BACKGROUND: Epstein-Barr virus (EBV) is a double-stranded DNA oncogenic virus. Several types of solid tumors, such as nasopharyngeal carcinoma, EBV-associated gastric carcinoma, and lymphoepithelioma-like carcinoma of the lung, have been linked to EBV infection. Currently, several TCR-T-cell therapies for EBV-associated tumors are in clinical trials, but due to the suppressive immune microenvironment of solid tumors, the clinical application of TCR-T-cell therapy for EBV-associated solid tumors is limited. Figuring out the mechanism by which EBV participates in the formation of the tumor immunosuppressive microenvironment will help T cells or TCR-T cells break through the limitation and exert stronger antitumor potential.
METHODS: Flow cytometry was used for analyzing macrophage differentiation phenotypes induced by EBV-infected and EBV-uninfected tumors, as well as the function of T cells co-cultured with these macrophages. Xenograft model in mice was used to explore the effects of M2 macrophages, TCR-T cells, and matrix metalloprotein 9 (MMP9) inhibitors on the growth of EBV-infected tumors.
RESULTS: EBV-positive tumors exhibited an exhaustion profile of T cells, despite the presence of a large T-cell infiltration. EBV-infected tumors recruited a large number of mononuclear macrophages with CCL5 and induced CD163+M2 macrophages polarization through the secretion of CSF1 and the promotion of autocrine IL10 production by mononuclear macrophages. Massive secretion of MMP9 by this group of CD163+M2 macrophages induced by EBV infection was an important factor contributing to T-cell exhaustion and TCR-T-cell therapy resistance in EBV-positive tumors, and the use of MMP9 inhibitors improved the function of T cells cocultured with M2 macrophages. Finally, the combination of an MMP9 inhibitor with TCR-T cells targeting EBV-positive tumors significantly inhibited the growth of xenografts in mice.
CONCLUSIONS: MMP9 inhibitors improve TCR-T cell function suppressed by EBV-induced M2 macrophages. TCR-T-cell therapy combined with MMP9 inhibitors was an effective therapeutic strategy for EBV-positive solid tumors.

BACKGROUND: While Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade is a potent antitumor treatment strategy, it is effective in only limited subsets of patients with cancer, emphasizing the need for the identification of additional immune checkpoints. Butyrophilin 1A1 (BTN1A1) has been reported to exhibit potential immunoregulatory activity, but its ability to function as an immune checkpoint remains to be systematically assessed, and the mechanisms underlying such activity have yet to be characterized.
METHODS: BTN1A1 expression was evaluated in primary tumor tissue samples, and its ability to suppress T-cell activation and T cell-dependent tumor clearance was examined. The relationship between BTN1A1 and PD-L1 expression was further characterized, followed by the development of a BTN1A1-specific antibody that was administered to tumor-bearing mice to test the amenability of this target to immune checkpoint inhibition.
RESULTS: BTN1A1 was confirmed to suppress T-cell activation in vitro and in vivo. Robust BTN1A1 expression was detected in a range of solid tumor tissue samples, and BTN1A1 expression was mutually exclusive with that of PD-L1 as a consequence of its inhibition of Janus-activated kinase/signal transducer and activator of transcription signaling-induced PD-L1 upregulation. Antibody-mediated BTN1A1 blockade suppressed tumor growth and enhanced immune cell infiltration in syngeneic tumor-bearing mice.
CONCLUSIONS: Together, these results confirm that the potential of BTN1A1 is a bona fide immune checkpoint and a viable immunotherapeutic target for the treatment of individuals with anti-PD-1/PD-L1 refractory or resistant disease, opening new avenues to improving survival outcomes for patients with a range of cancers.

Enzalutamide, a next-generation antiandrogen agent, is approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). While enzalutamide has been shown to improve time to progression and extend overall survival in men with CRPC, the majority of patients ultimately develop resistance to treatment. Immunotherapy approaches have shown limited clinical benefit in this patient population; understanding resistance mechanisms could help develop novel and more effective treatments for CRPC. One of the mechanisms involved in tumor resistance to various therapeutics is tumor phenotypic plasticity, whereby carcinoma cells acquire mesenchymal features with or without the loss of classical epithelial characteristics. This work investigated a potential link between enzalutamide resistance, tumor phenotypic plasticity, and resistance to immune-mediated lysis in prostate cancer.
Models of prostate cancer resistant to enzalutamide were established by long-term exposure of human prostate cancer cell lines to the drug in culture. Tumor cells were evaluated for phenotypic features in vitro and in vivo, as well as for sensitivity to immune effector cell-mediated cytotoxicity.
Resistance to enzalutamide was associated with gain of mesenchymal tumor features, upregulation of estrogen receptor expression, and significantly reduced tumor susceptibility to natural killer (NK)-mediated lysis, an effect that was associated with decreased tumor/NK cell conjugate formation with enzalutamide-resistant cells. Fulvestrant, a selective estrogen receptor degrader, restored the formation of target/NK cell conjugates and increased susceptibility to NK cell lysis in vitro. In vivo, fulvestrant demonstrated antitumor activity against enzalutamide-resistant cells, an effect that was associated with activation of NK cells.
NK cells are emerging as a promising therapeutic approach in prostate cancer. Modifying tumor plasticity via blockade of estrogen receptor with fulvestrant may offer an opportunity for immune intervention via NK cell-based approaches in enzalutamide-resistant CRPC.

Anticancer immunotherapies, such as immune checkpoint inhibitors, bispecific antibodies, and chimeric antigen receptor T cells, have improved outcomes for patients with a variety of malignancies. However, most patients either do not initially respond or do not exhibit durable responses due to primary or adaptive/acquired immune resistance mechanisms of the tumor microenvironment. These suppressive programs are myriad, different between patients with ostensibly the same cancer type, and can harness multiple cell types to reinforce their stability. Consequently, the overall benefit of monotherapies remains limited. Cutting-edge technologies now allow for extensive tumor profiling, which can be used to define tumor cell intrinsic and extrinsic pathways of primary and/or acquired immune resistance, herein referred to as features or feature sets of immune resistance to current therapies. We propose that cancers can be characterized by immune resistance archetypes, comprised of five feature sets encompassing known immune resistance mechanisms. Archetypes of resistance may inform new therapeutic strategies that concurrently address multiple cell axes and/or suppressive mechanisms, and clinicians may consequently be able to prioritize targeted therapy combinations for individual patients to improve overall efficacy and outcomes.

Type I interferons (IFN-Is), secreted by hematopoietic cells, drive immune surveillance of solid tumors. However, the mechanisms of suppression of IFN-I-driven immune responses in hematopoietic malignancies including B-cell acute lymphoblastic leukemia (B-ALL) are unknown.
Using high-dimensional cytometry, we delineate the defects in IFN-I production and IFN-I-driven immune responses in high-grade primary human and mouse B-ALLs. We develop natural killer (NK) cells as therapies to counter the intrinsic suppression of IFN-I production in B-ALL.
We find that high expression of IFN-I signaling genes predicts favorable clinical outcome in patients with B-ALL, underscoring the importance of the IFN-I pathway in this malignancy. We show that human and mouse B-ALL microenvironments harbor an intrinsic defect in paracrine (plasmacytoid dendritic cell) and/or autocrine (B-cell) IFN-I production and IFN-I-driven immune responses. Reduced IFN-I production is sufficient for suppressing the immune system and promoting leukemia development in mice prone to MYC-driven B-ALL. Among anti-leukemia immune subsets, suppression of IFN-I production most markedly lowers the transcription of IL-15 and reduces NK-cell number and effector maturation in B-ALL microenvironments. Adoptive transfer of healthy NK cells significantly prolongs survival of overt ALL-bearing transgenic mice. Administration of IFN-Is to B-ALL-prone mice reduces leukemia progression and increases the frequencies of total NK and NK-cell effectors in circulation. Ex vivo treatment of malignant and non-malignant immune cells in primary mouse B-ALL microenvironments with IFN-Is fully restores proximal IFN-I signaling and partially restores IL-15 production. In B-ALL patients, the suppression of IL-15 is the most severe in difficult-to-treat subtypes with MYC overexpression. MYC overexpression promotes sensitivity of B-ALL to NK cell-mediated killing. To counter the suppressed IFN-I-induced IL-15 production in MYChigh human B-ALL, we CRISPRa-engineered a novel human NK-cell line that secretes IL-15. CRISPRa IL-15-secreting human NK cells kill high-grade human B-ALL in vitro and block leukemia progression in vivo more effectively than NK cells that do not produce IL-15.
We find that restoration of the intrinsically suppressed IFN-I production in B-ALL underlies the therapeutic efficacy of IL-15-producing NK cells and that such NK cells represent an attractive therapeutic solution for the problem of drugging MYC in high-grade B-ALL.

Emerging data suggest that patients with enzalutamide-treated prostate cancer with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment. Unfortunately, the Phase III IMbassador250 clinical trial revealed that the combination of atezolizumab (a PD-L1 inhibitor) and enzalutamide failed to extend overall survival in patients with castration-resistant prostate cancer (CRPC). However, the mechanisms underlying treatment failure remain unknown.
Human CRPC C4-2B cells and murine Myc-CaP cells were chronically exposed to increasing concentrations of enzalutamide and the cells resistant to enzalutamide were referred to as C4-2B MDVR and Myc-CaP MDVR, respectively. The mechanisms of action in drug-resistant prostate cancer cells were determined using RNA sequencing analyses, RNA interference, real-time PCR, western blotting, and co-culturing technologies. Myc-CaP and Myc-CaP MDVR tumors were established in syngeneic FVB mice, and tumor-infiltrating leukocytes were isolated after enzalutamide treatment. The stained immune cells were determined by flow cytometry, and the data were analyzed using FlowJo.
Immune-related signaling pathways (interferon alpha/gamma response, inflammatory response, and cell chemotaxis) were suppressed in human enzalutamide-resistant prostate cancer cells. PD-L1 was overexpressed and negatively regulated by androgen receptor signaling in resistant cells and patient with CRPC cohorts. Enzalutamide treatment decreased CD8+ T-cell numbers but increased monocytic myeloid-derived suppressor cell (M-MDSC) populations and PD-L1 expression within murine Myc-CaP tumors. Similarly, chemotaxis and immune response-regulating signaling pathways were suppressed, and PD-L1 expression was also increased using enzalutamide-resistant Myc-CaP MDVR cells. Notably, MDSC populations were significantly increased in Myc-CaP MDVR orthotopic tumors compared with those in Myc-CaP parental tumors. Co-culturing bone marrow cells with Myc-CaP MDVR cells significantly promoted MDSC differentiation and shifted towards M2 macrophage skewing.
Our study suggests that immunosuppressive signaling can be promoted directly by enzalutamide-resistant prostate cancer cells and may be a potential means by which the efficacy of immune checkpoint inhibitors in enzalutamide-resistant prostate cancer is diminished.

Immune checkpoint inhibitors (ICIs) therapy targeting programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) shows promising clinical benefits. However, the relatively low response rate highlights the need to develop an alternative strategy to target PD-1/PD-L1 immune checkpoint. Our study focuses on the role and mechanism of annexin A1 (ANXA1)-derived peptide A11 degrading PD-L1 and the effect of A11 on tumor immune evasion in multiple cancers.
Binding of A11 to PD-L1 was identified by biotin pull-down coupled with mass spectrometry analysis. USP7 as PD-L1\'s deubiquitinase was found by screening a human deubiquitinase cDNA library. The role and mechanism of A11 competing with USP7 to degrade PD-L1 were analyzed. The capability to enhance the T cell-mediated tumor cell killing activity and antitumor effect of A11 via suppressing tumor immune evasion were investigated. The synergistic antitumor effect of A11 and PD-L1 mAb (monoclonal antibody) via suppressing tumor immune evasion were also studied in mice. The expression and clinical significance of USP7 and PD-L1 in cancer tissues were evaluated by immunohistochemistry.
A11 decreases PD-L1 protein stability and levels by ubiquitin proteasome pathway in breast cancer, lung cancer and melanoma cells. Mechanistically, A11 competes with PD-L1\'s deubiquitinase USP7 for binding PD-L1, and then degrades PD-L1 by inhibiting USP7-mediated PD-L1 deubiquitination. Functionally, A11 promotes T cell ability of killing cancer cells in vitro, inhibits tumor immune evasion in mice via increasing the population and activation of CD8+ T cells in tumor microenvironment, and A11 and PD-1 mAb possess synergistic antitumor effect in mice. Moreover, expression levels of both USP7 and PD-L1 are significantly higher in breast cancer, non-small cell lung cancer and skin melanoma tissues than those in their corresponding normal tissues and are positively correlated in cancer tissues, and both proteins for predicting efficacy of PD-1 mAb immunotherapy and patient prognosis are superior to individual protein.
Our results reveal that A11 competes with USP7 to bind and degrade PD-L1 in cancer cells, A11 exhibits obvious antitumor effects and synergistic antitumor activity with PD-1 mAb via inhibiting tumor immune evasion and A11 can serve as an alternative strategy for ICIs therapy in multiple cancers.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the only curative treatment option for a number of hemato-oncological disorders. In fact, allo-HSCT is considered as one of the most successful immunotherapies as its clinical efficacy is based on the donor T-cells\' capacity to control residual disease. This process is known as the graft-versus-leukemia (GvL) reaction. However, alloreactive T-cells can also recognize the host as foreign and trigger a systemic potentially life-threatening inflammatory disorder termed graft-versus-host disease (GvHD). A better understanding of the underlying mechanisms that lead to GvHD or disease relapse could help us to improve efficacy and safety of allo-HSCT. In recent years, extracellular vesicles (EVs) have emerged as critical components of intercellular crosstalk. Cancer-associated EVs that express the immune checkpoint molecule programmed death-ligand 1 (PD-L1) can suppress T-cell responses and thus contribute to immune escape. At the same time, it has been observed that inflammation triggers PD-L1 expression as part of a negative feedback network.Here, we investigated whether circulating EVs following allo-HSCT express PD-L1 and tested their efficacy to suppress the ability of (autologous) T-cells to effectively target AML blasts. Finally, we assessed the link between PD-L1 levels on EVs to (T-)cell reconstitution, GvHD, and disease relapse.We were able to detect PD-L1+ EVs that reached a peak PD-L1 expression at 6 weeks post allo-HSCT. Development of acute GvHD was linked to the emergence of PD-L1high EVs following allo-HSCT. Moreover, PD-L1 levels correlated positively with GvHD grade and declined (only) on successful therapeutic intervention. T-cell-inhibitory capacity was higher in PD-L1high EVs as compared with their PD-L1low counterparts and could be antagonized using PD-L1/PD-1 blocking antibodies. Abundance of T-cell-suppressive PD-L1high EVs appears to also impact GvL efficacy as patients were at higher risk for relapse. Finally, patients of PD-L1high cohort displayed a reduced overall survival.Taken together, we show that PD-L1-expressing EVs are present following allo-HSCT. PD-L1 levels on EVs correlate with their ability to suppress T-cells and the occurrence of GvHD. The latter observation may indicate a negative feedback mechanism to control inflammatory (GvHD) activity. This intrinsic immunosuppression could subsequently promote disease relapse.

The success of HER2-positive (HER2+) breast cancer treatment with trastuzumab, an antibody that targets HER2, relies on immune response. We demonstrated that TNFα induces mucin 4 (MUC4) expression, which shields the trastuzumab epitope on the HER2 molecule decreasing its therapeutic effect. Here, we used mouse models and samples from HER2+ breast cancer patients to unravel MUC4 participation in hindering trastuzumab effect by fostering immune evasion.
We used a dominant negative TNFα inhibitor (DN) selective for soluble TNFα (sTNFα) together with trastuzumab. Preclinical experiments were performed using two models of conditionally MUC4-silenced tumors to characterize the immune cell infiltration. A cohort of 91 patients treated with trastuzumab was used to correlate tumor MUC4 with tumor-infiltrating lymphocytes.
In mice bearing de novo trastuzumab-resistant HER2+ breast tumors, neutralizing sTNFα with DN induced MUC4 downregulation. Using the conditionally MUC4-silenced tumor models, the antitumor effect of trastuzumab was reinstated and the addition of TNFα-blocking agents did not further decrease tumor burden. DN administration with trastuzumab modifies the immunosuppressive tumor milieu through M1-like phenotype macrophage polarization and NK cells degranulation. Depletion experiments revealed a cross-talk between macrophages and NK cells necessary for trastuzumab antitumor effect. In addition, tumor cells treated with DN are more susceptible to trastuzumab-dependent cellular phagocytosis. Finally, MUC4 expression in HER2+ breast cancer is associated with immune desert tumors.
These findings provide rationale to pursue sTNFα blockade combined with trastuzumab or trastuzumab drug conjugates for MUC4+ and HER2+ breast cancer patients to overcome trastuzumab resistance.

Roughly half of all diffuse large B-cell lymphomas (DLBCLs) are infiltrated by large numbers of regulatory T-cells (Tregs). Although the presence of \'effector\' Tregs in particular is associated with an inferior prognosis in patients on standard rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) immunochemotherapy, the role of this cell type during lymphoma initiation and progression is poorly understood.
Here, we use tissue microarrays containing prospectively collected DLBCL patient specimens, as well as data from publicly available cohorts to explore the mutational landscape of Treg-infiltrated DLBCL. We further take advantage of a model of MYC-driven lymphoma to mechanistically dissect the contribution of Tregs to lymphoma pathogenesis and to develop a strategy of Treg-selective interleukin-2 (IL-2) starvation to improve immune control of MYC-driven lymphoma.
We find that all genetic DLBCL subtypes, except for one characterized by co-occurring MYD88/CD79 mutations, are heavily infiltrated by Tregs. Spectral flow cytometry and scRNA-sequencing reveal the robust expression of functional and immunosuppressive markers on Tregs infiltrating MYC-driven lymphomas; notably, we find that intratumoral Tregs arise due to local conversion from naïve CD4+ precursors on tumor contact. Treg ablation in Foxp3iDTR mice, or by antibody-mediated Treg-selective blockade of IL-2 signaling, strongly reduces the lymphoma burden. We identify lymphoma B-cells as a major source of IL-2, and show that the effects of Treg depletion are reversed by the simultaneous depletion of Foxp3-negative CD4+ T-cells, but not CD8+ T-cells or natural killer (NK) cells. The inhibition of ATP hydrolyzation and adenosine production by Tregs at least partly phenocopies the effects of Treg depletion. Treg depletion further synergizes with pro-apoptotic CD40 activation to sustain durable responses.
The combined data implicate Tregs as a potential therapeutic target in DLBCL, especially in combination with other immunotherapies.