Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow.

Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19low-BMPC are derived from follicular, while CD19high-BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19low- and CD19high-BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19high-BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observable in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response.

Urinary CD8+HLA-DR+ T Cell Abundance Non-invasively Predicts Kidney Transplant Rejection.

Early detection of kidney transplant (KT) rejection remains a challenge in patient care. Non-invasive biomarkers hold high potential to detect rejection, adjust immunosuppression, and monitor KT patients. So far, no approach has fully satisfied requirements to innovate routine monitoring of KT patients. In this two-center study we analyzed a total of 380 urine samples. T cells and tubular epithelial cells were quantified in KT patients with graft deterioration using flow cytometry. Epigenetic urine cell quantification was used to confirm flow cytometric results. Moreover, a cohort of KT patients was followed up during the first year after transplantation, tracking cell subsets over time. Abundance of urinary cell counts differed in patients with and without rejection. Most strikingly, various T cell subsets were enriched in patients with T cell-mediated rejection (TCMR) compared to patients without TCMR. Among T cell subsets, CD8+HLA-DR+ T cells were most distinctive (AUC = 0.91, Spec.: 95.9%, Sens.: 76.5%). Epigenetic analysis confirmed T cell and tubular epithelial cell quantities as determined by flow cytometry. Urinary T cell abundance in new KT patients decreased during their first year after transplantation. In conclusion urinary T cells reflect intrarenal inflammation in TCMR. T cell subsets yield high potential to monitor KT patients and detect rejection. Hereby we present a promising biomarker to non-invasively diagnose TCMR.

Noninvasive Diagnosis of Acute Rejection in Renal Transplant Patients Using Mass Spectrometric Analysis of Urine Samples: A Multicenter Diagnostic Phase III Trial.

Timely recognition and treatment of acute kidney graft rejection is important to prevent premature graft failure. A predefined urinary marker set for acute T cell-mediated rejection (TCMR) containing 14 peptides was tested for this purpose in a multicenter in-place validation study. Methods: Three hundred twenty-nine prospectively collected and 306 archived urine samples from 11 transplant centers in Germany, France, and Belgium were examined. Samples were taken immediately before a biopsy, performed for graft dysfunction within the first transplant year. Primary outcomes were sensitivity and specificity of the marker set for the diagnosis of biopsy-proven acute TCMR, with prespecified thresholds of 83% for sensitivity and 70% for specificity. Results: Eighty-two patients (13%) had acute TCMR grade I-III. In relation to the biopsy diagnosis of TCMR, the sensitivity of the urine test was 0.66 (95% confidence interval, 0.56-0.76) and the specificity 0.47 (95% confidence interval, 0.43-0.51), with an area under the curve (AUC) of 0.60. The different TCMR grades I-III were not reflected by the marker set, and borderline TCMR was not specifically detected. Secondary independent masked assessment of biopsies consented by 2 pathologists revealed an interobserver kappa value of 0.49 for diagnosing TCMR, compared with the local center's diagnosis. Using this consensus diagnosis, the AUC of the urine test was 0.63 (sensitivity 0.73, specificity 0.45). Post hoc optimization of the marker set improved the diagnostic performance in the study cohort (AUC 0.67) and in an independent patient cohort (AUC 0.69). Conclusions: This study illustrates the difficulty of proteomics-based diagnosis of TCMR and highlights the need for rigorous independent in-place validation and optimization of diagnostic biomarkers.

SARS-CoV-2 in severe COVID-19 induces a TGF-ß-dominated chronic immune response that does not target itself.

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-ß, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-ß. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-ß, and is distracted from itself.

Single-cell transcriptomes of murine bone marrow stromal cells reveal niche-associated heterogeneity.

Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non-overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long-term maintenance of immune cells.

The regulation of interferon type I pathway-related genes RSAD2 and ETV7 specifically indicates antibody-mediated rejection after kidney transplantation.

CONTEXT: Antibody-mediated rejection (ABMR) after kidney transplantation (KTx) remains the crucial obstacle to successful long-term graft function. The identification of gene signatures involved in ABMR could grant the basis for better prevention and treatment strategies. OBJECTIVE: The identification of gene signatures in whole blood cells specific for ABMR after KTx. MATERIALS AND METHODS: Total RNA from blood cells of 16 kidney-transplanted patients with ABMR, stable graft function (SGF), and with T-cell-mediated rejection (TCMR) was isolated. Gene expression was determined by high-throughput sequencing followed by validation and analyses of differentially expressed candidates on mRNA level and on protein level in a large patient cohort (n = 185) in patients with SGF, urinary tract infection (UTI), borderline rejection (BL), TCMR, ABMR, and interstitial fibrosis and tubular atrophy. RESULTS: From the 570 genes detected, 111 discriminated ABMR from SGF and TCMR. A distinct enrichment of interferon (IFN) type I and type II signature gene set was observed. The expression of candidate genes IFIT1, ETV7, and RSAD2 distinguished ABMR patients from patients with SGF and also TCMR, whereas ETV7 and RSAD2 differentiated ABMR also from BL. CONCLUSION: The IFN-inducible genes ETV7 and RSAD2 represent specific biomarkers for ABMR episodes after KTx.

MicroRNA regulation in blood cells of renal transplanted patients with interstitial fibrosis/tubular atrophy and antibody-mediated rejection.

Interstitial fibrosis/tubular atrophy (IFTA) is associated with reduced allograft survival, whereas antibody-mediated rejection (ABMR) is the major cause for renal allograft failure. To identify specific microRNAs and their regulation involved in these processes, total RNA from blood cells of 16 kidney transplanted (KTx) patients with ABMR, stable graft function (SGF) and with T-cell mediated rejection (TCMR) was isolated. MicroRNA expression was determined by high-throughput sequencing. Differentially expressed candidate microRNAs were analyzed with RT-PCR in patients with SGF (n = 53), urinary tract infection (UTI) (n = 17), borderline rejection (BL) (n = 19), TCMR (n = 40), ABMR (n = 22) and IFTA (n = 30). From the 301 detected microRNAs, 64 were significantly regulated between the three cohorts. Selected candidate microRNAs miR-223-3p, miR-424-3p and miR-145-5p distinguished TCMR and ABMR from SGF, but not from other pathologies. Most importantly, miR-145-5p expression in IFTA patients was significantly downregulated and displayed a high diagnostic accuracy compared to SGF alone (AUC = 0.891) and compared to SGF, UTI, BL, TCMR and ABMR patients combined (AUC = 0.835), which was verified by cross-validation. The identification of miR-145-5p as IFTA specific marker in blood constitutes the basis for evaluating this potentially diagnostic microRNA as biomarker in studies including high numbers of patients and different pathologies and also the further analysis of fibrosis causing etiologies after kidney transplantation.

The selective biomarker IL-8 identifies IFTA after kidney transplantation in blood cells.

Cellular and antibody-mediated rejection processes and also interstitial fibrosis/tubular atrophy (IFTA) lead to allograft dysfunction and loss. The search for accurate, specific and non-invasive diagnostic tools is still ongoing and essential for successful treatment of renal transplanted patients. Molecular markers in blood cells and serum may serve as diagnostic tools but studies with high patient numbers and differential groups are rare. We validated the potential value of several markers on mRNA level in blood cells and serum protein level in 166 samples from kidney transplanted patients under standard immunosuppressive therapy (steroids±mycophenolic acid±calcineurin inhibitor) with stable graft function, urinary tract infection (UTI), IFTA, antibody-mediated rejection (ABMR), and T-cell-mediated rejection (TCMR) applying RT-PCR and ELISA. The mRNA expression of RANTES, granulysin, granzyme-B, IP-10, Mic-A and Interferon-γ in blood cells did not distinguish specifically between the different pathologies. We furthermore discovered that the mRNA expression of the chemokine IL-8 is significantly lower in samples from IFTA patients than in samples from patients with stable graft function (p<0.001), ABMR (p<0.001), Borderline (BL) TCMR (p<0.001), tubulo-interstitial TCMR (p<0.001) and vascular TCMR (p<0.01), but not with UTI. Serum protein concentrations of granzyme-B, Interferon-γ and IL-8 did not differ between the patient groups, RANTES concentration was significantly different when comparing UTI and ABMR (p<0.01), whereas granulysin, Mic-A and IP-10 measurement differentiated ongoing rejection or IFTA processes from stable graft function but not from each other. The measurement of IL-8 mRNA in blood cells distinguishes clearly between IFTA and other complication after kidney transplantation and could easily be used as diagnostic tool in the clinic.

Free microRNA levels in plasma distinguish T-cell mediated rejection from stable graft function after kidney transplantation.

The potential diagnostic value of circulating free miRNAs in plasma compared to miRNA expression in blood cells for rejection processes after kidney transplantation is largely unknown, but offers the potential for better and timely diagnosis of acute rejection. Free microRNA expression of specific blood cell markers was measured in 160 plasma samples from kidney transplant patients under standard immunosuppressive therapy (steroids±mycophenolic acid±calcineurin inhibitor) with stable graft function, urinary tract infection, interstitial fibrosis and tubular atrophy, antibody-mediated rejection (ABMR), Borderline (Banff3), tubulo-interstitial (Banff4-I) and vascular rejection (Banff4-II/III) applying RT-PCR. The expression levels of specific microRNAs miR-15B, miR-103A and miR-106A discriminated patients with stable graft function significantly (p-values 0.001996, 0.0054 and 0.0019 resp.) from patients with T-cell mediated rejection (TCMR) and from patients with urinary tract infection (p-values 0.0001, <0.0001 and 0.0001, resp.). A combined measurement of several microRNAs after multivariate logistic regression improved the diagnostic value supported by subsequent cross-validation. In conclusion, the measurement of circulating microRNAs in plasma from patients with renal transplants distinguishes TCMR and urinary tract infection from stable graft function. In contrast to miRNA expression measurement in blood cells it does not allow a discrimination from ABMR or interstitial fibrosis and tubular atrophy.

Identification of T Cell-Mediated Vascular Rejection After Kidney Transplantation by the Combined Measurement of 5 Specific MicroRNAs in Blood.

BACKGROUND: MicroRNAs (miRNAs, miR) hold important roles in the posttranscriptional regulation of gene expression. Their function has been correlated with kidney disease, and they might represent a new class of biomarkers for frequent evaluation of renal graft status. We analyzed their potential in identifying severe T cell-mediated vascular rejection (TCMVR) (Banff 4-II/III) in kidney transplanted patients. METHODS: Microarray experiments and semiquantitative real-time reverse transcription polymerase chain reaction were performed with total RNA isolated from blood cells of kidney graft recipients. Initial microarray analysis revealed 23 differentially expressed miRNAs distinguishing patients with TCMVR from patients with stable grafts. From these, we validated and further determined the expression of 6 differentially expressed miRNAs and 2 control miRNAs in 161 samples from patients with T cell-mediated rejection (Banff 3-Borderline, Banff 4-I/II/III), Banff-2 antibody-mediated rejection, Banff-5 interstitial fibrosis/tubular atrophy, in samples from stable patients and in samples from patients with urinary tract infection using real-time reverse transcription polymerase chain reaction. RESULTS: Expression levels of all 6 candidate miRNAs were significantly downregulated in blood of TCMVR patients compared to the other groups and displayed high sensitivities and specificities for diagnosing TCMVR. The combination of 5 miRNAs, identified by an unbiased multivariate logistic regression followed by cross-validation, enhanced the sensitivity and specificity for the diagnosis of TCMVR after renal transplantation. CONCLUSIONS: The combined measurement of miRNA-15B, miRNA-16, miRNA-103A, miRNA-106A, and miRNA-107 may help to better identify TCMVR after renal transplantation in a precise and clinically applicable way.

Conversion to Belatacept based regimen does not change T-cell phenotype and function in renal transplantation.

Belatacept offers a new option for renal allograft recipients who are suffering from side effects of calcineurin inhibitors or mTOR inhibitors,which may result in renal and extrarenal benefits.We prospectively performed flow cytometric immunophenotyping with a T-cell panel. In total we were able to fully investigate the immunophenotypic change in 8 patients before and after conversion from calcineurin inhibitor (n = 5) or mTOR inhibitor (n=2) to Belatacept or additional administration (n=1). Cells were analysed pre conversion, 1 month, 3 months, 6 months and 12 months after first Belatacept administration. The percentage of central memory, naïve, effector memory and terminally differentiated effector memory CD4+ and CD4− T-cells was determined. CD28, CD25 and CD69 expression on CD4+ and CD4− T-cells was measured ex vivo and also after 3 days of mitogen stimulation. Intracellular cytokines IFNgamma and IL-2 were measured after polyclonal cellular stimulation. The expression of activation markers and intracellular cytokines as well as the percentage of T-cell subsets did not change significantly during the observation period compared to the time-point pre conversion. Therefore the conversion of calcineurin inhibitor or mTOR inhibitor to Belatacept seems to have no obvious impact on the immunophenotype of T-cells in patients after kidney transplantation.

miR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim.

Repeatedly activated T helper 1 (Th1) cells present during chronic inflammation can efficiently adapt to the inflammatory milieu, for example, by expressing the transcription factor Twist1, which limits the immunopathology caused by Th1 cells. Here, we show that in repeatedly activated murine Th1 cells, Twist1 and T-bet induce expression of microRNA-148a (miR-148a). miR-148a regulates expression of the proapoptotic gene Bim, resulting in a decreased Bim/Bcl2 ratio. Inhibition of miR-148a by antagomirs in repeatedly activated Th1 cells increases the expression of Bim, leading to enhanced apoptosis. Knockdown of Bim expression by siRNA in miR-148a antagomir-treated cells restores viability of the Th1 cells, demonstrating that miR-148a controls survival by regulating Bim expression. Thus, Twist1 and T-bet not only control the differentiation and function of Th1 cells, but also their persistence in chronic inflammation.

Combined standard and novel immunosuppressive substances affect B-lymphocyte function.

A considerable fraction of renal transplanted patients is susceptible to humoral rejection. Today well-established therapy regimens are available to control antibody-mediated rejection in the short term. Nevertheless, donor-specific antibodies persist and graft function deteriorates over time. This might be due to insufficient maintenance immunosuppression - which always consists of two to three drugs with different mechanisms of action. Since T- and B-cell functions always depend on each other in the alloimmune response it is of interest to analyze the effects of combined standard and new immunosuppressive substances with T-cell inhibitory properties on B-cell function. The effectiveness of complementary administrations of sotrastaurin, mycophenolic acid and everolimus on the activation and function of human primary B-lymphocytes was tested. Everolimus and mycophenolic acid alone and in combination proved to be highly effective in suppressing B-cell activation, whereas the proteinkinase C inhibitor sotrastaurin had an unexpected and reverse impact on various B-cell functions when applied in combination with the mammalian target of rapamycin and the inosine monophosphate dehydrogenase inhibitor.

An evaluation of sirolimus in renal transplantation.

INTRODUCTION: Sirolimus is a powerful antiproliferative immunosuppressive drug approved for the prevention of kidney allograft rejection. By its unique mechanism of action, sirolimus provides a multitude of clinical potential and has been used effectively in different drug combinations. Extensive experience has been gained regarding the best timing of its application, side effect profile and potential benefits and limitations compared with other immunosuppressive drugs. AREAS COVERED: The authors evaluate the recent experience with sirolimus in kidney transplantation. Pivotal randomized controlled trials were used to provide an overview with special attention to pharmacokinetic and dynamic aspects of sirolimus, its current clinical use as well as perspectives for its future role. EXPERT OPINION: Sirolimus enriches the possibilities of immunosuppressive therapies after renal transplantation. Beneficial effects toward kidney function by allowing CNI sparing, lower incidence of malignancies and less viral infections have been suggested. Sirolimus should be used cautiously in de novo patients for reasons of wound healing. An early conversion to a sirolimus-based CNI-free regimen has shown promising results, whereas late conversion is more challenging. Finally, sirolimus-associated side effects are causing tolerability concerns and frequent discontinuations. Future research should aim to better define the therapeutic window and those patients most likely to benefit.

Identification and therapeutic management of highly sensitized patients undergoing renal transplantation.

Sensitization is generally referred to as the development of alloantibodies, specifically anti-human leukocyte antigen (HLA) immunoglobulin G (IgG) antibodies, most commonly caused by pregnancy, blood transfusion or a previous transplant. Despite being a well known phenomenon, there has not been a general consensus on its definition, monitoring or management. Today, 25% of the patients waitlisted for kidney transplant in the US have a panel reactive antibody (PRA) of >10% while, in the Eurotransplant zone, 14% have a PRA of >5%. Sensitized patients have more difficulty in finding a well HLA-matched donor, and have a higher risk of experiencing longer waiting times, more rejection episodes and eventually inferior long-term graft or patient survival. We review the currently available strategies in identifying and managing highly sensitized patients undergoing renal transplantation. We discuss the progress and limitations in laboratory techniques to elaborate on challenges in defining sensitized patients. The main management options (i.e. the Acceptable Mismatch Program, donor exchange programmes and the desensitization approach) and their mechanisms, related policies, advantages and outcomes, as well as medications and methods being investigated, are updated. In addition, particular emphasis is given to sensitization prevention, a practice that is neglected with our increasing ability to suppress the immune system.

Effects of sotrastaurin, mycophenolic acid and everolimus on human B-lymphocyte function and activation.

Humoral rejection processes may lead to allograft injury and subsequent dysfunction. Today, only one B-cell-specific agent is in clinical use and the effects of standard and new immunosuppressant substances on B-cell activation and function are not fully clarified. The impact of sotrastaurin, mycophenolic acid and everolimus on human B-lymphocyte function was assessed by analysing proliferation, apoptosis, CD80/CD86 expression and immunoglobulin and IL-10 production in primary stimulated B cells. In addition, B-cell co-cultures with pre-activated T cells were performed to evaluate the effect of the different immunosuppressive agents on T-cell-dependent immunoglobulin production. Sotrastaurin did not inhibit B-cell proliferation, CD80/CD86 expression, and IgG production and had only minor effects on IgM levels at the highest concentration administered. In contrast, mycophenolic acid and everolimus had strong effects on all B-cell functions in a dose-dependent manner. All immunosuppressive agents caused decreased immunoglobulin levels in T-cell-dependent B-cell cultures. The data provided here suggest that mycophenolic acid and everolimus, but not sotrastaurin, are potent inhibitors of human B-lymphocyte function and activation.

Lymphocyte signaling: regulation of FoxO transcription factors by microRNAs.

The Forkhead box O (FoxO) family of transcription factors is important for the maintenance of immunological homeostasis and tolerance by controlling the development and function of B and T lymphocytes. Because dysregulation in FoxO activity can result in chronic inflammation and autoimmunity, the transcriptional activity of FoxO proteins is tightly controlled and generally dependent on complex posttranslational modifications that lead either to their nuclear entry and subsequent activation or, alternatively, to their nuclear export. The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) axis represents the major pathway phosphorylating and thereby inactivating FoxO proteins. However, recent results have revealed an additional posttranscriptional mechanism of FoxO inactivation by microRNAs. The discovery of this molecular pathway may provide a new therapeutic avenue for the modulation of FoxO activity in immune-mediated diseases using either microRNA targeting antagomirs or synthetic microRNA mimics, a topic that is addressed in this review.

Evaluation of the novel protein kinase C inhibitor sotrastaurin as immunosuppressive therapy after renal transplantation.

IMPORTANCE OF THE FIELD: The prevalence of acute renal allograft rejection has decreased substantially in past decades due to new and more specific immunosuppressive compounds but improvements in long-term graft function have not been achieved. There is a large need for new immunosuppressive agents that lack toxicity of current agents such as calcineurin inhibitors but show high synergistic efficiency in preventing rejection processes. AREAS COVERED IN THIS REVIEW: This review summarizes data concerning the pharmacokinetics, pharmacodynamics and clinical efficacy of the new PKC inhibitor sotrastaurin with a focus on renal transplantation. The article contains information that has been presented at international transplant meetings and congresses and that has been published between 2006 and 2010. Additionally, current ongoing trials are described in detail. WHAT THE READER WILL GAIN: Immunosuppressive regimens after kidney transplantation consist of a combination of several agents in order to minimize drug toxicity. Therefore, the reader is presented with the most up-to-date/current developments in sotrastaurin applications in Phase I and II trials with emphasis on data maintained from studies that combined sotrastaurin with established agents such as mycophenolic acid and tacrolimus. TAKE HOME MESSAGE: Several trials are ongoing and planned to determine the optimal immunosuppressive regimen to benefit from sotrastaurin's distinct mechanism of action.

The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes.

After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor-mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo. Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell-mediated immune responses and open new therapeutic possibilities.