Identification of Distinct Unmutated Chronic Lymphocytic Leukemia Subsets in Mice Based on Their T Cell Dependency

Chronic lymphocytic leukemia (CLL) can be divided into prognostically distinct subsets with stereotyped or non-stereotyped, mutated or unmutated B cell receptors (BCRs). Individual subsets vary in antigen specificity and origin, but the impact of antigenic pressure on the CLL BCR repertoire remains unknown. Here, we employed IgH.TEμ mice that spontaneously develop CLL, expressing mostly unmutated BCRs of which ~35% harbor VH11-2/Vκ14-126 and recognize phosphatidylcholine. Proportions of VH11/Vκ14-expressing CLL were increased in the absence of functional germinal centers in IgH.TEμ mice deficient for CD40L or activation-induced cytidine deaminase. Conversely, in vivo T cell-dependent immunization decreased the proportions of VH11/Vκ14-expressing CLL. Furthermore, CLL onset was accelerated by enhanced BCR signaling in Siglec-G−/− mice or in mice expressing constitutively active Bruton's tyrosine kinase. Transcriptional profiling revealed that VH11 and non-VH11 CLL differed in the upregulation of specific pathways implicated in cell signaling and metabolism. Interestingly, principal component analyses using the 148 differentially expressed genes revealed that VH11 and non-VH11 CLL clustered with BCR-stimulated and anti-CD40-stimulated B cells, respectively. We identified an expression signature consisting of 13 genes that were differentially expressed in a larger panel of T cell-dependent non-VH11 CLL compared with T cell-independent VH11/Vκ14 or mutated IgH.TEμ CLL. Parallel differences in the expression of these 13 signature genes were observed between heterogeneous and stereotypic human unmutated CLL. Our findings provide evidence for two distinct unmutated CLL subsets with a specific transcriptional signature: one is T cell-independent and B-1 cell-derived while the other arises upon antigen stimulation in the context of T-cell help.


INTRODUCTION
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia characterized by an accumulation of monoclonal CD5 + mature B cells with low surface immunoglobulin (Ig) expression in peripheral blood (1).
CLL is a clinically and molecularly heterogeneous disease whereby progression is influenced by many factors. One-third of patients can be classified as stereotypic CLL, in which BCRs are highly similar between patients (2). The remaining two-third of CLL either lack or have limited similarity with stereotyped CLL BCRs. This classification provides strong molecular evidence for antigen selection in CLL pathogenesis (2). CLL can also be grouped based on IGHV mutational status (3,4). Significant (>2%) somatic hypermutation (SHM) is observed in patients with mutated CLL (M-CLL), who often develop indolent disease. SHM is absent in unmutated CLL (U-CLL) which evolves rapidly and has a less favorable prognosis (4). The SHM status provides a robust and stable prognostic marker, independently of clinical stage and other markers (5). Furthermore, it reinforces the role of selection by self-antigens or exogenous antigens in CLL pathogenesis. CLL cells show constitutive activation of several BCR downstream kinases, increasing leukemic cell survival in vitro (6). In support, small molecule inhibitors of BCR-associated kinases including Bruton's tyrosine kinase (Btk) have shown impressive clinical anti-tumor activity (7,8).
Few external antigens that potentially drive CLL in vivo have been identified; CLL cells were shown to display antigenindependent, cell-autonomous signaling mediated by autorecognition (9). Several reports have shown that U-CLL express polyreactive BCRs that bind with low affinity to various autoantigens generated during apoptosis or oxidation (10,11). In this respect, they resemble natural antibodies secreted by B-1 cells in mice. B-1 cells are a self-renewing CD5 + B cell population with remarkably restricted IGHV gene usage and low or no SHM (12). B-1 cells are thought to be generated based on positive selection, by virtue of their receptor specificities to self-antigens, independent of T-cell help (12). Adding to this complexity, the antigen specificity of U-CLL includes both T cell-independent (TI) and T cell-dependent (TD) antigens (11,13,14). On the other hand, M-CLL express BCRs that are believed to bind with high-affinity to auto-antigens and show activation of pathways associated with anergic B cells (15,16).
Differences regarding BCR reactivity have fueled several theories concerning the cellular origins of CLL. SHM status and transcription profiling indicated that U-CLL and M-CLL are derived from CD5 + CD27 − pre-and CD5 + CD27 + postgerminal center (GC) B cells, respectively (17,18). Extrafollicular or marginal zone (MZ) B cell responses, involving the activation of low-affinity B cells to TI antigens with low SHM, could also be relevant for CLL (19). Direct in vivo evidence for the TD or TI origin of CLL subgroups is still missing, mainly due to a lack of mouse models that spontaneously develop both stereotypic and non-stereotypic, mutated and unmutated CLL (20). In the widely studied Eµ-TCL1 model, CLL predominantly express unmutated stereotyped IghV11 or IghV12 BCRs (21). The IgH.TEµ CLL mouse model that we previously generated is based on sporadic expression of the SV40 large T oncogene in mature B cells (22). This was achieved by SV40 large T insertion in opposite transcriptional orientation into the IgH locus D H -J H region. In contrast to the Eµ-TCL1 model, IgH.TEµ mice mainly develop unmutated CLL with a diverse IghV repertoire, and at low frequencies mutated CLL (20,22). Because of their mixed sv129xC57BL/6 background, we used IgMa/IgMb allotype expression to define CLL incidence by the accumulation of >70% IgMb + B-cells (22,23). Aging IgH.TEµ mice show accumulation of monoclonal CLL-like CD5 + CD43 + IgM + IgD low CD19 + B cells around nine months of age. Although constitutive Btk signaling was not apparent in primary IgH.TEµ CLL cells, CLL development was dependent on Btk. Btk-mediated signaling enhanced leukemogenesis and Btk-deficiency led to a complete rescue from the disease (23). Moreover, primary CLL cells from IgH.TEµ mice or stable cell lines generated from these mice had detectable expression of p-Akt and substantial levels of p-S6, both of which function downstream of the BCR (23,24).
To address the impact of antigenic pressure on BCR selection in CLL, we analyzed the effects of defective T cell help and GC formation, as well as robust antigenic stimulation on CLL development in IgH.TEµ mice. We show that there are two distinct unmutated CLL subsets present in the IgH.TEµ mouse model. The V H 11-2/Vκ14-126-expressing CLL developed independently of T-cell help. Conversely, non-V H 11 CLL was TD and displayed a specific transcriptional signature associated with non-stereotypic U-CLL in human. These findings provide evidence for differential dependence on T cell help in unmutated CLL in mice and suggest that development of human U-CLL can also be T cell-dependent.

Mice
Mice (C57BL/6) deficient for Cd40l (25), Aicda (26) or Siglec-G (27), and Cd19-E-Btk-2 (28) transgenic mice were crossed to IgH.TEµ mice (F1 sv129xC57BL/6). CLL development was monitored every 3-6 weeks by screening peripheral blood for a monoclonal B cell expansion using flow cytometry. CLL formation was defined by accumulation of >70% IgMb + B-cells in the peripheral blood of the mice. Mice were sacrificed after detection of CLL. Mice were bred and kept in the Erasmus MC experimental animal facility and experiments were approved by the Erasmus MC committee of animal experiments.

Patients and Healthy Controls
Primary patient material was obtained from peripheral blood from CLL patients, while peripheral blood from healthy controls (>50 years of age) was obtained via Erasmus MC and via Sanquin blood bank (Rotterdam). Diagnostic and control samples were collected upon informed consent and anonymized for further use, following the guidelines of the Institutional Review Board, and in accordance with the declaration of Helsinki. The BCR characteristics of all CLL patients are included in Supplementary Table 5. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll Hypaque (GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions.
Naïve mature B cells were isolated from healthy control PBMCs using FACS-purification for CD19+CD27-IgD+ cells. The purity of naïve mature healthy B cell samples was >95% as determined by flow cytometry.

In vivo Immunizations
TD immune responses were induced by i.p. immunization. Primary immunizations were induced in 10-12-week-old mice with 100 µg TNP-KLH on alum. After 5 weeks this was followed by a secondary immunization with 100 µg TNP-KLH in PBS (28).

BCR Sequencing
Primer sequences and PCR condition were previously described (22,23). PCR products were directly sequenced using the BigDye terminator cycle sequencing kit with AmpliTaq DNA polymerase on an ABI 3130xl automated sequencer (Applied Biosystems). Sequences were analyzed using IMGT/V-Quest (http://www. imgt.org, using Ig gene nomenclature as provided by IMGT). All sequences were confirmed in at least one duplicate analysis.

Flow Cytometry Procedure
Preparation of single-cell suspensions of lymphoid organs and lysis of red blood cells were performed according to standard procedures. Cells were (in)directly stained in flow cytometry buffer (PBS, supplemented with 0.25% BSA, 0.5 mM EDTA and 0.05% sodium azide) using the following fluorochrome or biotinconjugated monoclonal antibodies or reagents: anti-B220 (RA3-

RNA-Sequencing
RNA was extracted from naive or activated splenic B cells, as well as from purified (using MACS-purification for CD19+ cells) primary tumors from IgH.TEµ mice with the RNeasy Micro kit (Qiagen) according to manufacturer's instructions. The TruSeq RNA Library Prep kit (Illumina) was used to construct mRNA sequencing libraries that were sequenced on an Illumina HiSeq 2500 (single read mode, 36 bp read length). Raw reads were aligned using Bowtie to murine transcripts (RefSeq database) from the University of California at Santa Cruz (UCSC) mouse genome annotation (NCBI37/mm9) (30). Differential gene expression analysis was performed using DESeq2 (31) with an adjusted P-value (false discovery rate; FDR) of P < 0.05. Log2-fold changes and FDR values as calculated by DESeq2 were used to generate a volcano plot using R (R studio version 1.1.383). Normalized gene expression levels quantified as reads per kilobase of a transcript per million mapped reads (RPKMs) were used for various clustering approaches (unsupervised hierarchical clustering, supervised clustering, and PCA) that were performed using R and PAST software (https:// folk.uio.no/ohammer/past/). Visualization of clustering analysis output was performed using R, PAST, and Java TreeView (32). Molecular pathway enrichments were obtained from the online MSigDB database. Gene expression data for anti-CD40 plus IL-4 stimulated follicular B-cells was obtained from previously reported data and downloaded from the Gene Expression Omnibus (GEO; accession number GSE77744) (33). RNA-Seq data generated in this study have been deposited in the GEO database (accession number GSE117713).

Quantitative Real Time PCR Analysis
Samples tested in qRT-PCR were from IgH.TEµ (7 V H 11 and 15 non-V H 11), from IgH.TEµ.Aicda −/− (4 V H 11 and 4 non-V H 11), and from IgH.TEµ.TD (4 non-V H 11) mouse groups. For quantitative RT-PCR analysis, TaqMan probes were employed. Probe Finder software (Roche Applied Science), the Universal Probe Library (Roche Applied Science) and Ensembl genome browser (http://www.ensembl.org/) were used for primer and probe design. Taqman Universal Master Mix II, was purchased from Thermo Fisher Scientific. Quantitative RT-PCR was performed by using the 7300 Real Time PCR system (Applied Biosciences) according to manufacturer's instructions. Gene expression was analyzed with an ABI Prism 7300 Sequence Detector and ABI Prism Sequence Detection Software version 1.4 (Applied Biosystems). Cycle-threshold levels were calculated for each gene and the housekeeping gene glyceraldehyde-3phosphate dehydrogenase (Gapdh) was used for normalization of the values. All primer sequences and probe numbers are listed in Supplementary Table 7.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism software (San Diego, California, USA) or R. The log rank test was used for calculating the level of significance for survival differences between mouse groups. The Chi-square test was used to determine the significance for BCR usage differences between different mouse groups. To evaluate differences in expression levels of different genes by qRT-PCR we used a Mann-Whitney U-test between two groups or a Kruskal-Wallis test corrected with Dunn's multiple comparison test for more than two groups.

IgH.TEµ Mice
To analyze the BCR repertoire, we aged a panel of IgH.TEµ mice and collected blood every 3-6 weeks to monitor CLL incidence. Hereby, CLL incidence was defined by the accumulation of >70% IgMb + B-cells, which displayed a CLL-like CD5 + CD43 + IgM + IgD low CD19 + phenotype (22,23). We performed sequencing analyses of Ig heavy (Igh) and light (Igl) chain transcripts and found that a substantial proportion (∼36%) of CLL in IgH.TEµ mice expressed stereotyped BCRs consisting of the V H 11-2 Igh chain, with similar Igh CDR3 length and amino acid sequences, and the Vκ14-126 Igl chain (22, 23) (Supplementary Table 1, Figures 1A,B). The V H 11/Vκ14 CLL mice exhibited an earlier disease onset compared with IgH.TEµ mice with non-stereotypic (non-V H 11) BCR (mean incidence age 184 days and 219 days, respectively, p = 0.0175) ( Figure 1C). In wild-type mice the V H 11-2/Vκ14-126 BCR is preferentially expressed by B-1 lymphocytes and shows specificity to phosphatidylcholine (PtC) (12). We could confirm PtC-binding specificity of V H 11-2 BCRs on CLL cells ( Figure 1D). V H 11 CLL showed decreased surface IgM expression and increased surface IgD expression compared to non-V H 11 CLL (Figures 1E,F). A major proportion (∼65%) of the remaining non-V H 11 CLL expressed a J558 V H 1-family BCR with heterogeneous CDR3 length, amino acid sequence and Igl chain usage (Supplementary Table 1). V H 1 CLL showed delayed disease onset (mean incidence age 231 days), compared with V H 11 CLL (Supplementary Figure 1).
In conclusion, based on Ig gene usage we could distinguish different subsets of unmutated IgH.TEµ CLL displaying differential disease onset.
Taken together, these findings indicate that V H 11/Vκ14expressing CLL arise independently of T cell help or GC formation, whereas non-V H 11 CLL is T celldependent and reduced in the absence of functional GCs in IgH.TEµ.Cd40l −/− and IgH.TEµ.Aicda −/− mice.

T-cell Dependent Antigenic Stimulation of B Cells in vivo Reduces V H 11/Vκ14 Usage in Unmutated IgH.TEµ CLL
To directly investigate whether antigenic stimulation in the context of T cell help affects CLL onset and the CLL BCR repertoire, we immunized IgH.TEµ mice with TNP-KLH coupled to alum (IgH.TEµ.TD, n = 20) to induce a TD B cell response. CLL onset did not differ between immunized and nonimmunized littermates (n = 56) (Figure 3A). At the age of ∼400 days, CLL incidence in IgH.TEµ.TD mice was ∼65% similar to non-immunized control IgH.TEµ mice (∼62%) ( Figure 3A).
In summary, we found that robust TD immunization favors development of non-V H 11 CLL.

Enhanced BCR Signaling Accelerates Disease Onset in IgH.TEµ Mice
Our findings provide evidence that T cell-derived activation or selection signals, in particular CD40L, shape the BCR repertoire of CLL in IgH.TEµ mice, but do not significantly affect disease onset or progression. It is therefore conceivable that in the IgH.TEµ mouse model, BCR-derived signals may be more decisive for disease progression.
To monitor the impact of BCR signaling strength on CLL development and IghV gene selection, we first crossed IgH.TEµ mice with E-Btk-2 transgenic mice. These mice express the constitutive active E41K-BTK mutant selectively in the B-cell lineage driven by the CD19 promoter (28). The E41K mutation enhances Btk membrane localization and thereby its activation by Syk or Src-family tyrosine kinases (35). E-Btk-2 mice show defective follicular B cell survival and a relative expansion of splenic B-1 cells (28). Flow cytometry analysis of E-Btk-2 B-1 cells did not reveal detectable PtC binding, indicating that V H 11 BCR expression was limited (data not shown).
To confirm that enhanced BCR signaling affects disease onset, we crossed IgH.TEµ mice on a Siglec-G deficient background (IgH.TEµ.Siglec-G −/− ). Siglec-G is a negative regulator of BCRmediated signaling that is expressed in all B cells (27). It is a potent inhibitor of BCR-induced Ca 2+ signaling and a key regulator of survival and selection of B-1 cells (36). In addition, Siglec-G-deficiency abrogates V H 11 usage in B-1 cells (36).

Transcriptome Profiling Identifies Unique Genes and Pathway Aberrations for V H 11/Vκ14 and non-V H 11 CLL IgH.TEµ Mice
To further explore the biological phenotype of the V H 11 and non-V H 11 CLL subsets, we performed genome-wide gene expression profiling on primary IgH.TEµ CLL (tumor load >95%) expressing either a V H 11 (n = 3) or a non-V H 11 (n = 3) BCR. As a reference we included resting unstimulated (un-B, n = 4) and anti-IgM stimulated (αIgM-B, n = 4) naïve splenic B cells from wild-type mice. Normalized gene expression values (see Methods for details) were used for principle component analysis (PCA). The first two principal components, which represented ∼70% of the total variation among the different samples analyzed, identified three separate clusters, corresponding to un-B, αIgM-B and primary IgH.TEµ CLL samples, indicating a strong correlation between biological replicates (Supplementary Figure 2). When we performed differential gene expression analysis (focusing only on genes passing a stringent statistical filter of Benjamini-Hochberg false discovery rate corrected P < 0.05), we found 148 differentially expressed genes ( Figure 5A; Supplementary Table 2). Of these genes, 59 genes were upregulated in V H 11 CLL and 89 genes were upregulated in non-V H 11 CLL. To identify biological processes that underlie the transcriptional differences between V H 11 and non-V H 11 CLL, we performed pathway enrichment analysis using the Molecular Signatures Database (MSigDB) (37). Genes upregulated in V H 11 CLL were functionally enriched for an interferon-mediated response, active Wnt signaling and constitutively active RAF1 signaling ( Figure 5B, Supplementary Table 3A). On the other hand, genes downregulated in V H 11 CLL were involved in quite diverse pathways, including interleukin-, epidermal growth factor receptor (EGFR)-, vascular endothelial growth factor (VEGF)-mediated signaling, metabolic processes, hypoxia and the UV radiation-induced stress response (Figure 5B, Supplementary Table 3A).
Taken together, these data suggest that in addition to a different origin, V H 11 and non-V H 11 CLL subsets display distinct transcriptional signatures, signifying differential activity of key signaling pathways.

Strong BCR Dependence of V H 11/Vκ14 CLL in IgH.TEµ Mice
Next, we performed a PCA of the 148 differentially expressed genes between V H 11 and non-V H 11 CLL. To investigate the impact of T-cell-independent BCR stimulation and T-celldependent CD40 stimulation on differential gene expression, we included RNA-Seq gene expression values of the 148 genes from the unstimulated and αIgM-stimulated B cells described above, as well as previously reported gene expression values from anti-CD40/IL-4 stimulated follicular B-cells (α-CD40/IL4-B) (33).
The first principal component (PC1) separated both CLL groups and the two stimulated B cell subsets from unstimulated B cells, suggesting IgH.TEµ CLL cells share a transcriptional signature related to activated B-cell phenotypes. Interestingly, PC2 revealed a strong similarity between αIgM-stimulated B cells and V H 11 CLL on one hand and between α-CD40/IL4-stimulated B cells and non-V H 11 CLL on the other hand ( Figure 6A). These findings indicate more prominent BCR stimulation in V H 11 than in non-V H 11 CLL B cells in vivo and are consistent with a dependence on T-cell help for non-V H 11 CLL.
To identify the gene signature underlying the clustering of αIgM-stimulated B cells and V H 11 CLL, as well as α-CD40/IL-4-stimulated B cells and non-V H 11 CLL, we performed hierarchical clustering analyses to separate the 148 genes into 4 clusters (Figure 6B, Supplementary Table 3B). Cluster 1 consists of 17 genes that were highly correlated between αIgM-stimulated B cells and V H 11 CLL and between α-CD40/IL- Table 3C) on this cluster revealed overrepresentation of genes involved in interferon response and KRAS signaling. Clusters 2 (35 genes) and Cluster 3 (56 genes) consist of genes that were highly correlated only between α-CD40/IL-4-stimulated B cells and non-V H 11 CLL or only between αIgM-stimulated B cells and V H 11 CLL, respectively. These clusters were enriched for interferon response/PI3K-AKT signaling genes (cluster 2) or UV response, epithelialmesenchymal transition, glycolysis, hypoxia, unfolded protein response genes (cluster 3) (Supplementary Table 3C). Finally, cluster 4 (enriched for genes involved in the reactive oxygen species pathway) represents genes with low or anti-correlated expression values between the stimulated B cells and CLL. Thus, genes from clusters 1 and 3 signify the clustering of αIgMstimulated B cells and V H 11 CLL, while genes from clusters 1 and 2 drive the clustering of α-CD40/IL-4-stimulated B cells and non-V H 11 CLL (Figure 6B). This analysis was further validated by computing the average correlation strength for each of the four gene clusters with PC2 from our PCA ( Figure 6B). Indeed, clusters 1 to 3 underlying the αIgM-B cells and V H 11 CLL and the α-CD40/IL-4-B cells and non-V H 11 CLL segregationand particularly cluster 1 genes-showed significantly stronger correlation values with PC2 than cluster 4 ( Figure 6C).
Expression of five of these 13 genes that were significantly upregulated in non-V H 11 CLL vs. V H 11 CLL (Ccdc88a, Clip3, Zcchc18, Chd3, Itm2a) was also evaluated in five mutated IgH.TEµ CLL, defined by <97% IghV germline identity [Supplementary Table 1 and ter Brugge et al. (22)]. Interestingly, qRT-PCR analysis showed that four out of five tested genes (except Itm2a) were expressed at low levels in mutated CLL, similar to V H 11 CLL (Figure 7B). Thus, non-V H 11 unmutated CLL in IgH.TEµ mice represent a unique subset that can be distinguished from V H 11 unmutated and from mutated CLL by a specific transcriptional signature. Furthermore, correlation analyses indicated that within the non-stereotypic subgroup in particular V H 1 CLL represents the most heterogeneous CLL subgroup in IgH.TEµ mice (n = 16; average spearman r, ρ = 0.280; Supplementary Figure 3). In these analyses we also found that expression of these five genes is positively correlated in V H 11 CLL (n = 15; average spearman r, ρ = 0.537) and in the small non-V H 11/non-V H 1 CLL subgroups (n = 6; average spearman r, ρ = 0.703) (Supplementary Figure 3).
To compute any parallel between stereotypic and heterogeneous U-CLL from patients and IgH.TEµ mice, we performed t-SNE clustering analysis on expression values for the 13 signature genes (Figure 7D, Supplementary Table 6).
Taken together, we conclude that differences in the expression of these signature genes in heterogeneous U-CLL, stereotyped U-CLL and M-CLL were partly overlapping between human CLL and the corresponding CLL subgroups in our IgH.TEµ CLL mouse model.  Supplementary Table 7) using dCT values obtained by qRT-PCR for non-sterotypic (#U-CLL, n = 10) and stereotypic (U-CLL, n = 10) human U-CLL and non-V H 11 (n = 21) and V H 11 (n = 14) CLL from IgH.TEµ mice, as indicated. Expression values were converted to Z-scores separately for mouse and human datasets to allow combined t-SNE analysis.

DISCUSSION
In this report, we investigated the role of antigenic pressure and BCR signaling thresholds on clonal selection of CLL cells in the IgH.TEµ CLL mouse model. We found that U-CLL tumors that develop in these mice can be classified into two different groups based on their IghV usage. The stereotypic V H 11-2/V κ14-126 CLL subset recognized the PtC self-antigen, developed independently of T cell help or GC formation and represented a somewhat more aggressive type of CLL. Proportions of V H 11/Vκ14-expressing CLL were increased in the absence of functional germinal centers in IgH.TEµ mice deficient for CD40L or activation-induced cytidine deaminase. Conversely, in vivo T cell-dependent immunization decreased the proportions of V H 11/Vκ14-expressing CLL. Mice were immunized at 10-12 weeks of age, with a secondary immunization at 15-17 weeks of age. In a proportion of mice at these time points, CLL cells become detectable in peripheral blood (Figure 1). In our immunization model the onset or frequency of CLL was not altered, but we cannot exclude that there will be effects on CLL onset or disease progression when immunizations are performed at a different age.
Consistent with the observed effects of defective germinal center function or robust T-cell dependent immunization on V H usage in CLL, PCA of a gene signature comprised of 148 genes differentially expressed between V H 11 and non-V H 11 CLL revealed that V H 11 and non-V H 11 CLL clustered with BCR-stimulated and anti-CD40-stimulated B cells, respectively.
The unmutated V H 11 CLL cells parallel B-1 cells, because these also have a restricted BCR repertoire, may recognize autoantigens including PtC, and produce natural IgM antibodies in the absence of T cell co-stimulation (12). In concordance, it was recently shown that peritoneal CD5 + B-1 cells generated early during fetal or neonatal development, increase in number over time and can progress into CLL in aged mice (47,48). Interestingly, CLL development in these mice was linked to the expression of a restricted BCR repertoire (V H Q52/V κ9 or V H 3609/V κ21, reactive toward non-muscle myosin-IIA or Thy-1, respectively) independent of CD40 signaling. Hereby, expression of the Eµ-TCL1 transgene enhanced aggressiveness of the disease.
Non-V H 11 CLL, on the other hand, consisted of tumors with heterogeneous IghV/IglV expression and CDR3 length, lacking affinity for PtC. Although these tumors were T-cell dependent, strongly reduced in the absence of functional GCs, their BCRs were not hypermutated (<3%). This is in line with findings in human U-CLL, indicating that U-CLL cells can recognize both TD and TI autoantigens that have relocated to the external cell surface during apoptosis (11,13,14). Our observations are also consistent with gene expression profiling studies suggesting that U-CLL reflect memory B cells (49). In contrast, more recent transcriptome analyses revealed that U-CLL resemble mature pre-GC CD5 + CD27 − B cells, while M-CLL resembles a distinct, previously unrecognized, CD5 + CD27 + post-GC B cell subset (18). Our findings imply that in mice unmutated CLL can be derived from (i) T cell-independent B-1 cells (e.g., PtCrecognizing V H 11-2/Vκ14-126) or (ii) from B cells that recognize their antigen in the presence of cognate T-cell help and are activated without SHM. This latter group of T cell-dependent unmutated CLL displayed an expression signature, as defined by 13 genes including the CCDC88A-CLIP3-ZCCHC18-CHD3-ITM2A module, that is not only different from TI unmutated CLL, but also from mutated CLL in the IgH.TEµ mouse model. Moreover, we found evidence that this expression signature may be partly associated with non-stereotypic human U-CLL, suggesting that the development of human U-CLL can also be TD. Such TD U-CLL may derive from B cells involved in an extra-follicular response or alternatively may be related to autoantibody producing B cells in mice that were shown to recognize TD antigens, mount a rapid IgM response and enter GCs, but do not develop into IgG-expressing plasma cells (50,51). Although our data suggest a role for T-cell help in human non-stereotypic U-CLL pathophysiology, further investigation is required to translate our findings to human disease. Such studies should include expression profiling of (1) large CLL patient cohorts containing a wide range of stereotypic and non-stereotypic U-CLL samples and (2) activated B cells that received various stimulations including anti-CD40.
Gene expression profiling revealed a set of genes that distinguish V H 11 from non-V H 11 CLL and are similarly regulated in BCR or CD40-stimulated cells, respectively. This observation probably reflects differences in supporting external cues: pathways induced by interleukin or growth factor-mediated signaling were specifically upregulated in non-V H 11 CLL. These include the regulator of G-protein signaling 16, Rgs16, which is upregulated in autoimmune B cells of BXD2 mice and enhances GC formation by the canonical NF-κB pathway, signifying the post-GC origin of non-V H 11 CLL (52,53). Second, the actinbinding protein Ccdc88a, which plays a role in cytoskeletal remodeling and cell migration following activation of Akt downstream of EGFR (54) and can also enhance Akt signaling (42,55). Third, integral membrane protein 2A (Itm2a) is a type II integral membrane protein that has been associated with an enhanced GATA3-mediated regulatory network in B ALL (56). Chd3 encodes a chromatin remodeler with unexplored function in lymphocytes.
On the other hand, Wnt-associated genes were specifically upregulated in V H 11 tumors, which is interesting because the BTK-inhibitor ibrutinib restrains Wnt signaling in CLL (57). Although the function of several other upregulated genes is currently unknown, Zcchc18 has been associated with a CLLspecific transcriptomic signature (42) and Clip3 was differentially regulated in a CLL patient undergoing spontaneous regression (58). Notably, many gene sets or pathways were active in both CLL subsets, including high expression levels of MET receptor tyrosine kinase, which prolongs CLL cell survival through STAT3 and AKT phosphorylation (40,59). This could contribute to the enhanced constitutive activation of the p-Akt/p-S6 pathway in IgH.TEµ CLL as reported previously (23,24). Additionally, genes involved in KRAS signaling were highly expressed in both CLL subsets, consistent with its essential role in B cell lymphopoesis (60), particularly for B-1 cells recognizing PtC (61).
Our data also indicated that availability of T cell help and GC formation did not affect tumor incidence or onset. In contrast, the finding of a significantly earlier CLL incidence of mainly the non-V H 11 type in IgH.TEµ.Siglec-G −/− and IgH.TEµ.E-Btk-2 mice suggests that BCR signaling thresholds are a key factor in determining CLL disease course. Yet, the appearance of V H 11 CLL in these mouse lines may indicate a substantial selective advantage of these clones, because in Siglec-G −/− and E-Btk-2 transgenic mice the frequency of PtC-recognizing cells within the B-1 cell population is very low (28,36).
In conclusion, we found that the formation of a major subset of unmutated CLL in IgH.TEµ mice is dependent on T cell signals. Our findings therefore provide a mechanistic explanation for the role of B-cell intrinsic factors, in particular BCR signaling, as well as extrinsic factors such as T cell help and support from the tumor microenvironment, in shaping the repertoire of CLL in mice. These findings are of potential clinical relevance, because B-cell extrinsic signals may reflect effective targets for novel therapeutic strategies in CLL patients.

AUTHOR CONTRIBUTIONS
SPS designed the research studies, performed experiments, analyzed the data, and wrote the manuscript. MdB, SP, RM, and MdA performed experiments and analyzed the data. RS analyzed RNA sequencing data and contributed to writing the manuscript. AL and LN contributed to the research design and the writing of the manuscript. RH contributed to the research design and the writing of the manuscript and supervised the study. All co-authors approved the final manuscript.

FUNDING
These studies were partly supported by the Dutch Cancer Society (KWF 2014-6564), the Association for International Cancer Research (10-562) to RH, NWO (to SPS) and a NWO Veni Fellowship (Grant No. 91617114) to RS.