing a causal link. Glomeruli of patients with FSGS lack DAF and stain positive for C3d, and urinary C3a positively correlates with the degree of proteinuria. Together, our data indicate that the development and progression of glomerulosclerosis involve loss of podocyte DAF, triggering local, complement-dependent, IL-1ß-induced podocyte injury, potentially identifying new therapeutic targets.