What if the missing ingredient in chronic lymphocytic leukemia research is not another drug, but the right neighborhood? By engineering stromal cells to deliver T-cell signals, this study makes CLL cells proliferate—and reveals a vulnerability that ordinary survival assays can miss.

Chronic lymphocytic leukemia depends on microenvironment interactions, with lymph nodes being the primary site of CLL cell proliferation. CLL cell birth rates in lymph nodes are zero point five to three percent per day. B-cell receptor crosslinking in vitro is insufficient to induce CLL proliferation, suggesting that other microenvironmental interactions are needed to drive proliferation.

The leading candidates are CLL and CD4-positive T-cell interactions. In immunodeficient mice, CLL cell engraftment depends on co-transplantation of activated T-cells. And in CLL, CD40 stimulation alone is not sufficient to drive proliferation; it must be combined with factors such as IL4 or IL21.

The lack of primary CLL cell proliferation in vitro makes this process difficult to study, even though it is fundamental for understanding disease progression and aggressiveness. Existing co-culture models often use stromal cells to overcome spontaneous CLL cell apoptosis or mimic adhesion-mediated drug resistance, but these models do not trigger proliferation.

Models that do induce proliferation usually add costly recombinant IL4 or IL21 to CD40L-expressing stromal cells, yet the CLL proliferation rate remains relatively low and highly variable. CXCR4 and CD5 distinguish CLL cells that have recently exited immune niches: CXCR4-dim, CD5-bright cells carry a fingerprint of microenvironmental interactions.

RNA profiling of ten paired samples revealed seven thousand three hundred ninety-three differentially expressed messenger RNAs. Pathway analysis identified T-cell factors, including CD40L and several interleukins, as key upstream gene-expression regulators in immune niches.

Three datasets overlapped in twelve factors. The model therefore focused on three canonical T-cell factors: CD40L, IL4, and IL21. The investigators genetically engineered the fibroblast-like HS5 cell line, which protects CLL lymphocytes from apoptosis but does not induce proliferation or produce T-cell factors.

Six HS5-derived cell lines were prepared with stable expression of CD40L, IL4, IL21, and their combinations. Their expression was verified, and the engineered cells activated the expected STAT and IKK signaling in CLL cells. Primary CLL cells from sixteen samples were co-cultured with six HS5 cell lines at a one-to-twenty HS5-to-CLL ratio to test pro-proliferative effects.

HS5 cells expressing CD40L and IL4 produced seven percent CLL proliferation on day seven, while cells expressing CD40L, IL4, and IL21 produced forty-four percent proliferation. Proliferation was measured by CFSE dilution, because Ki67 positivity indicated activation but not CLL cell division.

The triple-factor co-culture induced more proliferation than recombinant CD40L, IL4, and IL21. The result fits observations in normal B-cells that CD40L requires membrane presentation for an optimal effect. Figure one tests how engineered HS5 stromal cells activate and sustain primary CLL cells.

Immunoblots show IL21-, IL4-, and CD40L-associated signaling, while CFSE measurements report proliferation: seven percent of cells divided with HS5-CD40L-IL4 and forty-four percent with HS5-CD40L-IL4-IL21 by day seven. The panels also assess viability, spatial co-culture formats, and longer-term support, linking microenvironmental signals to CLL activation and expansion.

IL21 alone induced some CLL cell apoptosis, while IL4 co-cultures rescued spontaneous apoptosis. In combination with CD40L and IL4, IL21’s negative effect on viability was less pronounced, allowing all three factors to be used together. Viability was generally negatively correlated with proliferation rate, likely because of the metabolic burden associated with cell division.

Changing supportive cells or adding beta-mercaptoethanol could increase CLL survival. Lower apoptosis was also observed with HS5 cells having lower CD40L expression, while recombinant BAFF or APRIL produced no substantial pro-survival effect.

Overall, HS5-CD40L-IL4 produced reproducible, mild proliferation with a median of seven percent, whereas HS5-CD40L-IL4-IL21 produced high proliferation of about forty-four percent. For large drug screenings, the co-culture was downscaled into custom poly-dimethylsiloxane inserts with a five-microliter volume and an eight-square-millimeter surface.

Each microwell contained about two hundred fifty HS5-CD40L-IL4 cells and two thousand five hundred CLL cells—roughly twelve thousand times fewer cells than a standard twelve-well plate. CLL cells in the microwells had proliferation and viability identical to standard co-cultures, showing that the system remained robust at microliter scale.

The models also supported CLL proliferation and survival for more than two months, although total cell numbers did not significantly expand, indicating an ongoing balance between proliferation and apoptosis. To ask whether the model resembles an immune niche, primary CLL cells from four samples were profiled after three days on plastic, HS5-WT, or HS5-CD40L-IL4 cells.

HS5-WT had a surprisingly minor effect, with only ninety-four differentially expressed messenger RNAs compared with plastic. In contrast, HS5-CD40L-IL4 versus HS5-WT produced three thousand eight hundred thirty-three differentially expressed messenger RNAs. The altered program included MYC, NF-kappa-B, and E2F signatures previously defined from lymph-node CLL samples.

MYC induction was also verified at the protein level. The co-culture also included part of the BCR signaling signature despite lacking antigen, while IL4 induced a four point two-fold increase in cell-surface BCR. Figure two shows that adding CD40 ligand and interleukin four to the HS5 co-culture system substantially reshapes CLL-cell transcription, including MYC, NF-kappa-B, E2F, and BCR-associated signatures.

Adding interleukin twenty-one produces a related expression pattern, while immunoblotting and surface-IgM measurements provide protein-level validation. The overlap analyses also show significant similarity between these co-culture profiles and the CXCR4-dim, CD5-bright CLL subpopulation found in lymph nodes, supporting the model’s biological relevance.

Because absent CLL proliferation prevents anti-proliferative drug testing, the model was used to compare microenvironment-targeting inhibitors, including ibrutinib, idelalisib, and inhibitors of the RAF, MAPK, and ERK pathway. RNA profiling and immunoblotting showed ERK pathway activation in the co-culture, providing the reason to test this inhibitor group.

The BTK and PI3K inhibitors had only a mild effect, limited to some samples. In contrast, the wild-type RAF inhibitors LY3009120 and naporafenib inhibited proliferation in all cases, with at least a fifty percent reduction.

RAF inhibitors did not affect CLL viability, meaning their anti-proliferative activity would be missed by classical cytotoxicity screens. Figure three tests how RAF-pathway inhibition affects CLL proliferation in a supportive co-culture containing CD40L, interleukin four, and interleukin twenty-one.

The panels combine pathway and immunoblot data with seven-day proliferation and viability measurements, transcriptomic changes induced by two pan-RAF inhibitors, and evidence that LY3009120 alters phospho-P38 signaling. P38 inhibitors also affected cell-cycle progression, linking this downstream stress-kinase response to proliferation.

The two RAF inhibitors changed six hundred forty-seven and six hundred four messenger RNAs, respectively, in co-cultured CLL cells. Their effects largely overlapped, with three hundred fifty-nine identical messenger RNAs changed by both inhibitors. Only fifteen messenger RNAs differed between the two RAF inhibitor treatments, and gene-set enrichment analysis identified changes in GSK3, mTOR, EIF4, TOLL, and AKT pathways.

MEK and ERK inhibitors did not affect CLL proliferation, even though they act mainly within the same pathway. Unlike those inhibitors, RAF inhibitors very potently reduced AKT phosphorylation. LY3009120 also affected P38 phosphorylation, a mitogen-activated kinase involved in normal B-cell proliferation after BCR, IL4, and CD40 stimulation.

P38 inhibitors impaired CLL proliferation to some extent, but less potently than RAF inhibition. This supports a potent cytostatic effect from two chemically different pan-RAF inhibitors. The study’s model was designed to mimic T-cell signals and induce CLL proliferation without recombinant factors.

CD40L and IL4 produced mild proliferation of about seven percent, while CD40L, IL4, and IL21 produced a consistently high rate of about forty-four percent at day seven. These models mimic gene-expression changes in CLL cells interacting in immune niches and can be downscaled to microliter volumes.

That combination makes them suitable for anti-proliferative drug screening. The same supportive cells were also taken into immunodeficient NSG mice with primary CLL cells, where they led to B-cell lymphoproliferation and could simplify PDX model generation.

The next question was whether engineered HS5 cells could replace activated primary T-cell co-transplantation and simplify CLL patient-derived xenograft generation in NSG mice. The researchers aimed to transfer the co-culture model into NSG mice using engineered HS5 cells instead of activated primary T-cell co-transplantation.

Figure four shows the authors’ scaffold-based transplantation strategy for establishing human CLL tumors in NSG mice, using HS5-CD40L-IL4 cells with or without IL21 support. Human CD20 staining identifies B-cell engraftment in spleen and tumor samples, while spleen and tumor weights are plotted against time to death.

The survival curves summarize time to euthanasia for mice with extensive tumor growth, and FISH images from mice M20 and M29 detect the 13q14 deletion, supporting the CLL identity of the engrafted cells. To verify PDX tumor clonality, IGHV analysis was attempted in twenty-three mouse tumors, but an evaluable PCR product was obtained in only fourteen cases.

Two cases had a monoclonal IGHV rearrangement identical to the primary CLL sample, and two additional samples had two or three detectable clones, including one clone identical to the original CLL sample. The other ten samples had a different monoclonal or polyclonal IGHV. The study also tested for the thirteen-q-fourteen deletion originally identified in six patients’ samples to further validate clonal relationships.

The co-culture system induced MYC, NF-kappa-B, E2F, and BCR signatures defined from lymph-node CLL samples. Testing inhibitors of BTK, PI3K, RAF, P38, MEK, and ERK revealed that CLL proliferation was highly sensitive to RAF inhibitors. Pan-RAF inhibitors were not cytotoxic to CLL cells, so their activity would be missed in assays using purified CLL cells or models that do not induce high proliferation.

The results provide a rationale for further testing pan-RAF inhibitors in CLL, including possible combinations with venetoclax, because T-cell interactions provide resistance to that drug. Overall, the co-culture models are versatile tools for studying pro-proliferative CLL-T-cell interactions and identifying anti-proliferative inhibitors, with future potential for CRISPR screening using primary CLL cells.

Figure five summarizes how engineered HS5 cells create a supportive CLL model. Co-culture with CD40L and IL4 produced about seven percent CLL proliferation at day seven, while adding IL21 produced a median of forty-four percent; the system also reproduced lymph-node gene signatures involving MYC, NFκB, and E2F.

The figure highlights practical utility: RAF inhibitors block this proliferation, cultures can be miniaturized for screening, and the cells can support CLL engraftment on a three-dimensional collagen scaffold in NSG mice. The engineered co-culture model induces robust CLL cell proliferation through supportive cells expressing CD40L, IL4, and IL21.

It can be downscaled to microliter volumes for drug screening or upscaled to a PDX model. This is the first CLL xenograft model in which a genetically engineered cell line replaces primary T-cells. The study also shows for the first time that RAF inhibitors can disturb T-cell-factor-induced CLL proliferation.

Engineered HS5 cells expressing CD40L, IL4, and IL21 create a robust, niche-like CLL proliferation model that works in tiny drug-screening formats and PDX mice, while exposing pan-RAF inhibitors as potent cytostatic agents.