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Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a challenging respiratory disease for the global swine industry. Variations in the serotypes associated with clinical disease have been observed in different regions worldwide. This study aimed to provide an updated epidemiological assessment of A. pleuropneumoniae serotypes in Spain, incorporating bacterial characterization through serotyping and toxinotyping. Serotypes 9/11, 2, 4, 5, 17, and 13 were frequently identified in diseased animals. Furthermore, qPCR of lung samples from an outbreak, even when samples were pooled, emerged as a robust diagnostic tool, enabling the rapid detection of A. pleuropneumoniae and their serotypes without the need for microbiological isolation. This technology also facilitates serotype monitoring of apparently healthy herds through the testing of oral fluids. The study revealed the frequent simultaneous presence of diverse serotypes within a farm. Serotypes 1, 7, 10, 12, 18, and 19 were frequently found in subclinically infected animals but were rarely detected in acute pleuropneumonia outbreaks in the current study. These results provide valuable information for interpreting the potential virulence of the different serotypes in Spain. However, other predisposing factors and the immune status of the herds such as type of vaccines used when appropriate, should be carefully considered before drawing definitive conclusions. Nevertheless, the study offers valuable insights that underscore the necessity for detailed regional data to contribute toward a comprehensive understanding of the disease dynamics and toward formulating effective control measures for porcine pleuropneumonia.

Background The RANK pathway has been extensively investigated for its role in bone resorption; however, its significance extends beyond bone metabolism. Preclinical models suggest that inhibition of RANK signaling can prevent mammary tumor development by reducing proliferation and tumor cell survival. Additionally, both preclinical and clinical data support the ability of RANK pathway inhibitors to enhance the anti-tumor immune response. Methods D-BIOMARK is a prospective, randomized window-of-opportunity clinical trial assessing the biological effects of denosumab, a monoclonal antibody against RANKL, in patients with HER2-negative early breast cancer. The study aims to assess denosumab’s impact on breast tumor cell proliferation, apoptosis, and its potential to influence the tumor immune microenvironment. A total of 60 patients were enrolled and randomized 2:1 to receive two doses of single agent denosumab (120 mg one week apart) before surgery or to the control arm (no treatment). Fifty-eight patients were evaluated, 27 pre-menopausal and 31 post-menopausal women, 48 with luminal tumors and 10 with triple negative breast cancer. Paired tumor samples were collected to compare baseline (core biopsy) and surgical (surgical specimen) time points, as well as serum samples at both time points. Results Denosumab demonstrated its ability to reduce serum free RANKL levels (experimental p  < 0.001, control p  = 0.270). However, a reduction in tumor cell proliferation or cell survival was not observed. A denosumab-driven increase in tumor infiltrating lymphocytes (TILs) was observed (experimental p  = 0.001, control p  = 0.060), particularly in the luminal B-like population (experimental p  = 0.012, control p  = 0.070) and a similar trend in the TNBC group (experimental p  = 0.079, control p  = 0.237). Denosumab led to increased TILs in both pre-menopausal (experimental p  = 0.048, control p  = 0.639) and post-menopausal (experimental p  = 0.041, control p  = 0.062) women with luminal tumors. RANK protein expression in tumor and stroma was associated with markers of tumor aggressiveness but an increase in TILs was observed in the experimental arm, irrespectively of RANK and RANKL expression in tumor or stromal cells. Conclusions The D-BIOMARK trial suggests a potential role for denosumab as an immune-enhancing agent in early HER2-negative breast cancer. Although preoperative denosumab did not reduce tumor proliferation or increased apoptosis, it led to an increase in TILs, particularly in luminal B-like tumors. These findings underscore the importance of further investigation into the multifaceted aspects of the RANK pathway. Trial registration EudraCT number: 2016-002678-11 registered on June 15, 2018. ClinicalTrials.gov identifier: NCT03691311, retrospectively registered on September 04, 2018.

CD137 (4-1BB, Tnsfr9) is a member of the TNF-receptor (TNFR) superfamily without known intrinsic enzymatic activity in its cytoplasmic domain. Hence, akin to other members of the TNFR family, it relies on the TNFR-Associated-Factor (TRAF) family of adaptor proteins to build the CD137 signalosome for transducing signals into the cell. Thus, upon CD137 activation by binding of CD137L trimers or by crosslinking with agonist monoclonal antibodies, TRAF1, TRAF2, and TRAF3 are readily recruited to the cytoplasmic domain of CD137, likely as homo- and/or heterotrimers with different configurations, initiating the construction of the CD137 signalosome. The formation of TRAF2-RING dimers between TRAF2 molecules from contiguous trimers would help to establish a multimeric structure of TRAF-trimers that is probably essential for CD137 signaling. In addition, available studies have identified a large number of proteins that are recruited to CD137:TRAF complexes including ubiquitin ligases and proteases, kinases, and modulatory proteins. Working in a coordinated fashion, these CD137-signalosomes will ultimately promote CD137-mediated T cell proliferation and survival and will endow T cells with stronger effector functions. Current evidence allows to envision the molecular events that might take place in the early stages of CD137-signalosome formation, underscoring the key roles of TRAFs and of K63 and K48-ubiquitination of target proteins in the signaling process. Understanding the composition and fine regulation of CD137-signalosomes assembly and disassembly will be key to improve the therapeutic activities of chimeric antigen receptors (CARs) encompassing the CD137 cytoplasmic domain and a new generation of CD137 agonists for the treatment of cancer.

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8 N/C EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8 N/C EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8 N/C EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy. Cancer therapy using systemically administrated 4-1BB-targeting antibodies is often associated with severe toxicity due to the nonspecific activation of autoreactive T cells. Here, the authors have developed a trimeric antibody targeting both 4-1BB and EGFR, which activates T cells effectively and shows negligible cytotoxicity.

Mice can be genetically modified to mimic human diseases, providing valuable information about the genetic factors involved in B-cell malignancies. [...]Li et al.present a perspective based on their previous research (6) on lymphoma dissemination in mouse models of B-cell aggressive lymphomas. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Chronic lymphocytic leukemia (CLL)/Small lymphocytic lymphoma (SLL) is a heterogeneous disease consisting of at least two separate subtypes, based on the mutation status of the immunoglobulin heavy chain variable gene (IGHV) sequence. Exposure to antigens seems to play a role in malignant transformation and in the selection and expansion of more aggressive CLL clones. Furthermore, a biased usage of particular IGHV gene subgroups and the existence of stereotyped B-cell receptors (BCRs) are distinctive characteristics of human CLL. We have previously described that Traf2 DN/ BCL2 double-transgenic (tg, +/+ ) mice develop CLL/SLL with high incidence with aging. In this model, TNF-Receptor Associated Factor (TRAF)-2 deficiency cooperates with B cell lymphoma (BCL)-2 in promoting CLL/SLL in mice by specifically enforcing marginal zone (MZ) B cell differentiation and rendering B cells independent of BAFF for survival. In this report, we have performed the sequencing of the IGHV-D-J rearrangements of B cell clones from the Traf2 DN/ BCL2 -tg +/+ mice with CLL/SLL. The results indicate that these mice develop oligoclonal and monoclonal B cell expansions. Allotransplantation of the oligoclonal populations into immunodeficient mice resulted in the preferential expansion of one of the parental clones. The analysis of the IGHV sequences indicated that 15% were mutated (M) and 85% unmutated (UM). Furthermore, while the Traf2 DN/ BCL2 -tg -/- (wild-type), -/+ ( BCL2 single-tg) and +/- ( Traf2DN DN single-tg) littermates showed the expression of various IGHV gene subgroups, the CLL/SLL expanded clones from the Traf2 DN/ BCL2 -tg +/+ (double-transgenic) mice showed a more restricted IGHV gene subgroup usage and an overrepresentation of particular IGHV genes. In addition, the HCDR3-encoded protein sequence indicates the existence of stereotyped immunoglobulin (Ig) in the BCRs and strong similarities with BCR recognizing autoantigens and pathogen-associated antigens. Altogether, these results highlight the remarkable similarities between the CLL/SLL developed by the Traf2 DN/ BCL2 -tg +/+ mice and its human counterpart.

Furthermore, the next generation sequencing has opened new possibilities for forward and reverse genetic screenings to identify gene mutations involved in tumor development, progression, evasion and relapse, both in human and mice, shaping the field for an exciting future. A mutation in the MyD88 gene introducing a leucine in position 265 instead of a proline causing constitutive MyD88 dimerization and NFKB and JAK activation is found in a variety of human B cell neoplasms (2), including in most patients with WM (3).Schmidt et al.developed three genetically engineered conditional mouse models harboring floxed-Myd88L252P (the mouse homolog of the human L265P mutation), one with Cre under the control of CD19 (CD19-Cre mice), where Myd88L252P expression is enforced in all B cells, a second mouse strain with Cre under the control of Cγ1 promoter (Cγ1-Cre mice), thus limiting Myd88L252P expression to GC cell, and a third mouse line with restricted MyD88L252P expression to a few random B cells (CD19-CreERT2 mice). Dysregulation of c-MYC is a trademark of a variety of B-cell lymphomas, where translocations of this gene lead to overexpression of intact c-MYC protein (8). c-MYC translocation is a primary transformation event in Burkitt´s lymphoma but its occurrence as a secondary event in diffuse large B-cell lymphoma, plasmablastic lymphoma, mantle cell lymphoma, or double-hit lymphoma fuels the aggressiveness of these lymphomas.Ferrad et al.overview the various c-Myc-driven mouse models of lymphoma focusing on those mouse models of c-myc overexpression regulated by the two main enhancers in the Igh locus, namely, Eµ and 3´RR enhancer. In addition,Malaney et al.also overview the various transgenic mouse models of B cell lymphoma based on c-MYC upregulation, with particular emphasis on the heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a driver of B cell lymphoma through its role on c-Myc regulation. hnRNP K is a ssDNA and RNA binding protein that regulates a plethora of processes controlling transcription and translation (9) and its over- and under-expression is causative of cancer (10). hnRNP K has been shown to be upregulated in DLBCL and Burkitt´s lymphoma patients and the oncogenic role of hnRNP K was previously confirmed by the authors by means of a B cell specific Eµ-hnRNP K transgenic mice that develop B cell lymphomas with high latency and high incidence (11). hnRNP K’s oncogenic potential stems from its ability to regulate c-MYC expression at post-transcriptional and translational level, without requiring c-MYC translocations.

Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor, denosumab, in breast cancer patients, beyond the bone, is unclear. Aiming to select patients who may benefit from denosumab, we hereby analyzed RANK and RANKL protein expression in more than 2,000 breast tumors (777 estrogen receptor‐negative, ER − ) from four independent cohorts. RANK protein expression was more frequent in ER − tumors, where it associated with poor outcome and poor response to chemotherapy. In ER − breast cancer patient‐derived orthoxenografts (PDXs), RANKL inhibition reduced tumor cell proliferation and stemness, regulated tumor immunity and metabolism, and improved response to chemotherapy. Intriguingly, tumor RANK protein expression associated with poor prognosis in postmenopausal breast cancer patients, activation of NFKB signaling, and modulation of immune and metabolic pathways, suggesting that RANK signaling increases after menopause. Our results demonstrate that RANK protein expression is an independent biomarker of poor prognosis in postmenopausal and ER − breast cancer patients and support the therapeutic benefit of RANK pathway inhibitors, such as denosumab, in breast cancer patients with RANK + ER − tumors after menopause. Synopsis The analyses of RANK and RANKL expression in large cohorts of breast cancer samples and functional studies in RANK+ breast cancer patient‐derived xenografts (PDXs) revealed a key role for RANK in postmenopausal women with estrogen receptor negative (ER ‐ ) breast cancer. RANK biology and prognostic value in breast cancer is determined by ER status and menopause. RANK protein expression in tumor cells is associated with ER ‐ breast cancer and poor survival in ER ‐ and postmenopausal ER ‐ breast cancer patients. RANK protein expression in tumor cells is associated with poor survival in postmenopausal breast cancer patients independent of ER expression, tumor grade, stage and size. RANK expression associates with poor response to chemotherapy in ER ‐ breast cancer and RANKL inhibition improves response to taxanes in ER ‐ breast PDXs. Graphical Abstract The analyses of RANK and RANKL expression in large cohorts of breast cancer samples and functional studies in RANK+ breast cancer patient‐derived xenografts (PDXs) revealed a key role for RANK in postmenopausal women with estrogen receptor negative ER ‐ breast cancer.

Adoptive T cellular immunotherapies have emerged as relevant approaches for treating cancer patients who have relapsed or become refractory (R/R) to traditional cancer treatments. Chimeric antigen receptor (CAR) T-cell therapy has improved survival in various hematological malignancies. However, significant limitations still impede the widespread adoption of these therapies in most cancers. To advance in this field, six research groups have created the “NEXT Generation CART MAD Consortium” (NEXT CART) in Madrid’s Community, which aims to develop novel cell-based immunotherapies for R/R and poor prognosis cancers. At NEXT CART, various basic and translational research groups and hospitals in Madrid concur to share and synergize their basic expertise in immunotherapy, gene therapy, and immunological synapse, and clinical expertise in pediatric and adult oncology. NEXT CART goal is to develop new cell engineering approaches and treatments for R/R adult and pediatric neoplasms to evaluate in multicenter clinical trials. Here, we discuss the current limitations of T cell-based therapies and introduce our perspective on future developments. Advancement opportunities include developing allogeneic products, optimizing CAR signaling domains, combining cellular immunotherapies, multi-targeting strategies, and improving tumor-infiltrating lymphocytes (TILs)/T cell receptor (TCR) therapy. Furthermore, basic studies aim to identify novel tumor targets, tumor molecules in the tumor microenvironment that impact CAR efficacy, and strategies to enhance the efficiency of the immunological synapse between immune and tumor cells. Our perspective of current cellular immunotherapy underscores the potential of these treatments while acknowledging the existing hurdles that demand innovative solutions to develop their potential for cancer treatment fully.

TNF-Receptor Associated Factor (TRAF)-3 is a master regulator of B cell homeostasis and function. TRAF3 has been shown to bind and regulate various proteins involved in the control of innate and adaptive immune responses. Previous studies showed that TRAF3 overexpression renders B cells hyper-reactive to antigens and Toll-like receptor (TLR) agonists, while TRAF3 deficiency has been implicated in the development of a variety of B cell neoplasms. In this report, we show that transgenic mice overexpressing TRAF3 and BCL2 in B cells develop with high incidence severe lymphadenopathy, splenomegaly and lymphoid infiltrations into tissues and organs, which is the result of the growth of monoclonal and oligoclonal B cell neoplasms, as demonstrated by analysis of V DJ gene rearrangement. FACS and immunohistochemical analyses show that different types of mature B cell neoplasms arise in double-transgenic (tg) mice, all of which are characterized by the loss of surface IgM and IgD expression. However, two types of lymphomas are predominant: (1) mature B cell neoplasms consistent with diffuse large B cell lymphoma and (2) plasma cell neoplasms. The Ig isotypes expressed by the expanded B-cell clones included IgA, IgG, and IgM, with most having undergone somatic hypermutation. In contrast, mouse littermates representing all the other genotypes ( -/ -; +/ -, and -/ +) did not develop significant lymphadenopathy or clonal B cell expansions within the observation period of 20 months. Interestingly, a large representation of the HCDR3 sequences expressed in the -tg and -double-tg B cells are highly similar to those recognizing pathogen-associated molecular patterns and damage-associated molecular patterns, strongly suggesting a role for TRAF3 in promoting B cell differentiation in response to these antigens. Finally, allotransplantation of either splenocytes or cell-containing ascites from lymphoma-bearing TRAF3/BCL2 mice into SCID/NOD immunodeficient mice showed efficient transfer of the parental expanded B-cell clones. Altogether, these results indicate that TRAF3, perhaps by promoting exacerbated B cell responses to certain antigens, and BCL2, presumably by supporting survival of these clones, cooperate to induce mature B cell neoplasms in transgenic mice.