Molecular B-cell clonality assay in minor salivary glands as a useful tool for the lymphoma risk assessment in Sjögren's syndrome.

OBJECTIVES: Non-Hodgkin's lymphoma (NHL) risk assessment is crucial in Sjögren's syndrome (SS). We studied the prevalence of clonal immunoglobulin gene rearrangements in minor salivary glands (MSG) and their correlations with lymphoma occurrence and with previously established NHL predictors. METHODS: Molecular B-cell expansion was studied in fresh-frozen MSG of 207 patients with either suspected SS or with suspected lymphoma during SS, using a standardised multiplex PCR assay combined with heteroduplex analysis by microcapillary electrophoresis. The assignation of clonal cases was based on EuroClonality consortium guidelines. RESULTS: Among 207 studied patients, 31 (15%) had MSG monoclonal B-cell infiltration. Monoclonality was significantly more frequent in patients with SS (28/123, 22.8%) compared with patients without SS (3/84, 3.6%, P<0.001). Monoclonal B-cell infiltration in MSG of SS patients correlated significantly with ongoing salivary gland NHL, salivary gland swelling, CD4+ T-cell lymphopenia, rheumatoid factor (RF) activity, low complement levels and type 2 mixed cryoglobulinemia. The accumulation of biological risk factors was associated with a higher rate of MSG B-cell monoclonality given that patients with only positive RF had no probability of MSG B-cell monoclonality, RF-positive patients with 1 or 2 other risk factors had a 25.0% and 85.7% probability of MSG B-cell monoclonality, respectively. CONCLUSION: The detection of MSG monoclonal B-cell expansion by this easy-to-perform molecular assay is useful, both at the time of diagnosis and during the course of SS. Monoclonal B-cell expansion is associated with a subset of SS patients presenting either ongoing lymphoma or other established lymphoma predictive factors.

Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.

Immunophenotypic assessment of clonal plasma cells and B-cells in bone marrow and blood in the diagnostic classification of early stage monoclonal gammopathies: an iSTOPMM study.

Monoclonal gammopathy of undetermined significance (MGUS) is the earliest discernible stage of multiple myeloma (MM) and Waldenström's macroglobulinemia (WM). Early diagnosis of MG may be compromised by the low-level infiltration, undetectable to low-sensitive methodologies. Here, we investigated the prevalence and immunophenotypic profile of clonal (c) plasma cells (PC) and/or cB-lymphocytes in bone marrow (BM) and blood of subjects with a serum M-component from the iSTOPMM program, using high-sensitive next-generation flow cytometry (NGF), and its utility in the diagnostic classification of early-stage MG. We studied 164 paired BM and blood samples from 82 subjects, focusing the analysis on: 55 MGUS, 12 smoldering MM (SMM) and 8 smoldering WM (SWM). cPC were detected in 84% of the BM samples and cB-lymphocytes in 45%, coexisting in 39% of cases. In 29% of patients, the phenotypic features of cPC and/or cB-lymphocytes allowed a more accurate disease classification, including: 19/55 (35%) MGUS, 1/12 (8%) SMM and 2/8 (25%) SWM. Blood samples were informative in 49% of the BM-positive cases. We demonstrated the utility of NGF for a more accurate diagnostic classification of early-stage MG.

Research-based flow cytometry assays for pathogenic assessment in the human B-cell biology of gene variants revealed in the diagnosis of inborn errors of immunity: a Bruton's tyrosine kinase case-study.

Introduction: Inborn errors of immunity (IEI) are an expanding group of rare diseases whose field has been boosted by next-generation sequencing (NGS), revealing several new entities, accelerating routine diagnoses, expanding the number of atypical presentations and generating uncertainties regarding the pathogenic relevance of several novel variants. Methods: Research laboratories that diagnose and provide support for IEI require accurate, reproducible and sustainable phenotypic, cellular and molecular functional assays to explore the pathogenic consequences of human leukocyte gene variants and contribute to their assessment. We have implemented a set of advanced flow cytometry-based assays to better dissect human B-cell biology in a translational research laboratory. We illustrate the utility of these techniques for the in-depth characterization of a novel (c.1685G>A, p.R562Q) de novo gene variant predicted as probably pathogenic but with no previous insights into the protein and cellular effects, located in the tyrosine kinase domain of the Bruton's tyrosine kinase (BTK) gene, in an apparently healthy 14-year-old male patient referred to our clinic for an incidental finding of low immunoglobulin (Ig) M levels with no history of recurrent infections. Results and discussion: A phenotypic analysis of bone marrow (BM) revealed a slightly high percentage of pre-B-I subset in BM, with no blockage at this stage, as typically observed in classical X-linked agammaglobulinemia (XLA) patients. The phenotypic analysis in peripheral blood also revealed reduced absolute numbers of B cells, all pre-germinal center maturation stages, together with reduced but detectable numbers of different memory and plasma cell isotypes. The R562Q variant allows Btk expression and normal activation of anti-IgM-induced phosphorylation of Y551 but diminished autophosphorylation at Y223 after anti IgM and CXCL12 stimulation. Lastly, we explored the potential impact of the variant protein for downstream Btk signaling in B cells. Within the canonical nuclear factor kappa B (NF-κB) activation pathway, normal IκBα degradation occurs after CD40L stimulation in patient and control cells. In contrast, disturbed IκBα degradation and reduced calcium ion (Ca2+) influx occurs on anti-IgM stimulation in the patient's B cells, suggesting an enzymatic impairment of the mutated tyrosine kinase domain.

Flow cytometric assessment for minimal/measurable residual disease in B lymphoblastic leukemia/lymphoma in the era of immunotherapy.

Minimal/measurable residual disease (MRD) is the most important independent prognostic factor for patients with B-lymphoblastic leukemia (B-LL). MRD post therapy has been incorporated into risk stratification and clinical management, resulting in substantially improved outcomes in pediatric and adult patients. Currently, MRD in B-ALL is most commonly assessed by multiparametric flow cytometry and molecular (polymerase chain reaction or high-throughput sequencing based) methods. The detection of MRD by flow cytometry in B-ALL often begins with B cell antigen-based gating strategies. Over the past several years, targeted immunotherapy directed against B cell markers has been introduced in patients with relapsed or refractory B-ALL and has demonstrated encouraging results. However, targeted therapies have significant impact on the immunophenotype of leukemic blasts, in particular, downregulation or loss of targeted antigens on blasts and normal B cell precursors, posing challenges for MRD detection using standard gating strategies. Novel flow cytometric approaches, using alternative strategies for population identification, sometimes including alternative gating reagents, have been developed and implemented to monitor MRD in the setting of post targeted therapy.

Assessment of Plasmodium falciparum Antigen-Specific B Cells.

This protocol describes methods that exploit the specificity of binding between the B cell receptor and cognate antigen to detect and characterize Plasmodium-specific human B cells. Importantly, this approach allows for the isolation and study of B cells without activating the cells or requiring them to secrete antibodies. The protocol describes how antigen "probes" are generated and used in flow cytometry to identify and sort antigen-specific B cells, and includes methods for enrichment of antigen-specific B cells prior to flow cytometry. Finally, we detail techniques to optimize the exclusion of B cells that are not specific for the antigen of interest.

Assessment of plasmablast cells in immune thrombocytopenic purpura in children.

Primary immune thrombocytopenic purpura (ITP) is an autoimmune disorder with platelet destruction due to B- and T-cell dysregulation and antiplatelet autoantibodies production. Flow cytometry can be used to further characterize the B- and T-cell compartments involved in platelet destruction. This case-control study was to enumerate plasmablast cells in pediatric ITP patients and to correlate their levels with disease course. This study included 30 ITP patients and 10 controls. Identification and enumeration of Plasmablast were done by multicolor flow cytometry using specific antibody panels (CD19, CD27 & CD38) and sequential gating using FACSCanto flow cytometer and FlowJo software. We found that lymphocytes subpopulation in ITP patients and controls revealed increase in frequency of CD19 (B lymphocytes) in acute, persistent, and chronic ITP patients in comparison with controls (p < 0.001, 0.023, 0.001) respectively. Plasmablast cells could play a role in the pathogenesis of ITP and might guide therapy in ITP patients in the future.

Proportion versus absolute counts of B cell populations in the assessment of immunological profiles of kidney transplant recipients.

Immunophenotyping lymphocytes in kidney transplant recipients often raises questions as to whether proportions or absolute counts should be considered, especially for longitudinal assessment. Several studies conclude the pathophysiology of rejection based on proportions of naive and memory B cells. We compared the two analytical methods for B cells sampled from 82 clinically stable, adult kidney transplant recipients. Time post-transplant was analyzed both as a continuous variable and as tertiles (<1.5 years, 1.5-8 years, and > 8 years). B cells were stained for CD38 and IgD and were classified according to mature B cells (Bm) classification. The proportion of cells in the naive Bm2 compartment decreased by more than half in the late versus the early tertile, whereas the percentages of memory early Bm5 tripled and that of memory Bm5 cells doubled. In contrast, we observed a substantial reduction in naive B cell counts, but very stable memory B cell counts. Linear regressions showed that the absolute reduction in the Bm2 cell compartment was independent of age, sex, graft function, immunosuppression scheme, and rejection occurrence. In conclusion, the physiological reservoir of naive cells decreases over time post-transplant in kidney recipients, whereas that of memory B cells remains stable. Peripheral B subset percentages should be interpreted cautiously when analyzing pathophysiological processes.

Assessment of humoral and cellular immunity induced by the BNT162b2 SARS-CoV-2 vaccine in healthcare workers, elderly people, and immunosuppressed patients with autoimmune disease.

The development of vaccines to prevent SARS-CoV-2 infection has mainly relied on the induction of neutralizing antibodies (nAbs) to the Spike protein of SARS-CoV-2, but there is growing evidence that T cell immune response can contribute to protection as well. In this study, an anti-receptor binding domain (RBD) antibody assay and an INFγ-release assay (IGRA) were used to detect humoral and cellular responses to the Pfizer-BioNTech BNT162b2 vaccine in three separate cohorts of COVID-19-naïve patients: 108 healthcare workers (HCWs), 15 elderly people, and 5 autoimmune patients treated with immunosuppressive agents. After the second dose of vaccine, the mean values of anti-RBD antibodies (Abs) and INFγ were 123.33 U/mL (range 27.55-464) and 1513 mIU/mL (range 145-2500) in HCWs and 210.7 U/mL (range 3-500) and 1167 mIU/mL (range 83-2500) in elderly people. No correlations between age and immune status were observed. On the contrary, a weak but significant positive correlation was found between INFγ and anti-RBD Abs values (rho = 0.354, p = 0.003). As to the autoimmune cohort, anti-RBD Abs were not detected in the two patients with absent peripheral CD19+B cells, despite high INFγ levels being observed in all 5 patients after vaccination. Even though the clinical relevance of T cell response has not yet been established as a correlate of vaccine-induced protection, IGRA testing has showed optimal sensitivity and specificity to define vaccine responders, even in patients lacking a cognate antibody response to the vaccine.

Coformulation with Tattoo Ink for Immunological Assessment of Vaccine Immunogenicity in the Draining Lymph Node.

Characterization of germinal center B and T cell responses yields critical insights into vaccine immunogenicity. Nonhuman primates are a key preclinical animal model for human vaccine development, allowing both lymph node (LN) and circulating immune responses to be longitudinally sampled for correlates of vaccine efficacy. However, patterns of vaccine Ag drainage via the lymphatics after i.m. immunization can be stochastic, driving uneven deposition between lymphoid sites and between individual LN within larger clusters. To improve the accurate isolation of Ag-exposed LN during biopsies and necropsies, we developed and validated a method for coformulating candidate vaccines with tattoo ink in both mice and pigtail macaques. This method allowed for direct visual identification of vaccine-draining LN and evaluation of relevant Ag-specific B and T cell responses by flow cytometry. This approach is a significant advancement in improving the assessment of vaccine-induced immunity in highly relevant nonhuman primate models.

Next-Generation Sequencing-Based Clonality Assessment of Ig Gene Rearrangements: A Multicenter Validation Study by EuroClonality-NGS.

Ig gene (IG) clonality analysis has an important role in the distinction of benign and malignant B-cell lymphoid proliferations and is mostly performed with the conventional EuroClonality/BIOMED-2 multiplex PCR protocol and GeneScan fragment size analysis. Recently, the EuroClonality-NGS Working Group developed a method for next-generation sequencing (NGS)-based IG clonality analysis. Herein, we report the results of an international multicenter biological validation of this novel method compared with the gold standard EuroClonality/BIOMED-2 protocol, based on 209 specimens of reactive and neoplastic lymphoproliferations. NGS-based IG clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice.

Multiple Immunoglobulin κ Gene Rearrangements within a Single Clone Unraveled by Next-Generation Sequencing-Based Clonality Assessment.

Clonality assessment of the Ig heavy- and light-chain genes (IGH and IGK) using GeneScan analysis is an important supplemental assay in diagnostic testing for lymphoma. Occasionally cases with an IGK rearrangement pattern that cannot readily be assigned to a monoclonal lymphoma are encountered, whereas the occurrence of biclonal lymphomas is rare, and the result of the IGH locus of these cases is in line with monoclonality. Three such ambiguous cases were assessed for clonality using next-generation sequencing. Information on the sequences of the rearrangements, combined with knowledge of the complex organization of the IGK locus, pointed to two explanations that can attribute seemingly biclonal IGK rearrangements to a single clone. In two cases, this explanation involved inversion rearrangements on the IGK locus, whereas in the third case, the cross-reactivity of primers generated an additional clonal product. In conclusion, next-generation sequencing-based clonality assessment allows for the detection of both inversion rearrangements and the cross-reactivity of primers, and can therefore facilitate the interpretation of cases of lymphoma with complex IGK rearrangement patterns.

Cost-Effectiveness Analysis of Tisagenlecleucel for the Treatment of Pediatric and Young Adult Patients with Relapsed or Refractory B Cell Acute Lymphoblastic Leukemia in Japan.

Until recently, treatment options were relatively limited for children and young adults with relapsed or refractory (r/r) acute lymphoblastic leukemia (ALL). Tisagenlecleucel is a chimeric antigen receptor T cell (CAR-T) immunotherapy with promising efficacy and manageable safety that was approved in Japan in 2019 for the treatment of CD19-positive r/r B cell ALL (B-ALL). However, there is no publication assessing the cost-effectiveness of CAR-T in Japan. The objective of this study was to assess the cost-effectiveness of a tisagenlecleucel treatment strategy compared to a blinatumomab treatment strategy and a clofarabine combination treatment strategy (i.e., clofarabine + cyclophosphamide + etoposide) in Japan for pediatric and young adult patients up to 25 years of age with r/r B-ALL. A partitioned survival model with a lifetime horizon and monthly cycle was constructed from a Japanese public healthcare payer's perspective. Patients were distributed across the following partitioned health states: event-free survival (EFS), progressive disease, and death, which were informed by the EFS and overall survival (OS) data of respective clinical trials before year 5. For the tisagenlecleucel arm, a decision-tree structure was used to partition patients based on the infusion status; those who discontinued prior to receiving infusion were assigned efficacy and cost inputs of blinatumomab and those who received infusion were assigned efficacy and costs inputs based on tisagenlecleucel-infused patients. As trial data for blinatumomab and clofarabine ended before year 5, matching-adjusted indirect comparisons were used to extrapolate OS between the end of trial observation and up to year 5. All surviving patients followed the mortality risk of long-term ALL survivors without additional risk of disease relapse after year 5, regardless of initial treatment strategies. The model accounted for pretreatment costs, treatment costs, adverse event costs, follow-up costs, subsequent allogeneic hematopoietic stem cell transplantation costs, and terminal care costs. Incremental cost-effectiveness ratios (ICERs) per life-years (LYs) gained and ICERs per quality-adjusted life-years (QALYs) gained were evaluated using a 2% discount rate, and a threshold of ¥7.5 million was used to assess cost-effectiveness. Deterministic and probabilistic sensitivity analyses were performed. The total LYs (discounted) for tisagenlecleucel, blinatumomab, and clofarabine combination treatment strategies were 13.3, 4.0, and 2.7 years, respectively; the corresponding QALYs were 11.6, 3.1, and 2.1 years, respectively. The ICERs per QALY gained for tisagenlecleucel were ¥2,035,071 versus blinatumomab and ¥2,644,702 versus clofarabine combination therapy. Extensive sensitivity analyses supported the findings. Tisagenlecleucel is a cost-effective treatment strategy for pediatric and young adult patients with r/r B-ALL from a Japanese public healthcare payer's perspective.

Generation of a cost-effective cell line for support of high-throughput isolation of primary human B cells and monoclonal neutralizing antibodies.

The isolation of human monoclonal antibodies (mAbs) arising from natural infection with human pathogens has proven to be a powerful technology, facilitating the understanding of the host response to infection at a molecular level. mAbs can reveal sites of vulnerability on pathogens and illuminate the biological function of the antigenic targets. Moreover, mAbs have the potential to be used directly for therapeutic applications such as passive delivery to prevent infection in susceptible target populations, and as treatment of established infection. The isolation of antigen-specific B cells from vaccine trials can also assist in deciphering whether the desired B cells are being targeted by a given vaccine. Several different processes have been developed to isolate mAbs, but all are generally labor-intensive and result in varying degrees of efficiency. Here, we describe the development of a cost-effective feeder cell line that stably expresses CD40-ligand, interleukin-2 and interleukin-21. Sorting of single B cells onto a layer of irradiated feeder cells sustained antibody production that permits functional screening of secreted antibodies in a manner that enables subsequent recovery of B cells for recombinant antibody cloning. As a proof of concept, we show that this approach can be used to isolate B cells that secrete antibodies that neutralize human papilloma virus (HPV) from participants of an HPV vaccine study.

Maturation of T and B Lymphocytes in the Assessment of the Immune Status in COVID-19 Patients.

Cell response to novel coronavirus disease 19 (COVID-19) is currently a widely researched topic. The assessment of leukocytes population and the maturation of both B and T lymphocytes may be important in characterizing the immunological profile of COVID-19 patients. The aim of the present study was to evaluate maturation of B and T cells in COVID-19 patients with interstitial lesions on chest X-ray (COVID-19 X-ray (+)), without changes on X-ray (COVID-19 X-ray (-)) and in healthy control. The study group consisted of 23 patients divided on two groups: COVID-19 X-ray (+) n = 14 and COVID-19 X-ray (-) n = 9 and control n = 20. The flow cytometry method was performed. We observed a significantly higher percentage of plasmablasts and lower CD4+ lymphocytes in COVID-19 X-ray (+) patients than in COVID-19 X-ray (-) and control. In the COVID-19 X-ray (+) patients, there was a lower proportion of effector CD4+ T cells, naïve CD8+ T cells and higher central memory CD4+ cells and effector CD8+ T cells than control. The above results showed that the assessment of selected cells of B and T lymphocytes by flow cytometry can distinguish patients with COVID-19 and differentiate patients with and without changes on chest X-ray.

Multiplexed Functional Assessment of Genetic Variants in CARD11.

Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a "cloning-free" saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining.

To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.

Agammaglobulinemia: Epidemiology, Pathogenesis, Clinical Phenotype, Diagnosis, Prognosis and Management.

Agammaglobulinemia is a type of primary antibody deficiencies, characterized by severe reduction in serum level of all types of immunoglobulins level and absence of B cells in the peripheral blood. X-linked and various autosomal recessive/dominant mutations have been identified underlying the pathogenesis of this disorder. Affected patients present a broad range of clinical manifestations, including respiratory infections, gastrointestinal complications, Enterovirus infections, autoimmunity, and malignancies. This disease can be controlled by different therapeutic strategies. In this review, we describe different aspects of agammaglobulinemia such as epidemiology, pathogenesis, clinical phenotype, diagnosis, management, and prognosis of congenital agammaglobulinemia.

FB5P-seq: FACS-Based 5-Prime End Single-Cell RNA-seq for Integrative Analysis of Transcriptome and Antigen Receptor Repertoire in B and T Cells.

Single-cell RNA sequencing (scRNA-seq) allows the identification, characterization, and quantification of cell types in a tissue. When focused on B and T cells of the adaptive immune system, scRNA-seq carries the potential to track the clonal lineage of each analyzed cell through the unique rearranged sequence of its antigen receptor (BCR or TCR, respectively) and link it to the functional state inferred from transcriptome analysis. Here we introduce FB5P-seq, a FACS-based 5'-end scRNA-seq method for cost-effective, integrative analysis of transcriptome and paired BCR or TCR repertoire in phenotypically defined B and T cell subsets. We describe in detail the experimental workflow and provide a robust bioinformatics pipeline for computing gene count matrices and reconstructing repertoire sequences from FB5P-seq data. We further present two applications of FB5P-seq for the analysis of human tonsil B cell subsets and peripheral blood antigen-specific CD4 T cells. We believe that our novel integrative scRNA-seq method will be a valuable option to study rare adaptive immune cell subsets in immunology research.

Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection.

Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury.