Less is more: An analysis of venetoclax and hypomethylating agent post-induction treatment modifications in AML.

Venetoclax (Ven) combined with a hypomethylating agent (HMA) enhances survival in elderly/unfit acute myeloid leukemia (AML) patients, yet often necessitates regimen modifications due to intolerance. However, it is unclear how these modifications affect patient outcome. This retrospective cohort study evaluates the impact of post-induction HMA/Ven regimen modifications on disease progression and survival. This study reviewed 142 AML patients treated with HMA/Ven within the Northwell Health System from January 2019 to December 2022. To assess the impact of post-induction regimen modifications, patients were grouped according to median days between cycles (≤34 or ≥35 days cycle intervals) and median Ven days per cycle (≤14 or ≥15 days/cycle) based on only cycle 3 and beyond. Kaplan-Meier and Cox proportional hazard regression analyses were employed for univariate and multivariate assessments, respectively. There was no significant difference in median progression-free survival (mPFS)(11.6 vs 11.8 months, p = 0.73) or median overall survival (mOS)(15.1 vs 21.8 months, p = 0.16) between cycle interval groups. However, there was a clinically and statistically significant advantage in mPFS (15.8 vs 8.7 months, p = 0.01) and mOS (24.7 vs 11.3 months, p = 0.006) for patients with a median of ≤14 Ven days/cycle compared to ≥15 Ven days/cycle. Multivariate analysis demonstrated that ≤14 days of Ven for cycle 3 and beyond was an independent predictor of decreased mortality (HR 0.18, CI 0.07-0.48, p = 0.0007). Extended cycle intervals did not adversely affect mortality while reduced Ven duration per cycle post-induction was associated with improved survival in elderly AML patients.

Characterization of the Intraclonal Complexity of Chronic Lymphocytic Leukemia B Cells: Potential Influences of B-Cell Receptor Crosstalk with Other Stimuli.

Chronic lymphocytic leukemia (CLL) clones contain subpopulations differing in time since the last cell division ("age"): recently born, proliferative (PF; CXCR4DimCD5Bright), intermediate (IF; CXCR4IntCD5Int), and resting (RF; CXCR4BrightCD5Dim) fractions. Herein, we used deuterium (2H) incorporation into newly synthesized DNA in patients to refine the kinetics of CLL subpopulations by characterizing two additional CXCR4/CD5 fractions, i.e., double dim (DDF; CXCR4DimCD5Dim) and double bright (DBF; CXCR4BrightCD5Bright); and intraclonal fractions differing in surface membrane (sm) IgM and IgD densities. Although DDF was enriched in recently divided cells and DBF in older cells, PF and RF remained the most enriched in youngest and oldest cells, respectively. Similarly, smIgMHigh and smIgDHigh cells were the youngest, and smIgMLow and smIgDLow were the oldest, when using smIG levels as discriminator. Surprisingly, the cells closest to the last stimulatory event bore high levels of smIG, and stimulating via TLR9 and smIG yielded a phenotype more consistent with the in vivo setting. Finally, older cells were less sensitive to in vivo inhibition by ibrutinib. Collectively, these data define additional intraclonal subpopulations with divergent ages and phenotypes and suggest that BCR engagement alone is not responsible for the smIG levels found in vivo, and the differential sensitivity of distinct fractions to ibrutinib might account, in part, for therapeutic relapse.

Impact of the Types and Relative Quantities of IGHV Gene Mutations in Predicting Prognosis of Patients With Chronic Lymphocytic Leukemia.

Patients with CLL with mutated IGHV genes (M-CLL) have better outcomes than patients with unmutated IGHVs (U-CLL). Since U-CLL usually express immunoglobulins (IGs) that are more autoreactive and more effectively transduce signals to leukemic B cells, B-cell receptor (BCR) signaling is likely at the heart of the worse outcomes of CLL cases without/few IGHV mutations. A corollary of this conclusion is that M-CLL follow less aggressive clinical courses because somatic IGHV mutations have altered BCR structures and no longer bind stimulatory (auto)antigens and so cannot deliver trophic signals to leukemic B cells. However, the latter assumption has not been confirmed in a large patient cohort. We tried to address the latter by measuring the relative numbers of replacement (R) mutations that lead to non-conservative amino acid changes (Rnc) to the combined numbers of conservative (Rc) and silent (S) amino acid R mutations that likely do not or cannot change amino acids, "(S+Rc) to Rnc IGHV mutation ratio". When comparing time-to-first-treatment (TTFT) of patients with (S+Rc)/Rnc ≤ 1 and >1, TTFTs were similar, even after matching groups for equal numbers of samples and identical numbers of mutations per sample. Thus, BCR structural change might not be the main reason for better outcomes for M-CLL. Since the total number of IGHV mutations associated better with longer TTFT, better clinical courses appear due to the biologic state of a B cell having undergone many stimulatory events leading to IGHV mutations. Analyses of larger patient cohorts will be needed to definitively answer this question.

Activated CLL cells regulate IL-17F-producing Th17 cells in miR155-dependent and outcome-specific manners.

Chronic lymphocytic leukemia (CLL) results from expansion of a CD5+ B cell clone that requires interactions with other cell types, including T cells. Moreover, patients with CLL have elevated levels of circulating IL-17A+ and IL-17F+ CD4+ T (Th17) cells, with higher numbers of IL-17A+ Th17 cells correlating with better outcomes. We report that CLL Th17 cells expressed more miR155, a Th17-differentiation regulator, than control Th17 cells, despite naive CD4+ T (Tn) cell basal miR155 levels being similar in both. We also found that CLL cells directly regulated miR155 levels in Tn cells, thereby affecting Th17 differentiation, by documenting that coculturing Tn cells with resting or activated (Bact) CLL cells altered the magnitude and direction of T cell miR155 levels; CLL Bact cells promoted IL-17A+ and IL-17F+ T cell generation by an miR155-dependent mechanism, confirmed by miR155 inhibition; coculture of Tn cells with CLL Bact cells led to a linear correlation between the degree and direction of T cell miR155 expression changes and production of IL-17F but not IL-17A; and Bact cell-mediated changes in Tn cell miR155 expression correlated with outcome, irrespective of IGHV mutation status, a strong prognostic indicator. These results identify a potentially unrecognized CLL Bact cell-dependent mechanism, upregulation of Tn cell miR155 expression and subsequent enhancement of IL-17F+ Th17 generation, that favors better clinical courses.

Post-Transformation IGHV-IGHD-IGHJ Mutations in Chronic Lymphocytic Leukemia B Cells: Implications for Mutational Mechanisms and Impact on Clinical Course.

Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.

Myeloid-derived suppressor cell subtypes differentially influence T-cell function, T-helper subset differentiation, and clinical course in CLL.

Cancer pathogenesis involves the interplay of tumor- and microenvironment-derived stimuli. Here we focused on the influence of an immunomodulatory cell type, myeloid-derived suppressor cells (MDSCs), and their lineage-related subtypes on autologous T lymphocytes. Although MDSCs as a group correlated with an immunosuppressive Th repertoire and worse clinical course, MDSC subtypes (polymorphonuclear, PMN-MDSC, and monocytic, M-MDSCs) were often functionally discordant. In vivo, PMN-MDSCs existed in higher numbers, correlated with different Th-subsets, and more strongly associated with poor clinical course than M-MDSCs. In vitro, PMN-MDSCs were more efficient at blocking T-cell growth and promoted Th17 differentiation. Conversely, in vitro M-MDSCs varied in their ability to suppress T-cell proliferation, due to the action of TNFα, and promoted a more immunostimulatory Th compartment. Ibrutinib therapy impacted MDSCs differentially as well, since after initiating therapy, PMN-MDSC numbers progressively declined, whereas M-MDSC numbers were unaffected, leading to a set of less immunosuppressive Th cells. Consistent with this, clinical improvement based on decreasing CLL-cell numbers correlated with the decrease in PMN-MDSCs. Collectively, the data support a balance between PMN-MDSC and M-MDSC numbers and function influencing CLL disease course.

Postdischarge thromboembolic outcomes and mortality of hospitalized patients with COVID-19: the CORE-19 registry.

Thromboembolic events, including venous thromboembolism (VTE) and arterial thromboembolism (ATE), and mortality from subclinical thrombotic events occur frequently in coronavirus disease 2019 (COVID-19) inpatients. Whether the risk extends postdischarge has been controversial. Our prospective registry included consecutive patients with COVID-19 hospitalized within our multihospital system from 1 March to 31 May 2020. We captured demographics, comorbidities, laboratory parameters, medications, postdischarge thromboprophylaxis, and 90-day outcomes. Data from electronic health records, health informatics exchange, radiology database, and telephonic follow-up were merged. Primary outcome was a composite of adjudicated VTE, ATE, and all-cause mortality (ACM). Principal safety outcome was major bleeding (MB). Among 4906 patients (53.7% male), mean age was 61.7 years. Comorbidities included hypertension (38.6%), diabetes (25.1%), obesity (18.9%), and cancer history (13.1%). Postdischarge thromboprophylaxis was prescribed in 13.2%. VTE rate was 1.55%; ATE, 1.71%; ΑCM, 4.83%; and MB, 1.73%. Composite primary outcome rate was 7.13% and significantly associated with advanced age (odds ratio [OR], 3.66; 95% CI, 2.84-4.71), prior VTE (OR, 2.99; 95% CI, 2.00-4.47), intensive care unit (ICU) stay (OR, 2.22; 95% CI, 1.78-2.93), chronic kidney disease (CKD; OR, 2.10; 95% CI, 1.47-3.0), peripheral arterial disease (OR, 2.04; 95% CI, 1.10-3.80), carotid occlusive disease (OR, 2.02; 95% CI, 1.30-3.14), IMPROVE-DD VTE score ≥4 (OR, 1.51; 95% CI, 1.06-2.14), and coronary artery disease (OR, 1.50; 95% CI, 1.04-2.17). Postdischarge anticoagulation was significantly associated with reduction in primary outcome (OR, 0.54; 95% CI, 0.47-0.81). Postdischarge VTE, ATE, and ACM occurred frequently after COVID-19 hospitalization. Advanced age, cardiovascular risk factors, CKD, IMPROVE-DD VTE score ≥4, and ICU stay increased risk. Postdischarge anticoagulation reduced risk by 46%.

A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice.

Patient-derived xenograft models of chronic lymphocytic leukemia (CLL) can be created using highly immunodeficient animals, allowing analysis of primary tumor cells in an in vivo setting. However, unlike many other tumors, CLL B lymphocytes do not reproducibly grow in xenografts without manipulation, proliferating only when there is concomitant expansion of T cells. Here we show that in vitro pre-activation of CLL-derived T lymphocytes allows for a reliable and robust system for primary CLL cell growth within a fully autologous system that uses small numbers of cells and does not require pre-conditioning. In this system, growth of normal T and leukemic B cells follows four distinct temporal phases, each with characteristic blood and tissue findings. Phase 1 constitutes a period during which resting CLL B cells predominate, with cells aggregating at perivascular areas most often in the spleen. In Phase 2, T cells expand and provide T-cell help to promote B-cell division and expansion. Growth of CLL B and T cells persists in Phase 3, although some leukemic B cells undergo differentiation to more mature B-lineage cells (plasmablasts and plasma cells). By Phase 4, CLL B cells are for the most part lost with only T cells remaining. The required B-T cell interactions are not dependent on other human hematopoietic cells nor on murine macrophages or follicular dendritic cells, which appear to be relatively excluded from the perivascular lymphoid aggregates. Notably, the growth kinetics and degree of anatomic localization of CLL B and T cells is significantly influenced by intravenous versus intraperitoneal administration. Importantly, B cells delivered intraperitoneally either remain within the peritoneal cavity in a quiescent state, despite the presence of dividing T cells, or migrate to lymphoid tissues where they actively divide; this dichotomy mimics the human condition in that cells in primary lymphoid tissues and the blood are predominately resting, whereas those in secondary lymphoid tissues proliferate. Finally, the utility of this approach is illustrated by documenting the effects of a bispecific antibody reactive with B and T cells. Collectively, this model represents a powerful tool to evaluate CLL biology and novel therapeutics in vivo.

Musashi 2 influences chronic lymphocytic leukemia cell survival and growth making it a potential therapeutic target.

Progression of chronic lymphocytic leukemia (CLL) results from the expansion of a small fraction of proliferating leukemic B cells. When comparing the global gene expression of recently divided CLL cells with that of previously divided cells, we found higher levels of genes involved in regulating gene expression. One of these was the oncogene Musashi 2 (MSI2), an RNA-binding protein that induces or represses translation. While there is an established role for MSI2 in normal and malignant stem cells, much less is known about its expression and role in CLL. Here we report for the first time ex vivo and in vitro experiments that MSI2 protein levels are higher in dividing and recently divided leukemic cells and that downregulating MSI2 expression or blocking its function eliminates primary human and murine CLL and mature myeloid cells. Notably, mature T cells and hematopoietic stem and progenitor cells are not affected. We also confirm that higher MSI2 levels correlate with poor outcome markers, shorter time-to-first-treatment, and overall survival. Thus, our data highlight an important role for MSI2 in CLL-cell survival and proliferation and associate MSI2 with poor prognosis in CLL patients. Collectively, these findings pinpoint MSI2 as a potentially valuable therapeutic target in CLL.

CLL intraclonal fractions exhibit established and recently acquired patterns of DNA methylation.

Intraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.

Multiplex accurate sensitive quantitation (MASQ) with application to minimal residual disease in acute myeloid leukemia.

Measuring minimal residual disease in cancer has applications for prognosis, monitoring treatment and detection of recurrence. Simple sequence-based methods to detect nucleotide substitution variants have error rates (about 10-3) that limit sensitive detection. We developed and characterized the performance of MASQ (multiplex accurate sensitive quantitation), a method with an error rate below 10-6. MASQ counts variant templates accurately in the presence of millions of host genomes by using tags to identify each template and demanding consensus over multiple reads. Since the MASQ protocol multiplexes 50 target loci, we can both integrate signal from multiple variants and capture subclonal response to treatment. Compared to existing methods for variant detection, MASQ achieves an excellent combination of sensitivity, specificity and yield. We tested MASQ in a pilot study in acute myeloid leukemia (AML) patients who entered complete remission. We detect leukemic variants in the blood and bone marrow samples of all five patients, after induction therapy, at levels ranging from 10-2 to nearly 10-6. We observe evidence of sub-clonal structure and find higher target variant frequencies in patients who go on to relapse, demonstrating the potential for MASQ to quantify residual disease in AML.

Mechanism for IL-15-Driven B Cell Chronic Lymphocytic Leukemia Cycling: Roles for AKT and STAT5 in Modulating Cyclin D2 and DNA Damage Response Proteins.

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients.

Mechanistic Insights into CpG DNA and IL-15 Synergy in Promoting B Cell Chronic Lymphocytic Leukemia Clonal Expansion.

Malignant cell growth within patients with B cell chronic lymphocytic leukemia (B-CLL) is largely restricted to lymphoid tissues, particularly lymph nodes. The recent in vitro finding that TLR-9 ligand (oligodeoxynucleotide [ODN]) and IL-15 exhibit strong synergy in promoting B-CLL growth may be particularly relevant to growth in these sites. This study shows IL-15-producing cells are prevalent within B-CLL-infiltrated lymph nodes and, using purified B-CLL cells from blood, investigates the mechanism for ODN and IL-15 synergy in driving B-CLL growth. ODN boosts baseline levels of phospho-RelA(S529) in B-CLL and promotes NF-κB-driven increases in IL15RA and IL2RB mRNA, followed by elevated IL-15Rα and IL-2/IL-15Rß (CD122) protein. IL-15→CD122 signaling during a critical interval, 20 to 36-48 h following initial ODN exposure, is required for optimal induction of the cycling process. Furthermore, experiments with neutralizing anti-IL-15 and anti-CD122 mAbs indicate that clonal expansion requires continued IL-15/CD122 signaling during cycling. The latter is consistent with evidence of heightened IL2RB mRNA in the fraction of recently proliferated B-CLL cells within patient peripheral blood. Compromised ODN+IL-15 growth with limited cell density is consistent with a role for upregulated IL-15Rα in facilitating homotypic trans IL-15 signaling, although there may be other explanations. Together, the findings show that ODN and IL-15 elicit temporally distinct signals that function in a coordinated manner to drive B-CLL clonal expansion.

Romidepsin is effective and well tolerated in older patients with peripheral T-cell lymphoma: analysis of two phase II trials.

Peripheral T-cell lymphomas (PTCLs) are a rare group of lymphoid neoplasms with high relapse rates after initial therapy and poor prognosis. Most patients are aged ≥60 years and are often not candidates for aggressive salvage therapies. Romidepsin, a potent class I histone deacetylase inhibitor, has shown significant single-agent activity in relapsed/refractory PTCL. We evaluated the efficacy and tolerability of romidepsin in elderly patients in this setting. Ninety-five patients aged ≥60 years were identified from 2 prospective phase 2 registration trials of romidepsin, and comparative analyses were performed with younger patients from these trials. Response rates, progression-free survival, and overall survival were not statistically different for younger vs older patients. The toxicity profile in older and younger patients was similar in both trials. Romidepsin demonstrated similar efficacy and tolerability in younger and older patients and presents an attractive treatment option for relapsed/refractory PTCL regardless of age. TRIAL REGISTRATION: Clinicaltrials.gov identifiers: NCT00426764, NCT00007345.

Binding of CLL subset 4 B-cell receptor immunoglobulins to viable human memory B lymphocytes requires a distinctive IGKV somatic mutation.

Amino acid replacement mutations in certain CLL stereotyped B-cell receptor (BCR) immunoglobulins (IGs) at defined positions within antigen-binding sites strongly imply antigen selection. Prime examples of this are CLL subset 4 BCR IGs using IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements. Conspicuously and unlike most CLL IGs, subset 4 IGs do not bind apoptotic cells. By testing the (auto)antigenic reactivities of subset 4 IGs toward viable lymphoid-lineage cells and specific autoantigens typically bound by IGHV4-34+ IGs, we found IGs from both subset 4 and non-subset 4 IGHV4-34-expressing CLL cases bind naïve B cells. However, only subset 4 IGs react with memory B cells. Furthermore, subset 4 IGs do not bind DNA nor i or I carbohydrate antigens, common targets of IGHV4-34-utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively. Notably, we found that subset 4 IG binding to memory B lymphocytes depends on an aspartic acid at position 66 of FR3 in the rearranged IGKV2-30 gene; this amino acid residue is acquired by somatic mutation. Our findings illustrate the importance of positive and negative selection criteria for structural elements in CLL IGs and suggest that autoantigens driving normal B cells to become subset 4 CLL cells differ from those driving IGHV4-34+ B cells in other diseases.

Chronic lymphocytic leukemia immunoglobulins display bacterial reactivity that converges and diverges from auto-/poly-reactivity and IGHV mutation status.

Chronic lymphocytic leukemia (CLL) is an incurable leukemia of unknown etiology. Multiple studies suggest that the structure of the variable domains of the surface IGs on these cells, and signaling through them, play key roles in developing the disease. Hence, CLL appears to be driven by antigen-BCR interactions, and identifying the selecting antigens involved in this process is an important goal. We studied the antigen-binding characteristics of 23 CLL-derived, recombinantly-expressed IGs with 5 pathogenic bacteria, determining that CLL IGs differ in bacterial reactivity based on IGHV gene use, mutation status, and association with IGHD and IGHJ genes ("stereotypy"). Although most bacterial-reactive IGs followed the paradigm that IGHV-unmutated IGs were more auto-/poly-reactive, several did not. In addition, some CLL IGs were bacterial mono-reactive, and these displayed IGKV use biases. These findings are consistent with CLL B cells being driven into the leukemogenic process by bacterial as well as auto- antigens.

Chronic lymphocytic leukemia cells diversify and differentiate in vivo via a nonclassical Th1-dependent, Bcl-6-deficient process.

Xenografting primary tumor cells allows modeling of the heterogeneous natures of malignant diseases and the influences of the tissue microenvironment. Here, we demonstrate that xenografting primary chronic lymphocytic leukemia (CLL) B lymphocytes with activated autologous T cells into alymphoid mice results in considerable CLL B cell division and sizable T cell expansion. Nevertheless, most/all CD5+CD19+ cells are eventually lost, due in part to differentiation into antibody-secreting plasmablasts/plasma cells. CLL B cell differentiation is associated with isotype class switching and development of new IGHV-D-J mutations and occurs via an activation-induced deaminase-dependent pathway that upregulates IRF4 and Blimp-1 without appreciable levels of the expected Bcl-6. These processes were induced in IGHV-unmutated and IGHV-mutated clones by Th1-polarized T-bet+ T cells, not classical T follicular helper (Tfh) cells. Thus, the block in B cell maturation, defects in T cell action, and absence of antigen-receptor diversification, which are often cardinal characteristics of CLL, are not inherent but imposed by external signals and the microenvironment. Although these activities are not dominant features in human CLL, each occurs in tissue proliferation centers where the mechanisms responsible for clonal evolution operate. Thus, in this setting, CLL B cell diversification and differentiation develop by a nonclassical germinal center-like reaction that might reflect the cell of origin of this leukemia.