The tumor microenvironment (TME) plays an essential role in the development, growth, and survival of the malignant B-cell clone in chronic lymphocytic leukemia (CLL). Within the proliferation niches of lymph nodes, bone marrow, and secondary lymphoid organs, a variety of phenotypically and functionally altered cell types, including T cells, natural killer cells, monocytes/macrophages, endothelial and mesenchymal stroma cells, provide crucial survival signals, along with CLL-cellinduced suppression of antitumor immune responses. The B-cell receptor pathway plays a pivotal role in mediating the interaction between CLL cells and the TME. However, an increasing number of additional components of the multifactorial TME are being discovered. Although the majority of therapeutic strategies employed in CLL hitherto have focused on targeting the leukemic cells, emerging evidence implies that modulation of microenvironmental cells and CLL-TME interactions by novel therapeutic agents significantly affect their clinical efficacy. Thus, improving our understanding of CLL-TME interactions and how they are affected by current therapeutic agents may improve and guide treatment strategies. Identification of novel TME interactions may also pave the road for the development of novel therapeutic strategies targeting the TME. In this review, we summarize current evidence on the effects of therapeutic agents on cells and interactions within the TME. With a growing demand for improved and personalized treatment options in CLL, this review aims at inspiring future exploration of smart drug combination strategies, translational studies, and novel therapeutic targets in clinical trials.

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Background The immune pathogenesis underlying the diverse clinical course of COVID-19 is poorly understood. Currently, there is an unmet need in daily clinical practice for early biomarkers and improved risk stratification tools to help identify and monitor COVID-19 patients at risk of severe disease. Methods We performed longitudinal assessment of stimulated immune responses in 30 patients hospitalized with COVID-19. We used the TruCulture whole-blood ligand-stimulation assay applying standardized stimuli to activate distinct immune pathways, allowing quantification of cytokine responses. We further characterized immune cell subsets by flow cytometry and used this deep immunophenotyping data to map the course of clinical disease within and between patients. Results Here we demonstrate impairments in innate immune response pathways at time of COVID-19 hospitalization that are associated with the development of severe disease. We show that these impairments are transient in those discharged from hospital, as illustrated by functional and cellular immune reconstitution. Specifically, we identify lower levels of LPS-stimulated IL-1β, and R848-stimulated IL-12 and IL-17A, at hospital admission to be significantly associated with increasing COVID-19 disease severity during hospitalization. Furthermore, we propose a stimulated immune response signature for predicting risk of developing severe or critical COVID-19 disease at time of hospitalization, to validate in larger cohorts. Conclusions We identify early impairments in innate immune responses that are associated with subsequent COVID-19 disease severity. Our findings provide basis for early identification of patients at risk of severe disease which may have significant implications for the early management of patients hospitalized with COVID-19.

Background: For proliferation and survival, chronic lymphocytic leukemia (CLL) cells depend on interactions with cells and soluble factors present in the tumor microenvironment (TME). These interactions also increase expression of B-cell leukemia/lymphoma-2 (Bcl-2) proteins, including Bcl-XL, Mcl-1 and Bfl-1, thereby reducing drug sensitivity. In the VISION HOVON141 clinical trial, patients with relapsed or refractory CLL are treated with 2 cycles of Bruton tyrosine kinase inhibitor ibrutinib (IBR) lead-in followed by 13 cycles of IBR + Bcl-2 inhibitor venetoclax (VEN) combination before randomization. The combination of IBR + VEN may have synergistic anti-tumor effects, since IBR forces CLL cells from lymph node (LN) to the blood (PB) where they become fully dependent on Bcl-2, and succumb to VEN. To support a mechanistic basis for this premise, we investigated changes in expression of Bcl-2 proteins, effects of TME-mimicking signals, and venetoclax sensitivity before/after 2 months IBR treatment. Results: At baseline, lymph node emigrant cells (CXCR4dim/CD5high) have higher Bfl-1 expression, in addition to earlier reported Bcl-XL and Mcl-1 expression than cells immigrating back to the lymph node (CXCR4high/CD5dim)(Haselager et al., 2020). After 2 months of IBR treatment a clear reduction in all four pro-survival proteins was observed (N=17 p<0.001). Despite these changes, VEN sensitivity was not different in unstimulated PB CLL cells obtained at baseline versus at 2 months of IBR treatment (N=8; IC50=0.001µM). In contrast, CD40 stimulated CLL cells obtained at baseline are fully resistant to VEN (IC50>10µM), but unexectedly retained partial sensitivity to venetoclax after IBR treatment (IC50=0.05 µM, p-value <0.0001; Figure 1A). This suggested reduced CD40 signalling capacity and was accompanied by reduced ability to upregulate Bcl-XL, Mcl-1, and Bfl-1 expression. Indeed, cell surface expression, as well as level of CD40 activation as measured by CD95 induction, was clearly reduced after 2 months IBR. Importantly, these effects occurred in vivo, as IBR did not directly affect CD40 signaling in vitro. These data imply that under IBR treatment, when cells cannot (re-)enter LN sites, a factor is lacking that maintains or induces CD40 expression. In search of stimuli that can augment CD40 signaling capacity, it was found that BCR stimulation had no effect on CD40 expression. In contrast, TLR9 stimulation via CpG led to increased CD40 expression in CLL cells (N=7, p-value <0.0001)(Figure 1B). These findings align well with recent data which indicate a role for TLR9 signalling in vivo by unmethylated mitochondrial DNA in CLL (Kennedy et al., 2021). Taken together, these data provide a mechanistic explanation, by which a triad of signaling pathways not only involving BCR but also CD40 and TLR9 is interrupted by IBR (Figure 1C). Discussion/conclusion: IBR treatment broadly affects Bcl-2 family protein expression and CD40 signaling in vivo. The combined data indicate a novel aspect of IBR efficacy, namely its capacity to interrupt TLR9-induced CD40 upregulation, which normally primes CLL cells in the LN environment for drug resistance. This also suggests that drugs that inhibit TLR9 signaling may synergize with IBR. References Haselager, M. V., et al (2020). Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL. Blood, 136(25), 2918 Kennedy, E., et al (2021). TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target. Blood, 137(22), 3064 Figure 1 Figure 1. Disclosures Levin: Roche, Janssen, Abbvie: Other: Travel Expenses, Ad-Board. Westerweel: Novartis: Research Funding; Incyte: Consultancy; BMS / Celgene: Consultancy; Pfizer: Consultancy. Niemann: CSL Behring, Genmab, Takeda, Octapharma: Consultancy; Novo Nordisk Foundation: Research Funding; Abbvie, AstraZeneca, Janssen: Consultancy, Research Funding.