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Purpose:Using next-generation sequencing (NGS), we recently documented T-cell oligoclonality in treatment-naïve chronic lymphocytic leukemia (CLL), with evidence indicating T-cell selection by restricted antigens.Experimental Design:Here, we sought to comprehensively assess T-cell repertoire changes during treatment in relation to (i) treatment type [fludarabine-cyclophosphamide-rituximab (FCR) versus ibrutinib (IB) versus rituximab-idelalisib (R-ID)], and (ii) clinical response, by combining NGS immunoprofiling, flow cytometry, and functional bioassays.Results:T-cell clonality significantly increased at (i) 3 months in the FCR and R-ID treatment groups, and (ii) over deepening clinical response in the R-ID group, with a similar trend detected in the IB group. Notably, in constrast to FCR that induced T-cell repertoire reconstitution, B-cell receptor signaling inhibitors (BcRi) preserved pretreatment clones. Extensive comparisons both within CLL as well as against T-cell receptor sequence databases showed little similarity with other entities, but instead revealed major clonotypes shared exclusively by patients with CLL, alluding to selection by conserved CLL-associated antigens. We then evaluated the functional effect of treatments on T cells and found that (i) R-ID upregulated the expression of activation markers in effector memory T cells, and (ii) both BcRi improved antitumor T-cell immune synapse formation, in marked contrast to FCR.Conclusions:Taken together, our NGS immunoprofiling data suggest that BcRi retain T-cell clones that may have developed against CLL-associated antigens. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response.

Calcitriol, the biologically active form of vitamin D, modulates a plethora of cellular processes following its receptor ligation, namely the vitamin D receptor (VDR), a nuclear transcription factor that regulates the transcription of diverse genes. It has been proposed that vitamin D may play a role in prevention and treatment of cancer while epidemiological studies have linked vitamin D insufficiency to adverse disease outcome in chronic lymphocytic leukemia (CLL). Recently, we reported that VDR is functional in CLL cells after calcitriol supplementation, as well as after stimulation through both the calcitriol/VDR signaling system and other prosurvival pathways triggered from the tumor microenvironment. In this study, we aimed at investigating key molecules and signaling pathways that are altered after calcitriol treatment and are known to play a relevant role in CLL pathophysiology. CD19+ primary CLL cells were negatively selected from peripheral blood samples of patients that were treatment naïve at the time of sample collection. CLL cells were cultured in vitro with calcitriol or co-cultured with the HS-5 mesenchymal cell line for 24 hours. VDR+, CYP24A1+, phospho-ERK+ and phospho-NF-κB p65+ cells were determined by Flow Cytometry (FC). Total RNA was extracted from calcitriol-treated and non-treated CLL cells, while mRNA selection was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module. Library preparation for RNA-Sequencing (RNA-Seq) analysis was conducted with the NEBNext Ultra II Directional RNA Library Prep Kit. The libraries were paired-end sequenced on the NextSeq 500 Illumina platform. Differential expression analysis was performed using DESeq2; genes with log2FC>|1| and P≤0.05 were considered as differentially expressed. RNA-Seq analysis (n=6) confirmed our previous findings that the CYP24A1 gene is significantly upregulated by calcitriol, being the top upregulated gene, whereas the VDR gene remains unaffected by this treatment. Overall, 85 genes were differentially expressed in unstimulated versus calcitriol-treated cells, of which 28 were overexpressed in the latter thus contrasting the remaining 57 which showed the opposite pattern. Pathway enrichment and gene ontology (GO) analysis of the differentially expressed genes revealed significant enrichment in PI3K-Akt pathway and Toll-like receptor cascades, as well as in vitamin D metabolism and inflammatory response pathways. Additionally, flow cytometric analysis showed that calcitriol-treated CLL cells displayed increased pERKlevels (FD=1.3, p In conclusion, we provide evidence that the calcitriol/VDR system is active in CLL, modulating NF-κB and MAPK signaling as well as the expression of the CYP24A1 target gene. This observation is further supported by RNA-Seq analysis that confirms CYP24A1 upregulation and highlights new signaling pathways that need to be validated. Interestingly, the calcitriol/VDR system appears relatively unaffected by either stimulation or inhibition (ibrutinib) of microenvironmental signals that promote CLL cell survival and/or proliferation, indicating context-independent signaling capacity. Disclosures Kotsianidis: Celgene: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

The IGHV4-34 gene is intrinsically autoreactive due to carrying a germline(GL)-encoded (super)antigenic motif binding various self (and exogenous) antigens, while it is one of the few IGHV genes that contain a GL-encoded N-glycosylation (N-glyc) site. IGHV4-34 is overrepresented in chronic lymphocytic leukemia (CLL), particularly in cases expressing B cell receptor immunoglobulin (BcR IG) with a significant load of somatic hypermutation (SHM; 'mutated' CLL, M-CLL). Moreover, a large fraction of IGHV4-34 M-CLL cases are clustered in different stereotyped subsets, of which the best studied is subset #4, the largest within M-CLL, defined by the expression of IgG-switched IGHV4-34/IGKV2-30 BcR IG with a distinctive SHM imprint. Considerably smaller than subset #4 is subset #201, defined by the expression of IGHV4-34/IGLV1-44 BcR IG of the IgMD isotype. Subset #201 is noteworthy owing to recurrent replacement SHMs that frequently lead to the creation of novel N- glyc motifs within the VH domain. This may be functionally relevant, considering that N-linked glycosylation is a widespread post-translational modification that is largely SHM-induced during antigen-specific immune responses and can modulate antibody (Ab) affinity towards antigen. That said, nothing is yet known about the antigen reactivity of subset #201 BcR IG and whether/how it could be affected through SHM-induced changes of N-linked glycosylation. In order to obtain insight into this issue, 4 subset #201 clonotypic IGs were expressed as recombinant monoclonal Abs (mAbs) of the mu isotype in HEK293 human cells, in either the authentic SHM state ('wildtype', WT-mAbs) or after reverting specific SHMs that altered N-glyc sites (R-mAbs) by site-directed mutagenesis. Since not all N-glyc motifs are eventually glycosylated, we used the NetNglyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) for the prediction of N-glycan occupancy. Binding to MEC1 B CLL, Jurkat T and HEK293 cells was assessed by flow cytometry. Reactivity against nuclear Hep-2 cell extract, nDNA, actin, myosin, thyroglobulin (TG), β-amyloid, carbonic anhydrase, F(ab')2 and the non-self hapten trinitrophenyl was tested by ELISA. Non-subset #201 M-CLL mAbs (n=14, including 3 subset #4 mAbs), were used as controls. None of the subset #201 WT-mAbs displayed reactivity in any of the ELISAs. However, unlike most CLL mAbs, all subset #201 WT-mAbs bound to live MEC1 cells, while also exhibiting reactivity to HEK293 cells that was significantly higher when compared to non-subset #201 M-CLL (p=0.0095) or subset #4 (p=0.05); additionally, 1/4 subset #201 mAb displayed weak binding to Jurkat T cells. Three of 4 subset #201 mAbs bore a novel N-glyc site introduced by SHM in codons VL CDR1 36-38 of the clonotypic lambda light chains. Reversion to the GL in one such mAb resulted in enhanced binding to all 3 cell lines [fold change (FC) of binding of the R- vs WT-mAb to MEC1, Jurkat and HEK293: 1.3, 7.9 and 3.3, respectively) and in strong anti-TG activity. The GL-encoded N-glyc site in VH CDR2 57-59, that has been reported to be mostly unoccupied, was targeted by SHM in 2/4 subset #201 mAbs: reversion to GL decreased binding to both MEC1 and HEK293 cells (FC: -8 and -1.4 respectively). Finally, in 2/4 cases, SHM at codons VH FR3 67-68 inserted an N-glyc site that, however, is not predicted to acquire N-glycans. Reversion to GL enhanced the binding of one of these mAbs to MEC1 and HEK293 cells (FC: 2.1 and 5.6, respectively). The same mAb bore an additional predicted N-glyc site introduced by SHM at VH FR3 90-92; reversion of this change to GL augmented binding to both MEC1 and HEK293 cells (FC: 4.1 and 9.7, respectively). Double reversion of both aforementioned SHMs conferred further increased binding than any of the single reversions, implying a synergistic effect. Acquisition of novel N-glyc sites is not an intrinsic characteristic of either M-CLL in general or IGHV4-34 M-CLL in particular and its high incidence in subset #201 implies a selective process likely due to distinct (auto)antigenic pressure. Indeed, subset #201 mAbs exhibit an antigen reactivity profile that differs from that of typical polyreactive mAbs, including natural autoantibodies and other CLL mAbs, binding selectively to viable lymphoblastoid cell line cells and human HEK293 epithelial cells. These results further emphasize the importance of SHM in shaping the distinct (auto)antigenic recognition profile of CLL mAbs. Disclosures Chatzidimitriou: Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.