Distinct Signaling Profiles in Primary Splenic Small B-Cell Lymphomas

Splenic small B-cell lymphomas encompass a diverse group of rare entities that originate in or significantly involve the spleen, typically accompanied by bone marrow and peripheral blood involvement. Despite significant advancements in understanding their biology, the role of microenvironmental signals in shaping clonal behavior and clinical outcomes remains poorly understood, largely due to the scarcity of relevant primary patient samples. Here, we investigated for the first time the signaling capacity, both at the basal state and after triggering with microenvironmental stimuli, in primary splenic biopsy specimens of patients with splenic marginal zone lymphoma (SMZL; n=31), splenic diffuse red pulp lymphoma (SDPRL; n=7) and hairy cell leukemia variant (HCLv; n=10). Flow cytometric analysis of TLR1-10 expression in single-cell suspensions from splenectomy samples revealed distinct patterns, ranging from uniformly high in HCLv, to intermediate in SDRPL to generally lower yet variable expression in SMZL with significant differences between SMZL vs HCLv (i.e. TLR1, FD=8.6, p<0.001) and SDRPL vs HCLv (i.e. TLR5, FD=1.7, p<0.05). TLRs 2, 8 and 10 exhibited the lowest expression, whereas, TLR7 was highly expressed in all entities (>98% TLR7⁺ cells). Flow cytometry also disclosed: i) increased cell proliferation rate in HCLv vs SMZL (FD=6, p<0.01), assessed by Ki67 expression; ii) enhanced expression of the CD86 activation marker in HCLv vs SMZL (FD=3.2, p<0.01); and, iii) increased expression of the CD25 activation marker in SMZL vs either HCLv or SDRPL (FD=6.3 and FD=6.8, p<0.05, respectively). Next, we examined the functional capacity of TLRs after stimulation with specific ligands for the TLR1/2 and TLR2/6 heterodimer, TLR4 and TLR9 (Pam3CSK4, FSL-1, LPS and CpG, respectively). Both CD25 and CD86 were significantly upregulated in response to FSL-1, CpG and LPS stimulation in SMZL (FD=1.23-1.81, p<0.01), while only CD25 was upregulated in HCLv (FD=2.1, p<0.05); SDRPL was unaffected. In all entities, TLR triggering also resulted in enhanced proliferation of splenic B cells compared to unstimulated control cells, albeit in a heterogeneous manner. In particular, i) all cases strongly responded to FSL-1, particularly HCLv and SDRPL (FD=3.1, p<0.01 and FD=5.75, p<0.05, respectively); ii) both HCLv and SDRPL showed a moderate response to PAM3CSK4 (FD=1.2 and FD=2, p<0.05, respectively); and, iii) all cases showed augmented proliferation after triggering with CpG, particularly SMZL and SDRPL (FD=11.3, p<0.01 and FD=7.1, p<0.05, respectively). Cell viability, assessed by Annexin V, was markedly augmented in HCLv after CpG and LPS stimulation (FD=1.21 and FD=1.31, p<0.05, respectively), whereas, it was unaffected in SDRPL and SMZL. Following, we assessed signaling capacity after both isolated and combined BcR and TLR9 stimulation. SMZL cells displayed increased ERK and NF-κB phosphorylation after double stimulation (FD=1.44, p<0.01 and FD=1.96, p<0.05, respectively), while single BcR stimulation also induced ERK and NF-κB phosphorylation (FD=1.23, p<0.05 and FD=1.43, p=0.1, respectively), albeit to a lesser extent. Elevated pERK levels were also found in HCLv after single BcR and double BcR/TLR9 stimulation (FD=1.33 and FD=1.53, p<0.01, respectively). On the contrary, SDRPL cells did not present any change in phosphorylation status. Our prior studies in circulating SMZL B cells have implicated the histone methyltransferase EZH2 in the response to microenvironmental triggering. Here, using flow cytometry, we found that: i) EZH2 was expressed in all entities, albeit significantly higher in SDRPL and HCLv compared to SMZL (FD=1.64, p<0.01 and FD=1.83, p<0.001, respectively); ii) H3k27me3, the main target of EZH2, was highly (~95%) and uniformly expressed in all entities, indicating active EZH2; iii) BcR/TLR9 triggering resulted in significant upregulation of EZH2 expression in SMZL (FD=2.57, p<0.01); and, iv) neither SDRPL nor HCLv displayed significantly altered EZH2 expression after co-stimulation. Taken together, signaling through the BcR and TLRs is functional in splenic small B cell lymphomas, leading to activation of downstream pathways and increased proliferation. Nevertheless, each entity presents a particular signaling capacity with SMZL, in particular, appearing rather distinct from SDPRL and HCLv in terms of responsiveness to external triggering.