Effective targeting of cancer stem cells (CSCs) requires neutralization of self-renewal and chemoresistance, but these phenotypes are often regulated by distinct molecular mechanisms. Here we report the ability to target both of these phenotypes via CD55, an intrinsic cell surface complement inhibitor, which was identified in a comparative analysis between CSCs and nonCSCs in endometrioid cancer models. In this context, CD55 functions in a complement-independent manner and required lipid raft localization for CSC maintenance and cisplatin resistance. CD55 regulated self-renewal and core pluripotency genes via ROR2/JNK signaling and in parallel cisplatin resistance via lymphocyte-specific protein tyrosine kinase (LCK) signaling, which induced DNA repair genes. Targeting LCK signaling via saracatinib, an inhibitor currently undergoing clinical evaluation, sensitized chemoresistant cells to cisplatin. Collectively, our findings identify CD55 as a unique signaling node that drives self-renewal and therapeutic resistance through a bifurcating signaling axis and provides an opportunity to target both signaling pathways in endometrioid tumors.

Background The PD-1/PD-L1 axis represents a key mechanism of tumor immune evasion. However, its expression across different immune compartments and clinical implications in DLBCL patients remain unclear. Methods This study included 67 newly diagnosed DLBCL patients and 59 healthy controls. The levels of PD-1-positive T cells in the peripheral blood (PB) and bone marrow (BM) were assessed via flow cytometry. Furthermore, this study analyzed the correlation between PD-1 expression on PB and BM T-cell subsets and the clinicopathological features of DLBCL patients to elucidate their clinical implications. Results Compared with healthy controls, DLBCL patients presented significantly elevated PD-1 expression on T cells in both the PB and BM. Elevated PD-1 expression is significantly correlated with adverse clinical outcomes and poor prognostic factors, including advanced Ann Arbor stage (III-IV), a high International Prognostic Index (IPI 3-5), elevated lactate dehydrogenase (LDH>250 U/L) and the presence of B symptoms. A strong concordance in PD-1 levels was observed between paired PB and BM samples. Conclusion Increased PD-1 expression in the peripheral blood of DLBCL patients is correlated with PD-1 expression in the bone marrow. Our results demonstrate that elevated circulating PD-1+T cells are associated with high-risk, aggressive disease features. Importantly, our data support the potential utility of peripheral blood PD-1+T cells as a minimally invasive surrogate biomarker to reflect intramedullary T cell exhaustion. DLBCL patients with abundant circulating PD-1+T cells may have greater potential to respond to PD-1/PD-L1 blockade immunotherapy, although this hypothesis requires prospective validation. These findings warrant further clinical investigation.

Complement component 3 (C3) is an attractive therapeutic target for several inflammatory diseases; however, the strict primate specificity of emerging C3 inhibitors mandates the use of nonhuman primates (NHPs) for preclinical evaluation, creating significant ethical and economic barriers. Although we previously developed a human C3 complementary DNA knockin rat, the model was limited by low expression levels of human C3 and an inability to support gene-targeted therapies requiring genomic architecture. To overcome these limitations, we developed a second-generation humanized rat model by introducing the full-length human C3 gene onto a rat C3-deficient background. These rats exhibited significantly increased levels of human C3 in all tissues examined while maintaining normal renal function. We validated the utility of this new model using AMY-101, a clinical-stage, primate-specific C3 inhibitor, demonstrating its suppression of complement-mediated hemolysis both in vivo and in vitro. Notably, this inhibition primarily targeted the alternative pathway while sparing the classical pathway, consistent with the reported C3-bypass phenomenon. This second-generation C3 humanized rat model represents a robust, cost-effective preclinical platform for evaluating diverse human C3-targeted therapeutics, thereby reducing reliance on NHP models.

During activation the T cell transmembrane receptor CD6 becomes incorporated into the T cell immunological synapse where it can exert both co-stimulatory and co-inhibitory functions. Given the ability of CD6 to carry out opposing functions, this study sought to determine how CD6 regulates early T cell activation in response to viral infection. Infection of CD6 deficient mice with a neurotropic murine coronavirus resulted in greater activation and expansion of CD4 T cells in the draining lymph nodes. Further analysis demonstrated that there was also preferential differentiation of CD4 T cells into T follicular helper cells, resulting in accelerated germinal center responses and emergence of high affinity virus specific antibodies. Given that CD6 conversely supports CD4 T cell activation in many autoimmune models, we probed potential mechanisms of CD6 mediated suppression of CD4 T cell activation during viral infection. Analysis of CD6 binding proteins revealed that infection induced upregulation of Ubash3a, a negative regulator of T cell receptor signaling, was hindered in CD6 deficient lymph nodes. Consistent with greater T cell activation and reduced UBASH3a activity, the T cell receptor signal strength was intensified in CD6 deficient CD4 T cells. These results reveal a novel immunoregulatory role for CD6 in limiting CD4 T cell activation and deterring CD4 T follicular helper cell differentiation, thereby attenuating antiviral humoral immunity.

Cancer chemotherapy-induced peripheral neuropathy (CIPN) is a devastating pain syndrome without effective therapies. We previously reported that rodents deficient in complement C3, the central component of complement activation cascade, showed a significantly reduced degree of paclitaxel-induced mechanical allodynia (PIMA), suggesting that complement is integrally involved in the pathogenesis of paclitaxel-induced CIPN. However, the underlying mechanism was unclear. We found that administration of paclitaxel induced C3 activation in serum and upregulated the expression of C3a and C5a receptors (C3aR and C5aR) on macrophages, but not neurons, in mouse dorsal root ganglion (DRG). Administration of C3aR or C5aR antagonist (C3aRA, C5aRA) significantly alleviated PIMA in both male and female mice. C3aRA or C5aRA reduced paclitaxel-mediated macrophage infiltration in DRG, a key step in the development of CIPN. Western blot and immunohistochemistry studies showed that C3aRA or C5aRA suppressed paclitaxel-induced NF-κB activation and TNFα production in DRG. Finally, C3aRA or C5aRA upregulated the anti-inflammatory cytokine IL-10 in DRG. Taking together, administration of C3aRA or C5aRA mitigated paclitaxel CIPN in rodents through regulating neuroinflammatory signals. Targeting the C3aR or C5aR neuroimmune pathways could be a novel therapeutic approach to alleviate this debilitating pain syndrome. Supported by NIH K08 CA228039. Neuroimmunology (NEUR)

Recent advances in the understanding of immune-mediated neurological disorders have led to a paradigm shift toward pathophysiology-directed therapies. Central to this progress is a deeper appreciation of the complement system, a key component of innate immunity, and its role in neuroinflammation. Complement activation, while essential for host defense and tissue homeostasis, has been implicated increasingly in a spectrum of central and peripheral neurological disorders where complement dysregulation contributes to inflammation, cellular damage, and disease progression. Breakthroughs in conditions such as myasthenia gravis and aquaporin-4 IgG-positive neuromyelitis optica spectrum disorder underscore the therapeutic potential of targeting complement pathways to improve patient outcomes. In this review, we provide a comprehensive overview of complement activation pathways, regulatory controls, and their involvement in various autoimmune neurological diseases. We also highlight current and emerging complement-targeted therapies, many of which are now completing or entering clinical trials. Together, these insights offer a holistic perspective on the complement system as both a contributor to and a target for intervention in neurological diseases.

INTRODUCTION:C3 is central for all complement activation pathways, thus making it an attractive therapeutic target. Many C3-targeted agents are under extensive development with one already approved for clinical use. However, most, if not all, C3 inhibitors are human or nonhuman primate C3-specific, making evaluating their efficacies in vivo before a clinical trial extremely difficult and costly.METHODS:We first studied the compatibility of human C3 in the rat complement system, then developed a C3 humanized rat using the CRISPR/Cas9 technology. We thoroughly characterized the resultant human C3 humanized rats and tested the treatment efficacy of an established primate-specific C3 inhibitor in a model of complement-mediated hemolysis in the C3 humanized rats.RESULTS:We found that supplementing human C3 protein into the C3-deficient rat blood restored its complement activity, which was inhibited by rat factor H or compstatin, suggesting that human C3 is compatible to the rat complement system. The newly developed C3 humanized rats appeared healthy and expressed human but not rat C3 without detectable spontaneous C3 activation. More importantly, complement-mediated hemolysis in the C3 humanized rats was also inhibited by compstatin both in vitro and in vivo.CONCLUSION:The successfully developed C3 humanized rats provided a much-desired rodent model to evaluate novel C3 inhibitors in vivo as potential drugs.

Cancer chemotherapy-induced neuropathic pain is a devastating pain syndrome without effective therapies. We previously reported that rats deficient in complement C3, the central component of complement activation cascade, showed a reduced degree of paclitaxel-induced mechanical allodynia (PIMA), suggesting that complement is integrally involved in the pathogenesis of this model. However, the underlying mechanism was unclear. Complement activation leads to the production of C3a, which mediates inflammation through its receptor C3aR1. In this article, we report that the administration of paclitaxel induced a significantly higher expression level of C3aR1 on dorsal root ganglion (DRG) macrophages and expansion of these macrophages in DRGs in wild-type (WT) compared with in C3aR1 knockout (KO) mice. We also found that paclitaxel induced less severe PIMA, along with a reduced DRG expression of transient receptor potential channels of the vanilloid subtype 4 (TRPV4), an essential mediator for PIMA, in C3aR1 KO than in WT mice. Treating WT mice or rats with a C3aR1 antagonist markedly attenuated PIMA in association with downregulated DRG TRPV4 expression, reduced DRG macrophages expansion, suppressed DRG neuron hyperexcitability, and alleviated peripheral intraepidermal nerve fiber loss. Administration of C3aR1 antagonist to TRPV4 KO mice further protected them from PIMA. These results suggest that complement regulates PIMA development through C3aR1 to upregulate TRPV4 on DRG neurons and promote DRG macrophage expansion. Targeting C3aR1 could be a novel therapeutic approach to alleviate this debilitating pain syndrome.

The assembly of tissue-damaging membrane attack complexes (MACs; C5b-9) is a major mechanism by which excessive complement activation causes diseases. We previously developed a mouse anti-human C6 monoclonal antibody (mAb) 1C9 that selectively inhibits the assembly of MACs in human and non-human primates. In this project, we found that 1C9 also cross-reacted with rat and guinea pig C6, and determined its binding domains on C6 using different truncated C6 proteins. We then humanized the anti-C6 mAb by molecular modeling and complementarity-determining region grafting. After screening a library of 276 humanized variants with different combinations of humanized light and heavy chains in biophysical assays, we identified clone 3713 with the best developability profile, and an increased affinity against C6 when compared with the parental 1C9 mAb. This humanized 3713 mAb inhibited human, monkey, and rat complement-mediated hemolysis in vitro, and more importantly, it significantly reduced complement-mediated hemolysis in vivo in rats. These results demonstrated the successful humanization of the anti-C6 mAb and suggested that the humanized 3713 mAb could be further developed as a new therapeutic that selectively targets MAC for certain complement-mediated pathological conditions.

AbstractPrenatal SARS-CoV-2 infection is associated with higher rates of pregnancy and birth complications, despite that vertical transmission rates are thought to be low. Here, multi-omics analyses of human placental tissues, cord tissues/plasma, and amniotic fluid from 23 COVID-19 mother-infant pairs revealed robust inflammatory responses in both maternal and fetal compartments. Pronounced expression of complement proteins (C1q, C3, C3b, C4, C5) and inflammatory cytokines (TNF, IL-1α, and IL-17A/E) was detected in the fetal compartment of COVID-19-affected pregnancies. While ~26% of fetal tissues were positive for SARS-CoV-2 RNA, more than 60% of fetal tissues contained SARS-CoV-2 ORF8 proteins, suggesting transplacental transfer of this viral accessory protein. ORF8-positive fetal compartments exhibited increased inflammation and complement activation compared to ORF8-negative COVID-19 pregnancies. In human placental trophoblasts in vitro, exogenous ORF8 exposure resulted in complement activation and inflammatory responses. Co-immunoprecipitation analysis demonstrated that ORF8 binds to C1q specifically by interacting with a 15-peptide region on ORF8 (C37-A51) and the globular domain of C1q subunit A. In conclusion, an ORF8-C1q-dependent complement activation pathway was identified in COVID-19-affected pregnancies, likely contributing to fetal inflammation independently of fetal virus exposure.

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating condition affecting an increasing number of cancer survivors worldwide. However, insights into its pathophysiology and availability of effective therapies remain lacking. Dorsal root ganglia (DRG) have been studied as a key component of chemotherapeutic drug toxicity and a potential therapeutic target for CIPN treatment. This comprehensive review aims to synthesize, summarize, and correlate the results of both preclinical and clinical studies relevant to the pathophysiology and management of CIPN in relation to the DRG. Design: Review. A thorough literature search was conducted using the terms ‘dorsal root ganglion’ and ‘chemotherapy-induced peripheral neuropathy’, along with appropriate variations. Searched databases included PubMed, EMBASE, Medline, Cochrane Library, Wiley Library, and Web of Science. Inclusion criteria targeted all English language, peer-reviewed original research from the inception of these databases to the present year. Review articles, book chapters, and other nonoriginal publications were excluded. Of 134 relevant studies identified, the majority were preclinical studies elucidating how various chemotherapeutic agents, especially taxanes, disrupt neurotransmission, inflammatory processes, and apoptotic pathways within sensory neurons of DRG. Not only do these effects correlate with the presentation of CIPN, but their disruption has also been shown to reduce CIPN symptoms in preclinical models. However, clinical studies addressing DRG interventions are very limited in number and scope at this time. These results reveal various pathways within DRG that may be effective targets for CIPN treatment. While limited, clinical studies do offer promise in the utility of DRG neuromodulation in managing painful CIPN. In the future, clinical trials are needed to assess interventions aimed at these neuronal and nonneuronal pathological targets to better treat this complex condition.

The T cell marker CD6 regulates both T cells and target cells during inflammatory responses by interacting with its receptors. However, only a few receptors binding to the extracellular domains of CD6 have been identified, and cellular events induced by CD6 engagement with its receptors in target cells remain poorly understood. In this study, we identified CD44 as a novel CD6 receptor by proximity labeling and confirmed the new CD6-CD44 interaction by biochemical and biophysical approaches. CD44 and the other 2 known CD6 receptors, CD166 and CDCP1, were distributed diffusely on resting retinal pigment epithelium (RPE) cells but clustered together to form a receptor complex upon CD6 binding. CD6 stimulation induced dramatic remodeling of the actomyosin cytoskeleton in RPE cells mediated by activation of RhoA, and Rho-associated kinase signaling, resulting in increased myosin II phosphorylation. Such actomyosin activation triggered the disassembly of tight junctions responsible for RPE barrier integrity in a process that required all components of the tripartite CD6 receptor complex. These data provided new insights into the mechanisms by which CD6 mediates T cell-driven disruption of tissue barriers during inflammation. CD44 is a new receptor of CD6 and CD6 interacts with all 3 identified receptors to regulate cytoskeleton redmodeling.

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell–cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

T cell lymphomas (TCL) are heterogeneous, aggressive, and have few available targeted therapeutics. In this study, we determined that CD6, an established T cell marker, was expressed at high levels on almost all examined TCL patient specimens, suggesting that CD6 could be a new therapeutic target for this life-threatening blood cancer. We prepared a CD6-targeted antibody-drug conjugate (CD6-ADC) by conjugating monomethyl auristatin E (MMAE), an FDA-approved mitotic toxin, to a high-affinity anti-human CD6 monoclonal antibody (mAb). In contrast to both the unconjugated anti-CD6 mAb, and the non-binding control ADC, CD6-ADC potently and selectively killed TCL cells in vitro in both time- and concentration-dependent manners. It also prevented the development of tumors in vivo in a preclinical model of TCL. More importantly, systemic or local administration of the CD6-ADC or its humanized version, but not the controls, significantly shrank established tumors in the preclinical mouse model of TCL. These results suggest that CD6 is a novel therapeutic target in TCLs and provide a strong rationale for the further development of CD6-ADC as a promising therapy for patients with these potentially fatal lymphoid neoplasms.

Summary of the immunohistochemical staining results