Franziska Brauneck

Immunotherapy in acute myeloid leukemia (AML) is constrained by a hostile bone marrow niche. Here we identify atypical memory B cells (B _atyp ) as a suppressive population within this microenvironment. Across single cell and flow cytometry cohorts, B _atyp were expanded in AML, and their molecular and phenotypic profiles resembled those of atypical B cells associated with chronic inflammatory conditions. B _atyp were polarized within the AML microenvironment, and this was amplified by CAR T cell–AML interactions. Mechanistically, polarization was dependent on Toll-like receptor, interferon and CD70–CD27 signaling. B _atyp suppressed T cell and CAR T cell responses via PD-1-and TGF-β-R signaling, and were enriched in non-responding patients with AML and myeloma. CD180-directed CAR T cells, targeting an antigen shared by B _atyp and AML blasts, resisted suppression in vitro and cleared disease in B cell–humanized xenografts while sparing hematopoietic stem cells, with B cell engagement further enhancing their expansion. CAR T cells thus reinforce their own suppressive niche through B _atyp polarization, a constraint that can be relieved by dual blast- and B cell-targeting strategies.

Background: Bone metastases (BM) represent one of the most common sites of metastasis. The study aimed to compare the composition of immune cell infiltration from aspirates of different BM prior to systemic therapy. Method: Phenotypic and functional analyses were conducted via multiparametric flow cytometry (MFC) on BM-derived aspirates obtained from patients with breast cancer (BC, n = 6), patients with prostate cancer (PC, n = 5), patients with non-small-cell lung cancer (NSCLC) (n = 7), patients with myeloma (MM, n = 10) and bone aspirates from age-matched non-malignant controls (NMC, n = 10). Results: Across all tumors aspirates the fraction of CD8⁺ T cells was reduced. In contrast, infiltration by immunosuppressive CD56⁺CD16⁻NK and CD163⁺CD86⁺ M2-like macrophages was increased in BM compared to NMC aspirates. BM-derived CD8⁺ T cells aberrantly co-expressed TIGIT with PVRIG or CD39. Similarly, BM-derived cytotoxic NK cells co-expressed TIGIT and PVRIG. In addition, BM-derived M2-like macrophages exhibited an increased subset of cells co-expressing either TIGIT and PVRL4 or CD112 and CD155. Using a myeloma model, functional in vitro studies showed that blockade of TIGIT and CD39 leads to increased PBMC-mediated lysis of myeloma cells. Conclusion: The study shows that an altered immune cell composition is present in BM across the different tumor entities. Additionally, molecules of the TIGIT checkpoint as well as of the purinergic pathway are aberrantly expressed by BM-infiltrating CD8⁺ T cells, NK cells and macrophages and also functionally relevant for tumor cell lysis.

Comprehensive characterization of AML-associated T cells during disease progression is essential to identify relevant immune escape mechanisms and new immunotherapeutic approaches. Investigating the processes that lead to an immunosuppressive environment under progression of AML is difficult in humans, because by the time of diagnosis the disease is often progressed far beyond the initial stages. Therefore, to investigate T-cell phenotypes during progression a C57BL/6 mouse model was used. The CD3⁺ T cells were characterized by performing multiparametric flow analyses at different time points (day 0 = healthy mice, day 7, day 14, and day 21). The study revealed that the spleen is highly infiltrated by reg CD4⁺ T cells at day 21 of AML progression. These spleen-infiltrating reg CD4⁺ T cells mainly showed an effector memory differentiation with high expression and co-expression of the checkpoint molecules TIGIT, PD-1, OX40, and the two ectoenzymes CD39 and CD73. Substantial expression of the checkpoint molecules was restricted to the central memory and effector memory compartments. Furthermore, functional evaluation of TIGIT was performed. Blocking TIGIT resulted in a significantly increased lysis of C1498 AML cells in cocultures with AML-primed CD3⁺ T cells. Together these data confirm that the expression of the checkpoint receptor TIGIT is relevant for dysfunction of AML-associated T cells and, thus, represents a suitable target for future immunotherapeutic approaches.

Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3⁺ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8⁺ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1^high CD8⁺ T cell population was detected, which differed from PD-1^lowCD8⁺ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8⁺ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the “don’t eat me” molecule CD24 on tumor cells. Additionally, ascites-derived CD24⁺EpCAM⁺ tumor cells showed a higher frequency of CD39⁺ or CD73⁺ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8⁺ T cells.

Introduction: Tumor-associated macrophages (TAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype and function of these cells. The present study aims to characterize macrophages in high-grade serous ovarian cancer (HGSOC).Methods: Phenotype and expression of co-regulatory markers were assessed on TAMs derived from malignant ascites (MA) or peripheral blood (PB) by multiparametric flow cytometry. Samples were obtained from HGSOC patients (n=29) and healthy donors (HDs, n=16). Additional expression analysis was performed by RNAseq (n=192). Correlation with clinically relevant parameters was conducted and validated by a second patient cohort (n=517). Finally, the role of TIGIT in repolarization and phagocytosis was investigated in vitro.Results: Expression of the M2-associated receptors CD163, CD204, and CD206, as well as of the co-regulatory receptors TIGIT, CD226, TIM-3, and LAG-3 was significantly more frequent on macrophages in HGSOC than in HDs. CD39 and CD73 were broadly expressed on (mainly M2) macrophages, but without a clear clustering in HGSOC. CD163 mRNA levels were higher in TAMs from patients with residual tumor mass after surgery and associated with a shorter overall survival. In addition, TIGIT expression was associated with a higher tumor grading, indicating a prognostic relevance of M2 infiltration in HGSOC. TIGIT blockade significantly reduced the frequency of M2 macrophages. Moreover, combined blockade of TIGIT and CD47 significantly increased phagocytosis of ovarian cancer cells by TAMs in comparison to a single blockade of CD47.Conclusion: Combined blockade of TIGIT and CD47 represents a promising approach to enhance anti-CD47-facilitated phagocytosis.

The search for new and effective treatment targets for cancer immunotherapy is an ongoing challenge. Alongside the more established inhibitory immune checkpoints, a novel potential target is CD73. As one of the key enzymes in the purinergic signalling pathway CD73 is responsible for the generation of immune suppressive adenosine. The expression of CD73 is higher in tumours than in the corresponding healthy tissues and associated with a poor prognosis. CD73, mainly by the production of adenosine, is critical in the suppression of an adequate anti-tumour immune response, but also in promoting cancer cell proliferation, tumour growth, angiogenesis, and metastasis. The upregulation of CD73 and generation of adenosine by tumour or tumour-associated immune cells is a common resistance mechanism to many cancer treatments such as chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Therefore, the inhibition of CD73 represents a new and promising approach to increase therapy efficacy. Several CD73 inhibitors have already been developed and successfully demonstrated anti-cancer activity in preclinical studies. Currently, clinical studies evaluate CD73 inhibitors in different therapy combinations and tumour entities. The initial results suggest that inhibiting CD73 could be an effective option to augment anti-cancer immunotherapeutic strategies. This review provides an overview of the rationale behind the CD73 inhibition in different treatment combinations and the role of CD73 as a prognostic marker.

Background: Leukemia-associated macrophages (LAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype, function, and plasticity of these cells. The present study provides an extensive characterization of macrophages in patients with acute myeloid leukemia (AML). Methods: The phenotype and expression of coregulatory markers were assessed on bone marrow (BM)-derived LAM populations, using multiparametric flow cytometry. BM and blood aspirates were obtained from patients with newly diagnosed acute myeloid leukemia (pAML, n=59), patients in long-term remission (lrAML, n=8), patients with relapsed acute myeloid leukemia (rAML, n=7) and monocyte-derived macrophages of the blood from healthy donors (HD, n=17). LAM subpopulations were correlated with clinical parameters. Using a blocking anti-T-cell immunoreceptor with Ig and ITIM domains (TIGIT) antibody or mouse IgG2α isotype control, we investigated polarization, secretion of cytokines, and phagocytosis on LAMs and healthy monocyte-derived macrophages in vitro. Results: In pAML and rAML, M1 LAMs were reduced and the predominant macrophage population consisted of immunosuppressive M2 LAMs defined by expression of CD163, CD204, CD206, and CD86. M2 LAMs in active AML highly expressed inhibitory receptors such as TIGIT, T-cell immunoglobulin and mucin-domain containing-3 protein (TIM-3), and lymphocyte-activation gene 3 (LAG-3). High expression of CD163 was associated with a poor overall survival (OS). In addition, increased frequencies of TIGIT⁺ M2 LAMs were associated with an intermediate or adverse risk according to the European Leukemia Network criteria and the FLT3 ITD mutation. In vitro blockade of TIGIT shifted the polarization of primary LAMs or peripheral blood-derived M2 macrophages toward the M1 phenotype and increased secretion of M1-associated cytokines and chemokines. Moreover, the blockade of TIGIT augmented the anti-CD47-mediated phagocytosis of AML cell lines and primary AML cells. Conclusion: Our findings suggest that immunosuppressive TIGIT⁺ M2 LAMs can be redirected into an efficient effector population that may be of direct clinical relevance in the near future.

Recently new treatments for acute myeloid leukemia (AML) emerged, including regimens like CPX-351 and cladribine with cytarabine and daunorubicin (DA + C), demonstrating improved survival in patient subsets. This retrospective analysis is comparing the outcome of 124 patients treated with cytarabine and daunorubicin (DA; n = 54), CPX-351 (n = 26) and DA + C (n = 44). Complete response rate following one cycle of therapy was increased in DA + C (62%) compared to CPX-351 (42%) and DA (50%). CPX-351 demonstrated a significant increased survival post allogenic stem cell transplantation against DA (hazard ratio (HR): 4.9; 95% confidence interval (95%CI): 1.1–21, p = 0.03). Median survival was reached for DA (5.6 years) but not for DA + C or CPX-351. Subgroup analysis showed that AML with myelodysplasia-related changes and therapy-related AML treated with CPX-351 had increased survival compared to DA (HR: 5.2; 95%CI: 1.2–22; p = 0.03). Our findings point twoards a CPX-351 superiority. However, the use of DA + C should be further evaluated in comparative studies.

Phenotypic characterization of γδ T cells in the MALs (malignant ascites lymphocytes), TILs (tumor infiltrating lymphocytes), and PBLs (peripheral blood lymphocytes) of ovarian cancer (OvCA) patients is lacking. Therefore, we quantified γδ T cell prevalence in MAL, TIL, and PBL specimens from n = 18 OvCA patients and PBL from age-matched healthy donors (HD, n = 14). Multicolor flow cytometry was performed to evaluate the expression of inhibitory receptors (TIGIT, PD-1 and TIM-3), stimulatory receptors (Ox40), and purinergic ectoenzymes (CD39 and CD73) on γδ T cell subsets. We identified an abundant infiltration of Vδ1 T cells in the MALs and TILs. These cells varied in their differentiation: The majority of Vδ1 TILs displayed an effector memory (EM) phenotype, whereas Vδ1 MALs had a more mature phenotype of terminally differentiated effector memory cells (TEMRA) with high CD45RA expression. TIGIT and TIM-3 were abundantly expressed in both MALs and PBLs, whereas Vδ1 TILs exhibited the highest levels of PD-1, CD39, and Ox40. We also observed specific clusters on mature differentiation stages for the analyzed molecules. Regarding co-expression, Vδ1 TILs showed the highest levels of cells co-expressing TIGIT with PD-1 or CD39 compared to MALs and PBLs. In conclusion, the Vδ1 T cell population showed a high prevalence in the MALs and primary tumors of OvCA patients. Due to their (co-)expression of targetable immune receptors, in particular TIGIT with PD-1 and CD39 in TILs, Vδ1 T cell-based approaches combined with the inhibition of these targets might represent a promising strategy for OvCA.

This study aimed to characterize different natural killer (NK) cell phenotypes on bone marrow and peripheral blood cells from acute myeloid leukemia (AML) patients and healthy donors (HDs). Our data show that CD56^dimCD16⁻ and CD56^brightCD16⁻ NK cells represent the predominant NK cell subpopulations in AML, while the CD56^dimCD16⁺ NK cells are significantly reduced compared to HDs. Moreover, TIGIT⁺ and PVRIG⁺ cells cluster on the CD56^dimCD16⁺ subset whereas CD39⁺ and CD38⁺ cells do so on CD56^brightCD16⁻ NK cells in AML. Furthermore, functional effects of (co-)blockade of TIGIT and CD39 or A2AR on NK cell functionality were analyzed. These experiments revealed that the single blockade of the TIGIT receptor results in an increased NK-92 cell-mediated killing of AML cells in vitro. Combined targeting of CD39 or A2AR significantly augments the anti-TIGIT-mediated lysis of AML cells. Our data indicate that distinct NK cell subsets in AML exhibit different immunosuppressive patterns (via the TIGIT/PVRIG receptors and the purinergic pathway). In summary, we conclude that TIGIT, CD39, and A2AR constitute relevant inhibitory checkpoints of NK cells in AML patients. A combinatorial blockade synergistically strengthens NK-92 cell-mediated cytotoxicity. As inhibitors of TIGIT, CD39, and A2AR are clinically available, studies on their combined use could be conducted in the near future.