Tumor-activated lymph node fibroblasts suppress T cell function in diffuse large B cell lymphoma

Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of LN fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identified the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network expressing elevated fibroblast-activated protein (FAP). RNA-Seq analyses revealed that exposure to DLBCL reprogrammed key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen-presentation molecules. Functional assays showed that DLBCL-activated FRCs (DLBCL-FRCs) hindered optimal TIL and chimeric antigen receptor (CAR) T cell migration. Moreover, DLBCL-FRCs inhibited CD8+ TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8+ TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrated the potential to target inhibitory FRCs to rejuvenate interacting TILs. Cotreating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented antilymphoma TIL cytotoxicity. Our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis, and optimizing immunotherapy for patients

Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia

NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis

Diffuse Large B Cell Lymphoma Cell Line U2946 : Model for MCL1 Inhibitor Testing

Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8; 14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1(pos)/BCL2(neg)) cells. In contrast to BCL2(pos) DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members

Inhibition of BCL2 and MCL1: effect on apoptosis.

<p>Apoptosis was assessed by Annexin-V staining, 24 h after addition of a BCL2 inhibitor (ABT-263) or an MCL1 inhibitor (A-1210477). The MCL1 inhibitor triggered apoptosis in the MCL1^pos/BCL2^neg cell line U-2946, BCL2 inhibition induced apoptosis in the MCL1^pos/BCL2^pos cell line U-2932. Although expressing MCL1, cell line U-2932 was not responsive to MCL1 inhibition, suggesting dominant function of BCL2. The data were reproduced in two independent experiments.</p

Cytogenetic analysis of U-2946 cells.

<p>A) Classical and B) Spectral Karyotyping shows key oncogenomic rearrangements present in U-2946 cells. BAC clones used for FISH are shown along with corresponding genomic copy number plots. Blue arrows show t(8;14)(q24;q32), red arrows der(8)t(8;8)(p22;q24), yellow arrows der(9)t(2;9) and purple arrows add(17)(p11). Note differences between cells in A and B reflecting clonal heterogeneity indicated by stars. C-G) Image depicts analysis of t(8;14) by chromosome painting (C), and single locus FISH using probes for IGH (D) and MYC (E). Positions of BAC clones are shown for IGH (F) and MYC loci (G). Note partial duplication of the terminal der(14) long arm region including IGH/MYC (arrow) present in a subclone (C). H-M) Analysis of MCL1/MAP2K3 coamplification: gold signals from MCL1 clone (RP11-54A4), barely visible at native loci (arrows), are intense on der(17) due to amplification (H). Similarly, green signals from 17p11 clone (RP11-64J19) covering MAP2K3 are amplified, while red signals (RP11-160E2) from the neigboring hemizygously deleted region are present on the unrearranged chromosome only. Chromosome painting shows localization of amplicon to 17p (K). Genomic copy number plots show consistency with FISH amplification data for both MCL1 (L) and MAP2K3 (M). N-O) Cytoscan HD array analysis revealed prominent amplification of 1q21.3 (N) and 17p11.2 (O).</p

Inhibition of BCL2 and MCL1: effect on proliferation.

<p>The effect of A) A-1210477 (MCL1 inhibitor) and B) ABT-263 (BCL2 inhibitor) on proliferation of DLBCL cell lines was assessed by 3H-thymidine uptake (d2). Cell lines OCI-LY1 and RI-1 are MCL1^neg/BCL2^pos, cell line U-2932 is MCL1^pos/BCL2^pos and cell line U-2946 is MCL1^pos/BCL2^neg. Especially note the significant reduction of 3H-thymidine incorporation in cell line U-2946 after addition of the MCL1 inhibitor.</p

Expression of BCL2, BCL6, BCLXL, MAP2K3, MCL1, and MYC in DLBCL cell lines.

<p>A) qRT-PCR and B) Western blot analysis was performed to assess the expression of oncogenes in U-2946 and other DLBCL cell lines. The bars in A) indicate means with standard deviation (n = 3). The ct values for BCL-W in qRT-PCR were high (>30) in all cell lines tested explaining absence of specific bands in Western blot analysis (not shown). Note the high-level expression of MCL1 (red) in cell lines U-2946 and U-2932.</p

Immunoprofile of patient and–derived cell line U-2946.

<p>Immunoprofile of patient and–derived cell line U-2946.</p

Phosphorylation of the MAP2K3/p38 pathway.

<p>Expression and phosphorylation of MAP2K3 and p38 in U-2946 and five other DLBCL cell lines was determined by Western blot analysis. Note that overexpression of MAP2K3 was not accompanied by high phosphorylation of the MAP2K3 target in cell line U-2946.</p