IntroductionHumoral immunodeficiency is frequently observed in patients affected by chronic lymphocytic leukemia (CLL). The reasons for the compromised B-cell responses are only partly understood. We hypothesized that the normal residual B cells in CLL patients are negatively influenced in their functionality by soluble factors produced by CLL cells.MethodsWe performed functional B-cell assays to analyze the influence of serum from CLL patients or conditioned medium from CLL cells cultivated with stromal cells on B cells from healthy donors. Serum from healthy individuals or conditioned medium from normal B cells was used as controls, respectively. We stimulated the B cells in a T-cell-dependent fashion and incubated them in the respective serum or conditioned medium for 5 days. We measured plasma cell differentiation, proliferation, cell death, and B-cell activation, the latter using the markers CD80, CD86, and CD25.ResultsWe did not detect significant differences supporting the hypothesis that CLL-derived soluble factors negatively influence the proliferation, activation, or cell death levels of normal B cells from healthy individuals in in vitro coculture.DiscussionOur analysis suggests no major direct inhibitory effects of CLL-derived soluble factors on normal mature B cells. Thus, it is likely that other factors lead to the known B-cell dysfunction in CLL.

Dual productive B-cell receptor (BCR) rearrangements have been repeatedly reported for chronic lymphocytic leukemia (CLL), but the standard population-based PCR analyses cannot distinguish whether these are bi-clonal CLL, or a monoclonal CLL with bi-allelic productive rearrangements. We investigated CLL cells by combined single-cell RNA and BCR sequencing. We identified two CLL clones using different immunoglobulin (Ig) heavy-chain V region genes (IGHV) genes and distinct Ig lambda light chains. One clone is classified as Ig unmutated the other as mutated. The two CLL clones have distinct transcriptomes: Numerous genes were differentially expressed, with genes typical for unmutated or mutated CLL showing the expected representation in the two clones. Using PCR, cloning and Sanger sequencing of the IGHV rearrangements we detected both CLL clones over a period of three years without clinical progression of the CLL and thus giving insights into the disease biology of multi-clonal CLL.

Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 2331 DLBCL and memory states in MZL, we observed high variability in maturation state proportions between samples. For example, ABC DLBCL and FL tumors frequently contained both GC and post-GC subpopulations. Transcription factor mediators of the B cell maturation process remained active in malignancy. Copy number variation was observed between maturation states of the same tumor, indicating genetic variants can arise at sequential stages of the B cell maturation process in existent malignancy. Intratumor maturation states typically occupied distinct spatial niches. Although nodal architecture disruption and microenvironmental variation were observed across tumors, tumor cells often maintained proximity with follicular dendritic and follicular helper T cells, key mediators of the affinity maturation process.

Intratumor heterogeneity is intrinsic to cancer evolution and treatment resistance, although it is typically considered distinct from the differentiation processes driving cell-type and cancer-type diversity. Nodal B cell non-Hodgkin lymphomas are tied to distinct stages of B cell maturation. Through a single-cell multi-omic and spatial atlas of diffuse large B cell, mantle cell, follicular, and marginal zone lymphomas in addition to reactive lymph nodes in 51 patients, we uncovered co-existing B cell maturation states within the same tumors. These intratumor maturation states emerged from the same clone, revealing ongoing differentiation from the cell-of-origin. Maturation state composition varied across and within disease entities, with tumors encompassing mixed cell-of-origin clinical subtypes. Intratumor maturation states inhabited unique spatial niches, maintaining their maturation-associated cellular interactions and regulatory networks while harboring distinct genetic variant expression patterns. Our findings show that cell-type differentiation remains plastic in cancer and is central to tumor variation and evolution. ![Figure][1] ### Competing Interest Statement G.P.N. is a co-founder and stockholder of Akoya Biosciences, Inc. and inventor on patent US9909167 (On-slide staining by primer extension). [1]: pending:yes

Introduction: PAP is due to accumulation of phospholipids and lipoproteins in the alveoli. WLL is still the treatment of choice. Light microscopy examination of WLL fluid reveals a moderate to high lymphocytosis but lymphocyte subpopulations have not been further characterized at yet. Aims: To investigate T lymphocyte subsets in WLL fluid from patients with PAP. Methods: From the first recovered fluid portion during WLL, samples of 50 mL each were collected and analyzed fresh. Pancoll density-gradient centrifugation was performed to separate mononuclear (MN) cells. MACS® enrichment was used to separate T lymphocytes from remaining MN cells and acellular particles after adding anti-CD3 microbeads to the MN cell layer. Flow cytometry for T cell immunophenotyping was performed by using a 9-color antibody panel and a FACSAria™ Fusion cytometer (Becton Dickinson, Germany). Zombie UV™ fluorescent dye was used to assess cell viability. T lymphocyte counts were expressed as proportion of CD3+ viable lymphocyte counts. Results: WLL fluid from 3 consecutive PAP patients (2 M, 1 F, age 44±12) who underwent therapeutic WLL was analyzed. The mean T lymphocyte count was 87±1%. CD4+ T cells were the dominant T cell subset across all samples (73±7%), followed by CD8+ cytotoxic T cells (19±7%). Th17.1 cells were the most frequent Th cell subset (33±14%), followed by Th1 cells (29±2%), Tregs (17±6%), Th17 cells (4±2%), and Th2 cells (0.7±0.2%). Conclusions: Our study identified different T lymphocyte subsets, mostly related to autoimmunity, in WLL fluid obtained from PAP patients. Flow cytometry may be used to unravel pathogenetic mechanisms of this rare disease.

Improved classification of rare lymphoid neoplasms would be aided by a deeper understanding of their underlying molecular features and is important for diagnosis, prognosis and therapy. Tumor entities classified within the WHO category of splenic B cell lymphomas and leukemias often exhibit heterogenous, transecting features, and include hairy cell leukemia (HCL), splenic diffuse red pulp lymphoma (SDRPL), splenic marginal zone lymphoma (SMZL), and the newly described WHO entity, splenic B cell lymphoma/leukemia with prominent nucleoli (SBLPN); the latter including patients formerly classified as HCL-variant (HCL-V). Genome-wide epigenetic information provides a tumor cell fingerprint combining cell-of-origin and tumor-specific events. Here we used DNA methylation to perform an unbiased molecular subclassification and to explore novel biological aspects of these patients. Samples from patients with a pathological diagnosis of HCL, HCL-V, SDRPL and SMZL (made prior to the 5 th WHO revision and ICC classifications) were obtained from 19 institutions across 9 countries, totaling 367 patients. Cells were FACS-purified where necessary and DNA was analyzed by 450/850K Illumina DNA methylation arrays. Genetic mutations were assessed by whole-exome or targeted sequencing, IGHV-D-J sequences by Sanger sequencing, and copy number alterations (CNAs) by Illumina arrays. The 1000 most variable CpG methylation sites were used for k-means clustering. Recursive feature elimination/random forest algorithms were used to develop a classifier for DNA methylation-based subgroups with 98% accuracy. Unsupervised clustering of 197 patients diagnosed with HCL, HCL-V or SDRPL revealed 5 distinct DNA methylation (M) subgroups ( Figure 1). Subgroup assignment was stable throughout longitudinal sampling (including pre/post-treatment) and consistent between splenic, bone marrow and PBMC derived cells. A subgroup with universally clonal BRAF-V600E mutations and majority diagnosed as HCL was termed the M-HCL subgroup ( Table 1). Four other groups termed M-SBLPN1-4 contained all HCL-V and SDRPL diagnosed samples and were devoid of BRAF-V600E mutations. M-SBLPN1 comprised MAP2K1 mutations (91%) and was enriched for CREBBP, ARIDIA and TERT-promoter mutations. These patients displayed an HCL-like immunophenotype (64.3% CD25+) with 1/3 diagnosed as HCL. M-SBLPN2 exhibited the highest prevalence of TP53 mutations and concomitant genomic instability. Patients in M-SBLPN1,2 were enriched in unmutated IGHV4-34 rearrangements. M-SBLPN3,4 subgroups displayed an immunophenotype more dissimilar to HCL, mutated IGHV genes, and enrichment of IGLL5, SYK and BIRC3 mutations. M-SBLPN4 contained the most SDRPL samples, suggesting it may represent the SDRPL entity retained by the WHO. We next uncovered that 29/170 SMZL patients displayed DNA methylation patterns mapping to M-SBLPN2-4. These patients were phenotypically and molecularly similar to SBLPN (70% displaying villous morphology and depleted in IGHV1-2*04, NOTCH2, KLF2 mutations), likely representing SMZL patients suggested for reassignment to SBLPN in the updated WHO classification. To elucidate molecular pathways governing the biology of M-SBLPN subgroups, transcription factor motif enrichment analysis in hypomethylated genomic regions revealed selective activation of AP-1 in M-HCL along with ETS in M-SBLPN1,2. Both transcription factors are downstream of MAPK signaling, consistent with activating BRAF and MAP2K1 mutations in these subgroups. However, we observed strong ETS enrichment in the absence of MAP2K1 in M-SBLPN along with mutual exclusivity of MAP2K1 and TP53 mutations, suggesting TP53 mutations are driving ETS activation. Although lymphoid neoplasms rarely exhibit TERT promoter mutations, 83% of M-SBLPN1 patients showed the c.-124C>T mutation commonly observed in other cancers producing an ETS binding site and ectopic TERT activation. ETS activation and gain of an ETS site by mutation implies oncogenesis involves aberrant TERT activation in this subgroup. In summary, we have developed a DNA methylation-based classifier that resolves 4 SBLPN subgroups with distinct molecular features, and reclassifies a subset of SMZL and HCL patients, adding further information to the updated WHO/ICC entities. We reveal distinct biological pathways operating in M-SBLPN subgroups that may aid targeted therapy approaches.

A hallmark of T cell dependent (TD) humoral immune responses is the generation of long–lived memory B cells. The generation of these cells occurs primarily in the germinal center (GC) reaction, where antigen–activated B cells undergo affinity maturation as a major consequence of the combined processes of proliferation, somatic hypermutation of their immunoglobulin V (IgV) region genes, and selection for improved affinity of their B–cell antigen receptors. As many B cells also undergo class–switching to IgG or IgA in these TD responses, there was traditionally a focus on class–switched memory B cells in both murine and human studies on memory B cells. However, it has become clear that there is also a large subset of IgM–expressing memory B cells, which have important phenotypic and functional similarities but also differences to class–switched memory B cells. There is an ongoing discussion about the origin of distinct subsets of human IgM+ B cells with somatically mutated IgV genes. We argue here that the vast majority of human IgM–expressing B cells with somatically mutated IgV genes in adults is indeed derived from GC reactions, even though a generation of some mostly lowly mutated IgM+ B cells from other differentiation pathways, mainly in early life, may exist.

In this review, we will summarize the growing body of knowledge on the age-related changes of human splenic B cell composition and molecular evidence of immune maturation and discuss the contribution of these changes on splenic protective function. From birth on, the splenic marginal zone (sMZ) contains a specialized B cell subpopulation, which recruits and archives memory B cells from immune responses throughout the organism. The quality of sMZ B cell responses is augmented by germinal center (GC)-dependent maturation of memory B cells during childhood, however, in old age, these mechanisms likely contribute to waning of splenic protective function.

Intratumor heterogeneity (ITH) refers to the coexistence of distinct cancer cell subpopulations within a single tumor, each with unique molecular and functional properties. Understanding the dynamics and evolution of ITH is crucial for predicting tumor progression and the development of therapy resistance. Here, we conducted a comprehensive investigation of ITH in Chronic Lymphocytic Leukemia (CLL) and identified a subpopulation that carried the CLL B-cell receptor rearrangement but contrasted from the CLL main population by low CD5 and high CD20 expression and highly mutated Ig-genes. This CLL-related memory B-cell population shared somatic driver mutations with the main CLL population but also exhibited exclusive somatic mutations. Phylogenetic analysis suggested a pathogenically early generation of these CLL-related B cells before monoclonal B lymphocytosis or CLL manifestation. Our data indicated that CLL-related B cells have self-replenishing potential, as they diminish upon treatment but recover indistinguishably on relapse. This contrasts with the main CLL population, which mostly represents a selective and novel expansion of the CLL-related B cells. This differentiation capacity into conventional CLL cells, and the expression of leukemic stem cell signatures further supported their tumorigenic capacity. We propose that these CLL-related B cells represent a pool of highly diversified, early-stage CLL precursor cells, which persist in the shape of “malignant memory B cells”. Longitudinal analyses of these CLL precursor cells suggested that they form a reservoir of malignant, leukemia-originating cells which contribute during disease progression to CLL outgrowth and clonal evolution. ### Competing Interest Statement The authors have declared no competing interest.

Topic: 20. Lymphoma Biology & Translational Research Background: Intratumor heterogeneity (ITH) is increasingly recognized as an essential factor in the variability of B cell non-Hodgkin lymphoma (B-NHL) patient outcomes. It is well established that distinct nodal B cell non-Hodgkin lymphoma (NBNHL) entities originate from different stages of the humoral affinity maturation process, but the drivers of ITH are not well understood. Aims: In this study, we aimed to characterize ITH in B-NHL and investigate its relationship with the nodal B cell affinity maturation process. Methods: 51 diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and non-malignant reactive lymph node (rLN) samples from distinct patients were sequenced with CITE-Seq (coupling single-cell RNA-seq with surface epitope profiling). Transcription factor activities and copy number variants (validated against targeted DNA sequencing) were inferred computationally from gene expression, while B cell receptor clones were determined with VDJ profiling. Spatial relationships were determined with highly multiplexed immunochemistry (CODEX). Informed by gene expression signatures from RNA sequencing of FACS-identified nodal B cell maturation states, a CITE-Seq reference map was constructed in reactive lymph nodes. This reference map was used to classify maturation states in the malignant samples with logistic regression, which were verified and refined by their maturation marker profiles. Using the shared features between CITE-Seq and CODEX panels, maturation states were similarly classified in the spatial data and their microenvironments were characterized with cellular neighborhood analysis. (Fig A) Results: As in the non-malignant context, variability in maturation states was also observed among malignant cells in most NBNHL tumors. BCR-sequencing and light chain restriction confirmed that different intra-tumor maturation states observed in samples were monoclonal, and therefore arose from the same cell of origin. Although commonalities in composition existed between samples within entities, such as an abundance of germinal center (GC) states in DLBCL and memory states in MZL, we observed high variability in maturation state proportions between samples. For example, ABC DLBCL and FL tumors frequently contained both GC and post-GC subpopulations. Transcription factor mediators of the B cell maturation process remained active in malignancy. Copy number variation was observed between maturation states of the same tumor, indicating genetic variants can arise at sequential stages of the B cell maturation process in existent malignancy. Intratumor maturation states typically occupied distinct spatial niches. Although nodal architecture disruption and microenvironmental variation were observed across tumors, tumor cells often maintained proximity with follicular dendritic and follicular helper T cells, key mediators of the affinity maturation process. Summary/Conclusion: Our findings reveal that B cell maturation is not halted during malignancy, but remains a continuous and divergent process central to inter- and intratumor variation. Intratumor maturation states can be subject to genetic variation and occupy distinct spatial niches while retaining the cellular interactions and signals which enable the maturation process. (Fig B)Keywords: B cell development, B cell lymphoma, B cell subsets, OMICS

Young children and older adults suffer from enhanced susceptibility to infections with blood-borne pathogens. An essential step towards immunity is the establishment of a splenic marginal zone (sMZ), which is immature at below 2 years of age. At approximately 5 years of age, an adult level of protection is reached but wanes again in older adults. Although the infant sMZ is thought to contain mostly naïve B cells, memory B cells are recruited to and recirculate from the sMZ throughout life, and class-switched sMZ B cells dominate in older adults. For a better resolution of naïve versus memory B-cell subset accumulation in the sMZ, we performed a single cell-based gene expression analysis of (CD21highIgMhigh) sMZ B cells among five healthy donors (age 3 to 48 years) and validated the sMZ B-cell subset composition by flow cytometry of 147 spleen biopsies (age 0 to 82 years). We identified a major sMZ B-cell subpopulation, which is abundant at birth but decreases with age. These cells lack CD27 expression but carry a weak-to-intermediate memory B-cell signature. These CD27neg sMZ B cells are either IGHV-unmutated or carry only a few IGHV mutations early in life but show average memory B-cell IGHV mutation frequencies (>3%) in adults. The activation and proliferation potential of CD27neg sMZ B cells is significantly above that of non-sMZ B cells already in children. Our study suggests that the human sMZ B-cell pool changes with age, encompassing a major population of lowly Ig-mutated CD27neg but antigen-experienced B cells early in life.

Human memory B cells (MBCs) are generated and diversified in secondary lymphoid tissues throughout the organism. A paired immunoglobulin (Ig)-gene repertoire analysis of peripheral blood (PB) and splenic MBCs from infant, adult, and elderly humans revealed that throughout life, circulating MBCs are comprehensively archived in the spleen. Archive MBC clones are systematically preserved and uncoupled from class-switching. Clonality in the spleen increases steadily, but boosts at midlife, thereby outcompeting small clones. The splenic marginal zone (sMZ) represents a primed MBC compartment, generated from a stochastic exchange within the archive memory pool. This is supported by functional assays, showing that PB and splenic CD21+ MBCs acquire transient CD21high expression upon NOTCH2-stimulation. Our study provides insight that the human MBC system in PB and spleen is composed of three interwoven compartments: the dynamic relationship of circulating, archive, and its subset of primed (sMZ) memory changes with age, thereby contributing to immune aging.

Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.

The human infant B cell system is considered premature or impaired. Here we show that most cord blood B cells are mature and functional as seen in adults, albeit with distinct transcriptional programs providing accelerated responsiveness to T cell-independent and T cell-dependent stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into antibody-secreting cells, thereby presumably limiting memory B cell formation. The neonatal Ig-repertoire is highly variable, but conserved, showing recurrent B cell receptor (BCR) clonotypes frequently shared between neonates. Our study demonstrates that cord blood B cells are not impaired but differ from their adult counterpart in a conserved BCR repertoire and rapid but transient response dynamics. These properties may account for the sensitivity of neonates to infections and limited effectivity of vaccination strategies. Humanized mice suggest that the distinctness of cord blood versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice. Still, our findings reveal overall limited comparability of human cord blood B cells and murine B-1 cells.Significance Statement Neonates and infants suffer from enhanced susceptibility to infections. Our study contrasts with the current concept of a premature or impaired B cell system in neonates, by showing that most cord blood B cells are mature and functional. However, their responses are rapid but provide only short-term protection, which may help to improve infant vaccination strategies. We propose an altered perspective on the early human B cell system, which looks similar to but functions differently from the adult counterpart. Finally, our analysis indicates that cord blood- and adult B cell development occur layered as in mice, but certain mouse models still may offer a limited view on human neonatal B cell immunity.### Competing Interest StatementThe authors have declared no competing interest.