Liver transplantation (LT) is the ultimate treatment for patients with end-stage liver disease or early hepatocellular carcinoma. In the context of LT, because of the unique immunological characteristics of human liver allograft, 5%-20% of selected LT recipients can achieve operational tolerance. Nonetheless, there remains a risk of rejection in LT patients. Maintaining immune homeostasis is thus crucial for improving clinical outcomes in these patients. In mechanism, several immune cells, including dendritic cells, Kupffer cells, myeloid-derived suppressor cells, hepatic stellate cells, regulatory B cells, and CD4 + regulatory T cells (Treg), contribute to achieving tolerance following LT. In terms of Treg, it plays a role in successfully minimizing immunosuppression or achieving tolerance post-LT while also reducing the risk of rejection. Furthermore, the gut microbiome modulates systemic immune functions along the gut-liver axis. Recent studies have explored changes in the microbiome and its metabolites under various conditions, including post-LT, acute rejection, and tolerance. Certain functional microbiomes and metabolites exhibit immunomodulatory functions, such as the augmentation of Treg, influencing immune homeostasis. Therefore, understanding the mechanisms of tolerance in LT, the role of Treg in tolerance and rejection, as well as their interactions with gut microbiome, is vital for the management of LT patients.

In solid organ transplantation, especially renal transplantation, for the induction of immune tolerance, accumulating evidence has revealed that Regulatory B cells (Breg) play a crucial role in stimulating immune tolerance, alleviating immune responses, and improving graft survival. We describe the heterogeneous nature of Bregs, focusing on their defining surface markers and regulatory functions. Meanwhile, the major cytokine secretion function and the correlation between Breg and Treg or other immune checkpoints to balance the immune responses are addressed. Furthermore, we summarized the intrinsic and extrinsic pathways or costimulatory stimuli for the differentiation from naïve B cells. More importantly, we summarized the progression of the immune tolerance induction role of Breg in solid organ (kidney, liver, heart, lung, and islet) transplantation. This is an up-to-date review from the origin of Breg to the function of Breg in solid organ transplantation and how it induces immune tolerance in both murine models and human solid organ transplantation.

B cells play a crucial role in kidney transplantation through antibody production and cytokine secretion. To better understand their impact on kidney transplantation, this retrospective study aimed to characterize circulating B-cell phenotypes and cytokine production in a cohort of kidney transplant patients to identify whether pretransplant donor-specific antibodies (DSAs) or biopsy-proven rejection is associated with different B-cell profiles.

Pretransplant cryopreserved peripheral blood mononuclear cells were obtained from 96 kidney transplant recipients, of whom 42 had pretransplant DSAs. The cells underwent surface marker staining using a 33-color spectral flow cytometry panel for B-cell phenotyping. Simultaneously, cells were stimulated for interleukin-10, tumor necrosis factor-α, and interleukin-6 production, and analyzed with a 6-color panel.

Rejection was linked to decreased naive B cells and increased plasmablasts, CD27+ memory B cells, and memory B-cell subsets (all P < 0.04) compared with no rejection. Cytokine-producing B cells and immune regulatory molecule expression showed no significant differences. Multivariate analysis identified resting memory B cells (CD27+CD21+) and pretransplant DSAs as significantly associated with rejection (P = 0.01; odds ratio [OR], 1.07; P = 0.02; OR, 3.10, respectively). Cox regression analysis revealed resting memory B cells were associated with early antibody-mediated rejection (P = 0.04; OR, 1.05).

B-cell subset distributions differed between patients with and without rejection. Resting memory B-cell frequency was associated with increased early antibody-mediated rejection risk, whereas cytokine production and immune checkpoint expression did not influence rejection. The results suggest that B-cell subset composition could aid in rejection risk assessment and serve as a potential pretransplant diagnostic parameter.

T cells are one of the main drivers of inflammatory bowel diseases (IBD). Infliximab (IFX) is used in the treatment of IBD as an anti-inflammatory drug to induce remission by neutralizing TNFα. We determined the individual chemokine/homing receptor and cytokine profile in pediatric IBD patients before and during IFX therapy to identify predictive biomarkers for therapy success. Peripheral blood CD4+ cells from pediatric patients with IBD were immunomagnetically isolated and either directly analyzed by FACS for cell distribution and chemokine/homing receptor expression or evaluated for cytokine production after in-vitro-stimulation. Twenty-one responders (RS) and 21 non-responders (NRS) were recruited. Before IFX therapy, flow cytometry revealed decreased percentages of naïve conventional T cells in pediatric IBD patients. The proportions of CD62-L+ T cells were decreased in both CD and UC therapy responders. The cytokine profile of T cells was highly altered in IBD patients compared to healthy controls (HC). During IFX therapy, the frequencies of conventional memory and regulatory memory T cells expanded in both cohorts. IFX response was marked by a decrease of α4β7+ and IFNγ+ memory T cells in both CD and UC. In contrast, frequencies of Lag-3+ T cells proved to be significantly increased in NRS. These observations were irrespective of the underlying disease. T cells of pediatric IBD patients display an activated and rather Th1/Th17-shifted phenotype. The increased expression of the checkpoint molecule Lag-3 on T cells of NRS resembles a more exhausted phenotype than in RS and HC which appeared to be a relevant predictive marker for therapy failure.

Breast cancer (BC) is the most frequently diagnosed malignancy among women. It is characterized by a high level of heterogeneity that emerges from the interaction of several cellular and soluble components in the tumor microenvironment (TME), such as cytokines, tumor cells and tumor-associated immune cells. Tumor necrosis factor (TNF) receptor 2 (TNFR2) appears to play a significant role in microenvironmental regulation, tumor progression, immune evasion, drug resistance, and metastasis of many types of cancer, including BC. However, the significance of TNFR2 in BC biology is not fully understood. This review provides an overview of TNFR2 biology, detailing its activation and its interactions with important signaling pathways in the TME (e.g., NF-κB, MAPK, and PI3K/Akt pathways). We discuss potential therapeutic strategies targeting TNFR2, with the aim of enhancing the antitumor immune response to BC. This review provides insights into role of TNFR2 as a major immune checkpoint for the future treatment of patients with BC.

Allergen-specific immunotherapy (AIT) induces immune tolerance, showing the highest success rate (>95%) for insect venom while a much lower chance for pollen allergy. However, the molecular switches leading to successful durable tolerance restoration remain elusive. The primary outcome of this observational study is the comprehensive immunological cellular characterization during the AIT initiation phase, whereas the secondary outcomes are the serological and Th2-cell-type-specific transcriptomic analyses. Here we apply a multilayer-omics approach to reveal dynamic peripheral immune landscapes during the AIT-initiation phase in venom allergy patients (VAP) versus pollen-allergic and healthy controls. Already at baseline, VAP exhibit altered abundances of several cell types, including classical monocytes (cMono), CD4+ hybrid type 1-type 17 cells (Th1-Th17 or Th1/17) and CD8+ counterparts (Tc1-Tc17 or Tc1/17). At 8-24 h following AIT launch in VAP, we identify a uniform AIT-elicited pulse of late-transitional/IL-10-producing B cells, IL-6 signaling within Th2 cells and non-inflammatory serum-IL-6 levels. Sequential induction of activation and survival protein markers also immediately occur. A disequilibrium between serum IL-6 and cMono in VAP baseline is restored at day seven following AIT launch. Our longitudinal analysis discovers molecular switches during initiation-phase insect-venom AIT that secure long-term outcomes. Trial number: NCT02931955.

Regulatory B cells (Bregs) are a functionally distinct B-cell subset involved in the maintenance of homeostasis and inhibition of inflammation. Studies, from the last two decades, have increased our understanding of cellular and molecular mechanisms involved in their generation, function, and to a certain extent phenotype. Current research endeavours to unravel the causes and consequences of Breg defects in disease, with increasing evidence highlighting the relevance of Bregs in promoting tumorigenic responses. Here we provide historical and emerging findings of the significance of Bregs in autoimmunity and transplantation, and how these insights have translated into the cancer field.

Pathophysiologic function of B cells in graft rejection has been well recognized in transplantation. B cells promote alloantigen-specific T-cell response and secrete antibodies that can cause antibody-mediated graft failures and rejections. Therefore, strategies targeting B cells, for example, B-cell depletion, have been used for the prevention of both acute and chronic rejections. Interestingly, however, recent mounting evidence indicates that subsets of B cells yet to be further identified can display potent immune regulatory functions, and they contribute to transplantation tolerance and operational tolerance in both experimental and clinical settings, respectively. In this review, we integrate currently available information on B-cell subsets, including T-cell Ig domain and mucin domain 1-positive transitional and T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domain-positive memory B cells, displaying immune regulatory functions, with a focus on transplantation tolerance, by analyzing their mechanisms of action. In addition, we will discuss potential T-cell Ig domain and mucin domain 1-positive and T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domain-positive B cell-based strategies for the enhancement of operational tolerance in transplantation patients.

The adverse effects of traditional pharmaceutical immunosuppressive regimens have been a major obstacle to successful allograft survival in vascularized composite tissue allotransplantation (VCA) cases. Consequently, there is a pressing need to explore alternative approaches to reduce reliance on conventional immunotherapy. Cell therapy, encompassing immune-cell-based and stem-cell-based regimens, has emerged as a promising avenue of research. Immune cells can be categorized into two main systems: innate immunity and adaptive immunity. Innate immunity comprises tolerogenic dendritic cells, regulatory macrophages, and invariant natural killer T cells, while adaptive immunity includes T regulatory cells and B regulatory cells. Investigations are currently underway to assess the potential of these immune cell populations in inducing immune tolerance. Furthermore, mixed chimerism therapy, involving the transplantation of hematopoietic stem and progenitor cells and mesenchymal stem cells (MSC), shows promise in promoting allograft tolerance. Additionally, extracellular vesicles (EVs) derived from MSCs offer a novel avenue for extending allograft survival. This review provides a comprehensive summary of cutting-edge research on immune cell therapies, mixed chimerism therapies, and MSCs-derived EVs in the context of VCAs. Findings from preclinical and clinical studies demonstrate the tremendous potential of these alternative therapies in optimizing allograft survival in VCAs.

Despite the growing number of elderly kidney transplant (Ktx) recipients, few studies have examined the effects of immunosuppression on their lymphocyte profiles.

We evaluated the early conversion from mycophenolate sodium (MPS) to everolimus (EVL) after rabbit antithymocyte globulin (rATG) 2 mg/kg induction in elderly kidney recipients. Three groups of KTx patients were compared: (a) Young (n=20, 36 ± 7 y) receiving standard immunosuppression (Group A1) (prednisone, tacrolimus, and MPS), (b) Elderly (n=35, 65 ± 3 y) receiving standard immunosuppression (Group B1), and (c) Elderly (n=16, 65 ± 3 y) with early (mean 30 d) conversion from MPS to EVL (Group B2). Naive, memory, and regulatory peripheral blood TCD4+ lymphocytes were quantified at 0, 30, and 365 d.

Results are reported as [mean(p25-p75)]. Young recipients had higher lymphocyte counts at baseline [2,100(1,630-2,400) vs. 1,310 (1,000-1,600)/mm3, p<0.0001] maintained higher counts within 365 d [1,850(1,590-2,120) vs. 1,130(460-1,325)/mm3, p=0.018 and vs. 1,410(805-1,895)/mm3, p=0.268]. Elderly recipients showed a decrease in lymphocytes within 30 d [1,310(1,000-1,600) vs. 910(700-1,198)/mm3, p=0.0012] with recovery within 365 d. The same pattern was observed in total lymphocytes and TCD4+ counts. Rabbit antithymocyte globulin induced a reduction in central memory T-cell percentages at 30 d in both young recipients [6.2(3.77-10.8) vs. 5.32(2.49-7.28)% of CD4+, p=0.036] and in elderly recipients [8.17(5.28-12.88) vs. 6.74(4.36-11)% of CD4+, p=0.05] on standard immunosuppression, returning to baseline at 365 d in elderly recipients but not in young recipients. Regulatory T CD39+ cells (Treg) percentages decreased at 30 d in elderly recipients [2.1(1.23-3.51) vs. 1.69(0.8-2.66)% of CD4+, p=0.0028] and in young recipients [1.29(0.45-1.85) vs. 0.84(0.18-1.82)% of CD4+, p=0.0038], returning to baseline at 365 d in elderly recipients [2.1(1.23-3.51) vs. 2.042(0.88-2.42)% of CD4+], but not in young recipients [1.29(0.45-1.85) vs. 0.86(0.7-1.34) % of CD4+]. The elderly everolimus conversion group did not show significant changes in cell profile over time or compared to elderly recipients with standard immunosuppression.

Aging favored the maintenance of Treg during the late transplantation period despite ongoing immunosuppression. Lymphocyte depletion due to rATG was more prominent in elderly recipients and affected memory subsets with a temporary reduction in central memory T cells. However, conversion to everolimus did not impact Treg profile. Reducing the dose of rATG in elderly recipients seems necessary for the expected lymphocyte changes with EVL to occur.

nEverOld Trial, identifier NTC01631058.

B cells can have a wide range of pro- and anti- inflammatory functions. A subset of B cells called regulatory B cells (Bregs) can potently suppress immune responses. Bregs have been shown to maintain immune homeostasis and modulate inflammatory responses. Bregs are an exciting cellular target across a range of diseases, including Breg induction in autoimmunity, allergy and transplantation, and Breg suppression in cancers and infection. Bregs exhibit a remarkable phenotypic heterogeneity, rendering their unequivocal identification a challenging task. The lack of a universally accepted and exclusive surface marker set for Bregs across various studies contributes to inconsistencies in their categorization. This review paper presents a comprehensive overview of the current understanding of the phenotypic and functional properties of human Bregs while addressing the persisting ambiguities and discrepancies in their characterization. Finally, the paper examines the promising therapeutic opportunities presented by Bregs as their immunomodulatory capacities have gained attention in the context of autoimmune diseases, allergic conditions, and cancer. We explore the exciting potential in harnessing Bregs as potential therapeutic agents and the avenues that remain open for the development of Breg-based treatment strategies.

Following transplantation, human CD4+T cells can respond to alloantigen using three distinct pathways. Direct and semi-direct responses are considered potent, but brief, so contribute mostly to acute rejection. Indirect responses are persistent and prolonged, involve B cells as critical antigen presenting cells, and are an absolute requirement for development of donor specific antibody, so more often mediate chronic rejection. Novel in vitro techniques have furthered our understanding by mimicking in vivo germinal centre processes, including B cell antigen presentation to CD4+ T cells and effector cytokine responses following challenge with donor specific peptides. In this review we outline recent data detailing the contribution of CD4+ T follicular helper cells and antigen presenting B cells to donor specific antibody formation and antibody mediated rejection. Furthermore, multi-parametric flow cytometry analyses have revealed specific endogenous regulatory T and B subsets each capable of suppressing distinct aspects of the indirect response, including CD4+ T cell cytokine production, B cell maturation into plasmablasts and antibody production, and germinal centre maturation. These data underpin novel opportunities to control these aberrant processes either by targeting molecules critical to indirect alloresponses or potentiating suppression via exogenous regulatory cell therapy.

Understanding immune cell dynamics in kidney transplantation may provide insight into the mechanisms of rejection and improve patient management. B cells have gained interest with a special relevance of the "regulatory" subsets and their graft outcome prognostic value. In this study, we aimed to prove that the direct immunophenotyping and target gene expression analysis of kidney transplant patients' fresh whole blood will help to identify graft rejection risk and assist in the monitoring of kidney transplanted patients.

We employed flow cytometry and qPCR techniques to characterize B and T cell subsets within fresh whole blood samples, with particular emphasis on transitional B cells (TrB) identified as CD19+CD24hiCD38hi. TrB are a relevant population in the context of kidney transplantation and are closely associated with regulatory B cells (Bregs) in humans. Patients were monitored, tracking pertinent clinical parameters and kidney-related events, including alterations in graft function and episodes of biopsy proven rejection.

Higher percentages of TrB cells at 3 months after transplantation were positively associated with better graft outcomes and lower biopsy-proven acute rejection risk. Furthermore, a novel panel of B cell regulatory associated genes was validated at 3 months post-transplantation by qPCR analysis of peripheral blood mononuclear cell (PBMC) mRNA, showing high predictive power of graft events and prognostic value.

These findings suggest that monitoring TrB may provide interesting patient management information, improve transplant outcomes, and allow for personalized drug regimens to minimize clinical complications.

To evaluate the synergistic potential of Focused Ultrasound (FUS) in conjunction with microbubbles (MB) and recombinant adeno-associated virus serotype 9 (rAAV9) vectors for targeted gene delivery to neuronal cells in rats, optimizing gene expression conditions and assessing any adverse effects.

The parameters for permeability enhancement of the rat's blood-brain barrier (BBB) were established using FUS+MB, with MRI scans and Evans Blue (EB) dye assisting in the evaluation. Rats underwent FUS-mediated transfection using rAAV9-Syn-EGFP vectors produced via a triple-transfection in HEK293T cells. Following this, the uptake and expression of GFP in targeted brain regions were evaluated using confocal fluorescence microscopy at various time intervals. Inflammatory responses post-FUS treatment were tracked by observing levels of GFAP, a marker for astrocytic activation, and TNF-α, a pro-inflammatory cytokine. Motor behavior effects post-intervention were gauged using the Rotarod test across multiple groups over a span of four weeks.

FUS+MB affected BBB permeability, with optimal results at 4 W for 200 s showing 85 % permeability and evident Gd-DTPA leakage. Settings beyond these resulted in tissue damage. Control groups exhibited a basal GFP expression of 2 % ± 0.5 %, whereas FUS+MB with rAAV-EGFP injections substantially increased GFP expression to about 67 % ± 6 % in targeted neurons. This GFP expression peaked at three weeks post-treatment and remained evident six months later. Following FUS treatment, both GFAP and TNF-α levels underwent fluctuations before eventually nearing their baseline values. The Rotarod test revealed no significant behavioral differences post-treatments among the groups.

Combining FUS+MB with rAAV offers an innovative approach to enhance therapeutic delivery to the central nervous system (CNS) by transiently adjusting BBB permeability.

Renal transplantation represents the foremost efficacious approach for ameliorating end-stage renal disease. Despite the current state of advanced renal transplantation techniques and the established postoperative immunosuppression strategy, a subset of patients continues to experience immune rejection during both the early and late postoperative phases, ultimately leading to graft loss. Consequently, the identification of immunobiomarkers capable of predicting the onset of immune rejection becomes imperative in order to facilitate early intervention strategies and enhance long-term prognoses. Upon reviewing the pertinent literature, we identified several indicators that could potentially serve as immune biomarkers to varying extents. These include the T1/T2 ratio, Treg/Th17 ratio, IL-10/TNF-α ratio, IL-33, IL-34, IL-6, IL-4, other cytokines, and NOX2/4.

Cutaneous T cell lymphomas (CTCLs) are skin cancers with poor survival rates and limited treatments. While immunotherapies have shown some efficacy, the immunological consequences of administering immune-activating agents to CTCL patients have not been systematically characterized. We apply a suite of high-dimensional technologies to investigate the local, cellular, and systemic responses in CTCL patients receiving either mono- or combination anti-PD-1 plus interferon-gamma (IFN-γ) therapy. Neoplastic T cells display no evidence of activation after immunotherapy. IFN-γ induces muted endogenous immunological responses, while anti-PD-1 elicits broader changes, including increased abundance of CLA+CD39+ T cells. We develop an unbiased multi-omic profiling approach enabling discovery of immune modules stratifying patients. We identify an enrichment of activated regulatory CLA+CD39+ T cells in non-responders and activated cytotoxic CLA+CD39+ T cells in leukemic patients. Our results provide insights into the effects of immunotherapy in CTCL patients and a generalizable framework for multi-omic analysis of clinical trials.

Donor-reactive memory cells represent a barrier to long-term kidney graft survival. A better understanding of regulatory mechanisms that counterbalance alloreactive memory responses may help to identify patients with operational tolerance. This prospective study investigated the equilibrium between memory T-cell subsets and regulatory T or B cells (Tregs, Bregs) in peripheral blood of kidney transplant recipients with operational tolerance (N = 8), chronic rejection (N = 8), and different immunosuppressive treatment regimens (N = 81). Patients on hemodialysis and healthy individuals served as controls (N = 50). In addition, the expression of Treg- and Breg-associated molecule genes was analyzed. Patients with chronic rejection showed a disrupted memory T-cell composition with a significantly higher frequency of circulating CD8+ terminally differentiated effector memory (TEMRA) T cells than patients with operational tolerance, patients on hemodialysis, or healthy controls (P < 0.001). Low frequency of CD8+ TEMRA and high frequency of Tregs and transitional Bregs were found in operationally tolerant patients. Consequently, operationally tolerant patients showed, as compared to all other transplant recipients with different immunosuppressive regiments, the lowest ratios between CD8+ TEMRA T cells and Tregs or Bregs (for both P < 0.001). Moreover, a specific peripheral blood transcription pattern was found in operationally tolerant patients with an increased expression of Breg- and Treg-associated genes CD22 and FoxP3 and a decreased FcγRIIA/FcγRIIB transcript ratio (for all P < 0.001). In conclusion, monitoring the balance between circulating CD8+ TEMRA T cells and regulatory cell subsets and their transcripts may help to distinguish transplant recipients with operational tolerance from recipients at risk of graft loss.

Regulatory B cells (Breg) modulate the immune response in diverse disease settings including transplantation. Despite the lack of a specific phenotypic marker or transcription factor, their significance in transplantation is underscored by their ability to prolong experimental allograft survival, the possibility for their clinical use as immune monitoring tools, and the exciting prospect for them to form the basis for cell therapy. Interleukin (IL)-10 expression remains the most widely used marker for Breg. Several Breg subsets with distinct phenotypes that express this "signature Breg cytokine" have been described in mice and humans. Although T-cell immunoglobulin and mucin family-1 is the most inclusive and functional marker that accounts for murine Breg with disparate mechanisms of action, the significance of T-cell immunoglobulin and mucin family-1 as a marker for Breg in humans still needs to be explored. Although the primary focus of this review is the role of Breg in clinical transplantation, the net modulatory effect of B cells on the immune response and clinical outcomes is the result of the balancing functions of both Breg and effector B cells. Supporting this notion, B-cell IL-10/tumor necrosis factor α ratio is shown to predict immunologic reactivity and clinical outcomes in kidney and liver transplantation. Assessment of Breg:B effector balance using their IL-10/tumor necrosis factor α ratio may identify patients that require more immunosuppression and provide mechanistic insights into potential therapies. In summary, current advances in our understanding of murine and human Breg will pave way for future definitive clinical studies aiming to test them for immune monitoring and as therapeutic targets.

Liver transplantation (LT) has become the most efficient treatment for pediatric and adult end-stage liver disease and the survival time after transplantation is becoming longer due to the development of surgical techniques and perioperative management. However, long-term side-effects of immunosuppressants, like infection, metabolic disorders and malignant tumor are gaining more attention. Immune tolerance is the status in which LT recipients no longer need to take any immunosuppressants, but the liver function and intrahepatic histology maintain normal. The approaches to achieve immune tolerance after transplantation include spontaneous, operational and induced tolerance. The first two means require no specific intervention but withdrawing immunosuppressant gradually during follow-up. No clinical factors or biomarkers so far could accurately predict who are suitable for immunosuppressant withdraw after transplantation. With the understanding to the underlying mechanisms of immune tolerance, many strategies have been developed to induce tolerance in LT recipients. Cellular strategy is one of the most promising methods for immune tolerance induction, including chimerism induced by hematopoietic stem cells and adoptive transfer of regulatory immune cells. The safety and efficacy of various cell products have been evaluated by prospective preclinical and clinical trials, while obstacles still exist before translating into clinical practice. Here, we will summarize the latest perspectives and concerns on the clinical application of cellular strategies in LT recipients.

The aim of the following review is to shed light on the putative role of regulatory B cells (Bregs) in various human diseases and highlight their potential prognostic and therapeutic relevance in humans. Regulatory B cells are a heterogeneous group of B lymphocytes capable of suppressing inflammatory immune reactions. In this way, Bregs contribute to the maintenance of tolerance and immune homeostasis by limiting ongoing immune reactions temporally and spatially. Bregs play an important role in attenuating pathological inflammatory reactions that can be associated with transplant rejection, graft-versus-host disease, autoimmune diseases and allergies but also with infectious, neoplastic and metabolic diseases. Early studies of Bregs identified IL-10 as an important functional molecule, so the IL-10-secreting murine B10 cell is still considered a prototype Breg, and IL-10 has long been central to the search for human Breg equivalents. However, over the past two decades, other molecules that may contribute to the immunosuppressive function of Bregs have been discovered, some of which are only present in human Bregs. This expanded arsenal includes several anti-inflammatory cytokines, such as IL-35 and TGF-β, but also enzymes such as CD39/CD73, granzyme B and IDO as well as cell surface proteins including PD-L1, CD1d and CD25. In summary, the present review illustrates in a concise and comprehensive manner that although human Bregs share common functional immunosuppressive features leading to a prominent role in various human immunpathologies, they are composed of a pool of different B cell types with rather heterogeneous phenotypic and transcriptional properties.

Although donor-specific antibody pre- and posttransplantation is routinely assessed, accurate quantification of memory alloreactive B cells that mediate recall antibody response remains challenging. Major histocompatibility complex (MHC) tetramers have been used to identify alloreactive B cells in mice and humans, but the specificity of this approach has not been rigorously assessed.

B-cell receptors from MHC tetramer-binding single B cells were expressed as mouse recombinant immunoglobulin G1 (rIgG1) monoclonal antibodies, and the specificity was assessed with a multiplex bead assay. Relative binding avidity of rIgG1 was measured by modified dilution series technique and surface plasmon resonance. Additionally, immunoglobulin heavy chain variable regions of 50 individual B-cell receptors were sequenced to analyze the rate of somatic hypermutation.

The multiplex bead assay confirmed that expressed rIgG1 monoclonal antibodies were preferentially bound to bait MHC class II I-E d over control I-A d and I-A b tetramers. Furthermore, the dissociation constant 50 binding avidities of the rIgG1 ranged from 10 mM to 7 nM. The majority of tetramer-binding B cells were low avidity, and ~12.8% to 15.2% from naive and tolerant mice and 30.9% from acute rejecting mice were higher avidity (dissociation constant 50 <1 mM).

Collectively, these studies demonstrate that donor MHC tetramers, under stringent binding conditions with decoy self-MHC tetramers, can specifically identify a broad repertoire of donor-specific B cells under conditions of rejection and tolerance.

Environmental factors such as diet and lifestyle have been shown to influence the development of some intestinal mucosal lesions that may be precursors of colorectal cancer (CRC). The presence of these alterations seems to be associated with misbalanced immunological parameter levels. However, it is still unclear as to which immunological parameters are altered in each phase of CRC development. In this work, we aimed to study the potential relationships of immunological and metabolic parameters with diet in a CRC-related lesion context. Dietary information was obtained using an annual semi-quantitative food-frequency questionnaire (FFQ) from 93 volunteers classified via colonoscopy examination according to the presence of intestinal polyps or adenocarcinoma. Cytokines, chemokines, and adipokines were determined from serum samples. We observed a reduction in adiponectin according to the damage to the mucosa, accompanied by an increase and decrease in C-X-C motif chemokine ligand 10 (CXCL10) and resistin, respectively, in CRC cases. The presence of aberrant crypt foci (ACF) in the polyp group was associated with higher tumor necrosis factor-alpha (TNF-α) concentrations. Vegetables were directly correlated with adiponectin and resistin levels, while the opposite occurred with red meat. A bioactive compound, soluble pectin, showed a negative association with TNF-α. Future dietary strategies could be developed to modulate specific immunological parameters in the context of CRC.

This study aims to reveal the relationship between regulatory B cell (Breg) subsets and chronic-active antibody-mediated rejection (c-aABMR) in renal transplant recipients. Our study involved 3 groups of participants: renal transplant recipients with biopsy-proven c-aABMR as the chronic rejection group (c-aABMR, n = 23), recipients with stable graft functions as the patient control group (PC; n = 11), and healthy volunteers (HV; n = 11). Breg subsets, immature/transitional B cells, plasmablastic cells, B10 cells, and BR1 cells were isolated from venous blood samples by flow cytometry. The median values of Breg frequencies in the total lymphocyte population were analyzed. There were no significant differences between the study groups for immature and/or transitional B cell frequencies. Plasmablastic cell frequencies of the c-aABMR group (7.80 [2.10-27.40]) and the PC group (6.00 [1.80-55.50]) were similar, but both of these values were significantly higher than the HVs' (3.40 [1.20-8.50]), (respectively, P = .005 and P = .039). B10 cell frequencies were also similar, comparing the c-aABMR (4.20 [0.10-7.40]) and the PC groups (4.10 [0.10-5.90]), whereas the HVs (5.90 [2.90-8.50]) had the highest B10 cell frequency with an only statistical significance against the PC group (respectively, P = .09 and P = .028). The c-aABMR and the PC groups were similar regarding BR1 cell frequencies. However, the HV group significantly had the highest frequency of BR1 cells (5.50 [2.80-10.80]) than the other groups (P < .001 for both). We demonstrated that frequencies of B10 and BR1 cells were higher in HVs than in transplant recipients, regardless of rejection state. However, there was no significant relation between Breg frequencies and the c-aABMR state.

Immune cell pattern-recognition receptors such as Toll-like receptors (TLRs) play important roles in the regulation of host responses to periodontal pathogens. Our previous studies have demonstrated that immune regulatory B cells were activated by TLRs and alleviated periodontitis inflammation and bone loss. The purpose of this study is to determine the role of TLR9 signaling in the activation and IL-10 production of the primed-immune B cells in vitro. Wild-type (WT) and TLR9 knockout (TLR9KO) mice (C57BL/6 background, n = 5) were pre-immunized intraperitoneally with 1 × 108 formalin-fixed P. gingivalis and boosted once with 1 × 107 formalin-fixed P. gingivalis. Isolated splenocytes and purified B cells from each mouse were cultured with 1 × 108 formalin-fixed P. gingivalis for 48 h. Immunocytochemistry was performed to detect CD45+ IL-10+ cells. Levels of IL-10 expression and secretion in splenocytes and B cells were detected using qRT-PCR and ELISA, respectively. After stimulation with fixed P. gingivalis, the percentage of CD45+ IL-10+ B cells and the level of IL-10 expression were significantly increased (p < 0.01) in splenocytes and purified B cells isolated from WT mice. However, these changes were not observed in splenocytes and purified B cells from TLR9KO mice when the cells were treated with fixed P. gingivalis. The percentage of CD45+ IL-10+ B cells was significantly reduced in splenocytes and purified B cells from TLR9KO mice compared to those from WT mice when challenged with P. gingivalis. IL-10 expression in B cells from TLR9KO mice was significantly decreased compared to those from WT mice at both the mRNA and protein levels. Additionally, P. gingivalis-induced up-regulation of TNF-α mRNA expressions were consistently observed in B cells from both WT and TLR9KO mice. P. gingivalis-induced B10 activation and IL-10 production during adaptive responses by primed B cells requires TLR9 signaling and can be achieved independent of T-cell help.

Borderline rejection (BL) in renal transplantation is associated with decreased allograft survival, yet many patients with BL maintain stable graft function. Identifying patients with early BL at risk for shortened allograft survival would allow for timely targeted therapeutic intervention aimed at improving outcomes. 851/1187 patients transplanted between 2013-18 underwent early biopsy (0-4 mos). 217/851 (25%) had BL and were compared to 387/851 without significant inflammation (NI). Serial surveillance and for-cause biopsies and seven-year follow-up were used to evaluate histological and clinical progression. To identify high-risk patients, we examined clinical/histological parameters using regression and non-linear dimensionality reduction (tSNE) and a biomarker based on peripheral blood transitional-1 B cell (T1B) IL-10/TNFα ratio. Compared to NI, early BL was associated with increased progression to late acute rejection (AR; 5-12 mos), premature interstitial fibrosis and tubular atrophy (IFTA) and decreased seven-year graft survival. However, decreased graft survival was limited to BL patients who progressed to late AR or IFTA, and was not influenced by treatment. Although tSNE clustered patients into groups based on clinical factors, the ability of these factors to risk stratify BL patients was modest. In contrast, a low T1B IL-10/TNFα ratio at 3 months identified BL patients at high risk for progression to AR (ROC AUC 0.87) and poor 7-yr graft survival (52% vs. 92%, p=0.003), while BL patients with a high ratio had similar graft survival to patients with NI (91%, p=NS). Thus, progressive early allograft inflammation manifested as BL that progresses to late AR in the first post-transplant year represents a high-risk clinical state for poor allograft outcomes. Such high-risk status can be predicted by the T1B IL-10/TNFα ratio before irreversible scarring sets in, thus allowing timely risk stratification.

Borderline allograft rejection can promote acute rejection and graft loss in some, but not all, patients. In this issue, Cherukuri et al. use a novel test based on peripheral blood transitional T1 B cells producing interleukin-10 and tumor necrosis factor-α, which identifies patients at high risk for poor outcomes. The potential mechanisms by which transitional T1 B cells might modulate alloreactivity need exploration, but following appropriate validation, this biomarker could risk stratify patients in need of early intervention.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous B cell neoplasm ranging from indolent to rapidly progressive disease. Leukemic cell subsets with regulatory properties evade immune clearance; however, the contribution of such subsets during CLL progression is not completely elucidated. Here, we report that CLL B cells crosstalk with their immune counterparts, notably by promoting the regulatory T (Treg) cell compartment and shaping several helper T (Th) subsets. Among various constitutively- and BCR/CD40-mediated factors secreted, tumour subsets co-express two important immunoregulatory cytokines, IL10 and TGFβ1, both associated with a memory B cell phenotype. Neutralizing secreted IL10 or inhibiting the TGFβ signalling pathway demonstrated that these cytokines are mainly involved in Th- and Treg differentiation/maintenance. In line with the regulatory subsets, we also demonstrated that a CLL B cell population expresses FOXP3, a marker of regulatory T cells. Analysis of IL10, TGFβ1 and FOXP3 positive subpopulations frequencies in CLL samples discriminated 2 clusters of untreated CLL patients that were significantly different in Tregs frequency and time-to-treatment. Since this distinction was pertinent to disease progression, the regulatory profiling provides a new rationale for patient stratification and sheds light on immune dysfunction in CLL.

The human immune system contains cells with either effector/memory or regulatory functions. Besides the well-established CD4+CD25hiCD127lo regulatory T cells (Tregs), we and others have shown that B cells can also have regulatory functions since their frequency and number are increased in kidney graft tolerance and B cell depletion as induction therapy may lead to acute rejection. On the other hand, we have shown that CD28-CD8+ T cells represent a subpopulation with potent effector/memory functions. In the current study, we tested the hypothesis that kidney allograft rejection may be linked to an imbalance of effector/memory and regulatory immune cells.

Based on a large cohort of more than 1000 kidney graft biopsies with concomitant peripheral blood lymphocyte phenotyping, we investigated the association between kidney graft rejection and the percentage and absolute number of circulating B cells, Tregs, as well as the ratio of B cells to CD28-CD8+ T cells and the ratio of CD28-CD8+ T cells to Tregs. Kidney graft biopsies were interpreted according to the Banff classification and divided into 5 biopsies groups: 1) normal/subnormal, 2) interstitial fibrosis and tubular atrophy grade 2/3 (IFTA), 3) antibody-mediated rejection (ABMR), 4) T cell mediated-rejection (TCMR), and 5) borderline rejection. We compared group 1 with the other groups as well as with a combined group 3, 4, and 5 (rejection of all types) using multivariable linear mixed models.

We found that compared to normal/subnormal biopsies, rejection of all types was marginally associated with a decrease in the percentage of circulating B cells (p=0.06) and significantly associated with an increase in the ratio of CD28-CD8+ T cells to Tregs (p=0.01). Moreover, ABMR, TCMR (p=0.007), and rejection of all types (p=0.0003) were significantly associated with a decrease in the ratio of B cells to CD28-CD8+ T cells compared to normal/subnormal biopsies. Taken together, our results show that kidney allograft rejection is associated with an imbalance between immune cells with effector/memory functions and those with regulatory properties.

A subset of B-cells with tolerogenic functions, termed B-regulatory cells or Bregs, is characterized by the expression of anti-inflammatory/tolerogenic cytokines, namely IL-10, TGF-β, and IL-35, that contribute to their regulatory functions. Breg regulation favors graft acceptance within a tolerogenic milieu. As organ transplantation invariably triggers inflammation, new insights into the crosstalk between cytokines with dual properties and the inflamed milieu are needed to tailor their function toward tolerance. Using TNF-α as a proxy of dual-function cytokines involved in immune-related diseases and transplantation settings, the current review highlights the multifaceted role of TNF-α. It focuses on therapeutic approaches that have revealed the complexity of TNF-α properties tested in clinical settings where total TNF-α inhibition has proven ineffective and often detrimental to clinical outcomes. To improve the efficacy of current TNF-α inhibiting therapeutics, we propose a three-prong strategy to upregulate the tolerogenic pathway engaging the TNFR2 receptor while simultaneously inhibiting the inflammatory mechanisms associated with TNFR1 engagement. When combined with additional administrations of Bregs-TLR that activate Tregs, this approach may become a potential therapeutic in overcoming transplant rejection and promoting graft tolerance.

B cells are classically considered solely as antibody-producing cells driving humoral immune responses to foreign antigens in infections and vaccinations as well as self-antigens in pathological settings such as autoimmunity. However, it has now become clear that B cells can also secrete a vast array of cytokines, which influence both pro- and anti-inflammatory immune responses. Indeed, similarly to T cells, there is significant heterogeneity in cytokine-driven responses by B cells, ranging from the production of pro-inflammatory effector cytokines such as IL-6, through to the release of immunosuppressive cytokines such as IL-10. In this review, focusing on human B cells, we summarize the key findings that have revealed that cytokine-producing B cell subsets have critical functions in healthy immune responses and contribute to the pathophysiology of autoimmune diseases.

Over the past two decades, regulatory B cells (Breg cells or Bregs) have emerged as an immunosuppressive subset of B lymphocytes playing a key role in inflammation, infection, allergy, transplantation, and cancer. However, the involvement of Bregs in various pathological conditions of the gastrointestinal tract is not fully understood and is the subject of much recent research. In this review, we aimed to summarize the current state of knowledge about the origin, phenotype, and suppressive mechanisms of Bregs. The relationship between the host gut microbiota and the function of Bregs in the context of the disturbance of mucosal immune homeostasis is also discussed. Moreover, we focused our attention on the role of Bregs in certain diseases and pathological conditions related to the digestive tract, especially Helicobacter pylori infection, parasitic diseases (leishmaniasis and schistosomiasis), and gastrointestinal neoplasms. Increasing evidence points to a relationship between the presence and number of Bregs and the severity and progression of these pathologies. As the number of cases is increasing year by year, also among young people, it is extremely important to understand the role of these cells in the digestive tract.

Regulatory B cells (Bregs) play a prominent role in various disease settings. While progress has been hindered by the lack of a specific Breg marker, new findings highlight their role modulating the alloimmune response and promoting allograft survival.

Herein, we focus on the recent advances in Breg biology and their role in transplantation. We review studies showing that T-cell immunoglobulin and mucin domain 1 (TIM-1) is an inclusive and functional Breg marker in mice that may have human relevance. We highlight the utility of the B cell interleukin-10/tumor necrosis factor-alpha (IL-10/TNFα) ratio in identifying underlying immunological reactivity and predicting clinical outcomes in kidney transplantation. This may identify patients requiring more immunosuppression and provide insight into potential therapeutic approaches that can modulate the Breg: B effector cell (Beff) balance.

Emerging data support Bregs as potent modulators of immune responses in humans. Their ability to promote allograft survival must await development of approaches to expand Bregs in vitro/in vivo . The low IL-10/TNFα ratio reflecting decreased Breg/Beff balance, predicts acute rejection (AR) and poorer outcomes in renal transplantation. It remains to be determined whether this paradigm can be extended to other allografts and whether therapy aiming to correct the relative deficiency of Bregs will improve outcomes.

Brain death (BD) is characterized by a complex inflammatory response, resulting in dysfunction of potentially transplantable organs. This process is modulated by cytokines, which amplify graft immunogenicity. We have investigated the inflammatory response in an animal model of BD and analyzed the effects of thalidomide, a drug with powerful immunomodulatory properties.

BD was induced in male Lewis rats. We studied three groups: Control (sham-operated rats) (n = 6), BD (rats subjected to brain death) (n = 6) and BD + Thalid (BD rats treated with one dose of thalidomide (200 mg/Kg), administered by gavage) (n = 6). Six hours after BD, serum levels of urea and creatinine, as well as systemic and renal tissue protein levels of TNF-α and IL-6, were analyzed. We also determined the mRNA expression of ET-1, and macrophage infiltration by immunohistochemistry.

BD induced a striking inflammatory status, demonstrated by a significant increase of plasma cytokines: TNF-α (2.8 ± 4.3 pg/mL [BD] vs. 9.4 ± 2.8 pg/mL [Control]), and IL-6 (6219.5 ± 1380.6 pg/mL [BD] vs. 1854.7 ± 822.6 pg/mL [Control]), and in the renal tissue: TNF-α (2.5 ± 0.3 relative expression [BD] vs. 1.0 ± 0.4 relative expression [Control]; p < 0.05), and IL-6 (4.0 ± 0.4 relative expression [BD] vs. 1.0 ± 0.3 relative expression [Control]; p < 0.05). Moreover, BD increased macrophages infiltration (2.47 ± 0.07 cells/field [BD] vs. 1.20 ± 0.05 cells/field [Control]; p < 0.05), and ET-1 gene expression (2.5 ± 0.3 relative expression [BD] vs. 1.0 ± 0.2 relative expression [Control]; p < 0.05). In addition, we have observed deterioration in renal function, characterized by an increase of urea (194.7 ± 25.0 mg/dL [BD] vs. 108.0 ± 14.2 mg/dL [Control]; p < 0.05) and creatinine (1.4 ± 0.04 mg/dL [BD] vs. 1.0 ± 0.07 mg/dL [Control]; p < 0.05) levels. Thalidomide administration significantly reduced plasma cytokines: TNF-α (5.1 ± 1.4 pg/mL [BD + Thalid] vs. BD; p < 0.05), and IL-6 (1056.5 ± 488.3 pg/mL [BD + Thalid] vs. BD; p < 0.05), as well as in the renal tissue: TNF-α (1.5 ± 0.2 relative expression [BD + Thalid] vs. BD; p < 0.05), and IL-6 (2.1 ± 0.3 relative expression [BD + Thalid] vs. BD; p < 0.05). Thalidomide treatment also induced a significant decrease in the expression of ET-1 (1.4 ± 0.3 relative expression [BD + Thalid] vs. BD; p < 0.05), and macrophages infiltration (1.17 ± 0.06 cells/field [BD + Thalid] vs. BD; p < 0.05). Also thalidomide prevented kidney function failure by reduced urea (148.3 ± 4.4 mg/dL [BD + Thalid] vs. BD; p < 0.05), and creatinine (1.1 ± 0.14 mg/dL [BD + Thalid] vs. BD; p < 0.05).

The immunomodulatory properties of thalidomide were effective in decreasing systemic and local immunologic response, leading to diminished renal damage, as reflected in the decrease of urea and creatinine levels. These results suggest that use of thalidomide may represent a potential strategy for treating in BD kidney organ donors.

B cells play crucial roles in cell-mediated alloimmune responses. In vitro, B cells can support or regulate indirect T-cell alloreactivity in response to donor antigens on ELISpot and these patterns associate with clinical outcome. Previous reports of associations between B-cell phenotype and function have examined global phenotypes and responses to polyclonal stimuli. We hypothesized that studying antigen-specific B cells, using samples from sensitized patients, would inform further study to identify novel targets for intervention. Using biotinylated HLA proteins, which bind HLA-specific B cells via the B-cell receptor in a dose-dependent fashion, we report the specific phenotype of HLA-binding B cells and define how they associated with patterns of anti-HLA response in interferon-γ ELISpot. HLA-binding class-switched and IgM+CD27+ memory cells associated strongly with B-dependent interferon-γ production and appeared not suppressible by endogenous Tregs. When the predominant HLA-binding phenotype was naïve B cells, the associated functional ELISpot phenotype was determined by other cells present. High numbers of non-HLA-binding transitional cells associated with B-suppressed interferon-γ production, especially if Tregs were present. However, high frequencies of HLA-binding marginal-zone precursors associated with B-dependent interferon-γ production that appeared suppressible by Tregs. Finally, non-HLA-binding marginal zone precursors may also suppress interferon-γ production, though this association only emerged when Tregs were absent from the ELISpot. Thus, our novel data provide a foundation on which to further define the complexities of interactions between HLA-specific T and B cells and identify new targets for intervention in new therapies for chronic rejection.

Regulatory B cells (Bregs) suppress immune responses through the secretion of interleukin-10 (IL-10). This immunomodulatory capacity holds therapeutic potential, yet a definitional immunophenotype for enumeration and prospective isolation of B cells capable of IL-10 production remains elusive. Here, we simultaneously quantify cytokine production and immunophenotype in human peripheral B cells across a range of stimulatory conditions and time points using mass cytometry. Our analysis shows that multiple functional B cell subsets produce IL-10 and that no phenotype uniquely identifies IL-10+ B cells. Further, a significant portion of IL-10+ B cells co-express the pro-inflammatory cytokines IL-6 and tumor necrosis factor alpha (TNFα). Despite this heterogeneity, operationally tolerant liver transplant recipients have a unique enrichment of IL-10+, but not TNFα+ or IL-6+, B cells compared with transplant recipients receiving immunosuppression. Thus, human IL-10-producing B cells constitute an induced, transient state arising from a diversity of B cell subsets that may contribute to maintenance of immune homeostasis.