Fibroblastic reticular cells (FRCs) are known to inhabit T cell-rich areas of lymphoid organs where they function to coordinate T cell and dendritic cell interactions. However, in vivo manipulation of FRCs has been limited by a dearth of genetic tools targeting this lineage. Here, using a mouse model to conditionally ablate FRCs, we demonstrate their indispensable role in anti-viral T cell responses. Unexpectedly, FRC loss also attenuated humoral immunity due to impaired B cell viability and follicular organization. Follicle-resident FRCs established a favorable niche for B lymphocytes via production of the cytokine BAFF. Thus, our study indicates that adaptive immunity requires an intact FRC network and illuminates a subset of FRCs that controls B cell homeostasis and follicle identity.

Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1–4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of ‘better effector’ CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor β- and γ-chain (IL-2Rβγ)-biased agonists are currently being developed6–10. Here we show that IL-2Rβγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rβγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

Highlights d In myeloma cells, FAM46C boosts ER growth and Ig secretion beyond sustainability d The ER-expanding activity of FAM46C requires its interaction with FNDC3 proteins d FAM46C abundance is tightly regulated by UPS-operated proteolysis d p62 compensates for UPS insufficiency by sequestering FAM46C away from the ER

Diffuse large B cell lymphomas (DLBCL) are a highly heterogeneous subtype of Non Hodgkin Lymphoma (NHL), accounting for about 25% of NHL. Despite an increased progression-free survival upon therapy, 40–50% of patients develop relapse/refractory disease, therefore there remains an important medical need. T cell recruiting therapies, such as the CD20xCD3 T cell bi-specific antibody CD20-TCB (RG6026 or glofitamab), represent a novel approach to target all stages of DLBCL, especially those that fail to respond to multiple lines of treatment. We aimed for a better understanding of the molecular features related to the mode of action (MoA) of CD20-TCB in inducing Target/T cell synapse formation and human T cell recruitment to the tumor. To directly evaluate the correlation between synapse, cytokine production and anti-tumor efficacy using CD20-TCB, we developed an innovative preclinical human DLBCL in vivo model that allowed tracking in vivo human T cell dynamics by multiphoton intravital microscopy (MP-IVM). By ex vivo and in vivo approaches, we revealed that CD20-TCB is inducing strong and stable synapses between human T cell and tumor cells, which are dependent on the dose of CD20-TCB and on LFA-1 activity but not on FAS-L. Moreover, despite CD20-TCB being a large molecule (194.342 kDa), we observed that intra-tumor CD20-TCB-mediated human T cell-tumor cell synapses occur within 1 hour upon CD20-TCB administration. These tight interactions, observed for at least 72 hours post TCB administration, result in tumor cell cytotoxicity, resident T cell proliferation and peripheral blood T cell recruitment into tumor. By blocking the IFNγ-CXCL10 axis, the recruitment of peripheral T cells was abrogated, partially affecting the efficacy of CD20-TCB treatment which rely only on resident T cell proliferation. Altogether these data reveal that CD20-TCB’s anti-tumor activity relies on a triple effect: i) fast formation of stable T cell-tumor cell synapses which induce tumor cytotoxicity and cytokine production, ii) resident T cell proliferation and iii) recruitment of fresh peripheral T cells to the tumor core to allow a positive enhancement of the anti-tumor effect.

To cope with intrinsic and environmental stress, cancer cells rely on adaptive pathways more than non-transformed counterparts. Such non-oncogene addiction offers new therapeutic targets and strategies to overcome chemoresistance. In an attempt to study the role of adaptive pathways in acquired drug resistance in carcinoma cells, we devised a model of in vitro conditioning to three standard chemotherapeutic agents, cisplatin, 5-fluorouracil, and docetaxel, from the epithelial cancer cell line, HEp-2, and investigated the mechanisms underlying reduced drug sensitivity. We found that triple-resistant cells suffered from higher levels of oxidative stress, and showed heightened anti-stress responses, including the antioxidant Nrf2 pathway and autophagy, a conserved pleiotropic homeostatic strategy, mediating the clearance of aggregates marked by the adapter p62/SQSTM1. As a result, re-administration of chemotherapeutic agents failed to induce further accumulation of reactive oxygen species and p62. Moreover, autophagy proved responsible for chemoresistance through the avoidance of p62 accumulation into toxic protein aggregates. Indeed, p62 ablation was sufficient to confer resistance in parental cells, and genetic and pharmacological autophagic inhibition restored drug sensitivity in resistant cells in a p62-dependent manner. Finally, exogenous expression of mutant p62 lacking the ubiquitin- and LC3-binding domains, required for autophagic engulfment, increased chemosensitivity in TDR HEp-2 cells. Altogether, these findings offer a cellular system to investigate the bases of acquired chemoresistance of epithelial cancers and encourage challenging the prognostic and antineoplastic therapeutic potential of p62 toxicity.

Diffuse large B cell lymphomas (DLBCL) are a highly heterogeneous subtype of Non Hodgkin Lymphoma (NHL), accounting for about 25% of NHL [1]. Despite an increased progression-free survival upon therapy, 40-50% of patients develop relapse/refractory disease, therefore there remains an important medical need [2]. T cell recruiting therapies, such as the CD20xCD3 T cell bi-specific antibody CD20-TCB (RG6026 or glofitamab), represent a novel approach to target all stages of DLBCL, especially those that fail to respond to multiple lines of treatment [3, 4]. We aimed for a better understanding of the molecular features related to the mode of action (MoA) of CD20-TCB in inducing Target/T cell synapse formation and human T cell recruitment to the tumor. To directly evaluate the correlation between synapse, cytokine production and anti-tumor efficacy using CD20-TCB, we developed an innovative preclinical human DLBCL in vivo model that allowed tracking in vivo human T cell dynamics by multiphoton intravital microscopy (MP-IVM). By ex vivo and in vivo approaches, we revealed that CD20-TCB is inducing strong and stable synapses between human T cell and tumor cells, which are dependent on the dose of CD20-TCB and on LFA-1 activity but not on FAS-L. Moreover, despite CD20-TCB being a large molecule (194.342 kDa), we observed that intra-tumor CD20-TCB-mediated human T cell-tumor cell synapses occur within 1 hour upon CD20-TCB administration. These tight interactions, observed for at least 72 hours post TCB administration, result in tumor cell cytotoxicity, resident T cell proliferation and peripheral blood T cell recruitment into tumor. By blocking the IFNγ-CXCL10 axis, the recruitment of peripheral T cells was abrogated, partially affecting the efficacy of CD20-TCB treatment which rely only on resident T cell proliferation. Altogether these data reveal that CD20-TCB’s anti-tumor activity relies on a triple effect: i) fast formation of stable T cell-tumor cell synapses which induce tumor cytotoxicity and cytokine production, ii) resident T cell proliferation and iii) recruitment of fresh peripheral T cells to the tumor core to allow a positive enhancement of the anti-tumor effect.

Background: Substantial advances in our understanding of the biology of the incurable plasma cell (PC) malignancy multiple myeloma (MM) came from the study of the bone marrow (BM) microenvironment (BMME). Our previous work disclosed an essential role for autophagy in sustaining MM cell proliferation and survival. However, the control exerted on PC autophagy by the BMME and its pathophysiological significance are virtually unknown. Experimental design: We integrated ex vivo multiplexed phosphoprotein cell signaling analyses of primary MM and BMME cells, metabolomic profiling of patient-derived BM and peripheral plasma, and in vitro studies on human MM cell lines. Primary CD138+ MM and BMME CD138- cells were isolated from BM aspirates obtained from 35 clinically characterized MM patients and 60 proteins - representative of autophagy, cell survival, proliferation, protein degradation and translation pathways - were analyzed by Reverse Phase Protein Arrays (RPPA).Comprehensive metabolomics profiling was achieved by ultra-high performance liquid and gas chromatography followed by mass spectrometry (UHPLC/GC-MS) on ad hoc-collected B and peripheral plasma samples from patients at different disease stages (30 MGUS, 17 smoldering MM, 16 MM) and age-matched healthy donors (29). For in vitro studies, human MM lines were exposed to amino acid (AA) depletion and selected responses evaluated by quantitative RT-PCR and immunoblotting analyses. Results: First, RPPA revealed that patients with the most aggressive clinical presentation (refractoriness, short time to progression and active bone disease) displayed higher activity of the PI3K/AKT/mTOR pathway associated to higher expression of the autophagic proteins ATG5, LC3B and p62. In search for extrinsic stimuli capable of raising both mTOR and autophagic activity, we recapitulated such expression pattern in MM cells through selective starvation of tryptophan (Trp) and arginine (Arg), two AA endowed with distinctive immune regulatory activity. When exposed to Trp-free or Arg-free medium, human MM cell lines activated GCN2-mediated responses, increasing mTOR, p-S6RP, CHOP, p62 and Blimp-1 and immunoglobulin secretion; conversely, stable lentiviral p62 silencing reduced Blimp-1 and caused the in vitro extinction of MM cell lines within 10 days of culture. UHPLC/GC-MS metabolomics analysis of plasma samples revealed a progressively and significantly lower concentration of Trp and Arg associated with disease evolution and shorter progression-free survival. Conclusion: Taken together, our findings disclose a novel extrinsic circuit whereby reduced concentration of the essential immunoregulatory AA Trp and Arg sustains PC cell fitness and survival. Attesting to pathophysiologic relevance, this mechanism appears coopted by MM as a component of its vicious BMME. Note: This abstract was not presented at the meeting. Citation Format: Alessandra Romano, Floriana Cremasco, Antonella Chiechi, Francesca Paradiso, Enrico Milan, Francesca Fontana, Jose M. Manteiga, Francesco Di Raimondo, Alexander I. Spira, Amy Van Meter, Emanuel Chip Petricoin, Virginia Espina, Lance A. Liotta, Simone Cenci. Multiplexed phosphoprotein cell signaling analysis in multiple myeloma reveals a pro-survival pathway elicited by amino-acid starvation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5413. doi:10.1158/1538-7445.AM2017-5413

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