CD46, a human-specific complement receptor, regulates gene programs essential for T helper 1 (TH1) cell differentiation, yet how it exerts direct transcriptional control remains unclear. We show that the CD46 signaling domain cytoplasmic tail 1 (CYT-1) engages the transcription factor Sp1 in human CD4 T cells to dynamically modulate Sp1-DNA interactions. Beyond promoting TH1 cell induction, CD46-Sp1-controlled programs support naive CD4 T cell survival by maintaining nutrient transporter expression and suppressing the intrinsic caspase 9-caspase 3 apoptotic pathway. The CD46-Sp1 axis also restrains HIV transcription in infected CD4 T cells in vitro. Disruption of CYT-1-Sp1-regulated programs identifies T cells from individuals with HIV who exhibit incomplete viral suppression during antiretroviral therapy. Together, these findings define a human-specific transcriptional mechanism linking complement signaling to metabolic adaptation, apoptosis regulation, and antiviral defense, highlighting an unexpected role for CD46 in coordinating T cell homeostasis and host protection.

Background and hypothesis : Maladaptive repair drives progression from acute kidney injury (AKI) to chronic kidney disease. Injured proximal tubular cells overexpress complement C3 mRNA, but its functional role is not well understood. We hypothesized that complement proteins produced and active locally within proximal tubules drive maladaptive repair and remain inaccessible to plasma-targeted inhibitors. Methods : We investigated complement, encompassing extracellular, autocrine and intracellular pools, in human biopsies and mouse model of rhabdomyolysis-induced AKI (RIAKI). Hyperplex sequential immunofluorescence combined with RNAscope was used to localize complement proteins and transcripts in situ and immune infiltrate. C3 or factor B (FB) deficient mice and pharmacological FB inhibition were tested in a RIAKI model. Bulk transcriptomics and C3/CFB knockdown in tubular cells with rescue experiments dissected mechanisms in vivo and in vitro . Results : In human and experimental RIAKI, injured VCAM-1 proximal tubules showed reabsorption and local transcription of C3 and CFB in a subset of tubules, induced by inflammatory cytokines. Genetic deletion of C3 or Cfb attenuated AKI, reducing tubular necrosis, immune infiltration and signatures of epithelial-to-mesenchymal transition and maladaptive repair. Pharmacological FB inhibition reduced extracellular C3 activation but did not preserve renal function. In vitro , C3 or FB knockdown in tubular cells suppressed proliferation, inflammation and mTOR-linked metabolic pathways and was not rescued by exogenous purified protein, consistent with cell-intrinsic/intracellular mode of action. Conclusion : Locally produced complement in proximal tubules drives inflammatory and maladaptive repair programs during AKI. The dissociation between genetic deletion and pharmacological FB inhibition indicates that the relevant complement activity occurs in a compartment poorly accessible to systemic inhibitors and supports development of locally acting, tissue-penetrant or cell-permeable complement-targeted therapies for AKI.

Overexpression of complement genes in the tumor microenvironment, including Factor H (FH), is a strong predictor of poor prognosis in multiple cancers. Its canonical functions in the bloodborne complement cascade, though, cannot explain this prognostic impact. Here, we demonstrate that FH operates within the intracellular space in fibroblasts and tumor cells. By transcriptomics approach in patient tumors, cellular and biochemical assays we revealed that the prognostic impact of FH overexpression is mediated mainly by its cell-intrinsic functions in tumor-promoting fibroblasts and malignant cells. Intranuclear FH interacts with the cell cycle-transcription factor E2F3. FH also promotes proliferation by lowering the nuclear p53 pool. Moreover, in ccRCC cancer cells, FH also regulates cytoskeleton organization and cell morphology, potentially, via interaction with the actin capping CapZ complex. Therefore, complement FH acts as a multitasking effector, regulating cell cycle and actin polymerization, challenging the paradigm of extracellular space-restricted functioning, considered for many complement proteins.

Clear cell renal cell carcinoma (ccRCC) tumors display strong yet functionally suppressed immune infiltrate, through mechanisms that remain poorly defined. The contribution of the complement system to this microenvironment is understudied, in part because complement activation cannot be inferred from most omics-based methods. Using our integrated complementomics approach combining spatial imaging, single-cell and spatial transcriptomics, plasma profiling, and clinical datasets, we show that malignant cells facilitate complement activation through local C3 production, without formation of cytotoxic membrane attack complexes. This abortive activation reshapes the myeloid compartment by recruiting C5aR1 + macrophages, comprising C1q-enriched tumor-associated macrophages (TAM), linked to T-cell exhaustion. In primary ccRCC, C3 production, C3 deposition, and C5aR1+/C1q + TAM infiltration marks T-cell exhaustion and predicts poor prognosis in the absence of systemic therapy. In metastatic disease (BIONIKK trial), complement activity diverges by treatment context: elevated plasma C3a associates with resistance to anti-angiogenic therapy, whereas complement activation fragments (C4a, C4d) and strong C1qTAM infiltration identify patients responsive to combined anti-PD1/anti-CTLA-4 immune checkpoint inhibitors (ICI). Our findings suggest that C1qTAMs infiltrate tumors and promote T-cell exhaustion upon local complement activation, making this complement-driven tumor–macrophage–lymphoid crosstalk an interesting target to improve ICI efficacy. *Mikel Rezola, Idris Boudhabhay and Lubka T. Roumenina contributed equally to this study.

ABSTRACT:Extracellular heme, released during intravascular hemolysis in sickle cell disease (SCD) and hemolytic anemia, acts as a proinflammatory danger signal, requiring robust defense mechanisms. Previous studies identified G protein-coupled receptor (GPCR) signaling triggered by heme, but the specific receptor remained unknown. Transcriptomic analysis of bulk RNA sequencing of liver tissues from SCD and hemolytic mice (injection of phenylhydrazine) revealed GPCR signaling as a commonly enriched pathway. Unbiased screening of 241 GPCRs identified Hydroxycarboxylic Acid Receptor 2 (HCAR2/GPR109A), an anti-inflammatory receptor for niacin, as a novel heme sensor. Heme binding to human HCAR2 was validated using a functional reporter cell assay and direct interaction analyses via surface plasmon resonance and absorbance spectroscopy. In vivo, HCAR2 was upregulated in the liver of SCD and hemolytic mice, paralleling the expression of the heme-degrading enzyme heme oxygenase-1 (HO-1). HO-1 inhibition or heme injection further increased HCAR2 expression, indicating that heme acts as both a ligand and an inducer of HCAR2. These findings identify HCAR2 as a novel heme receptor and reveal a heme-HCAR2-HO-1 negative feedback loop involved in tissue protection in hemolytic diseases.

Immune cell autonomous complement expression and function emerges as pivotal regulator of tissue immunity and T cell biology. Specifically, CD4 T cell intrinsic engagement of CD46 during T cell stimulation is required for normal Th1 response. We identified CD109, a GPI anchored C3 complement-like protein amongst the top up-regulated CD46 signaling targets. A direct function for CD109 on T cells has not been described. We found that human CD4+ T cells lacking CD109 displayed significantly augmented IFN-g and IL-17 production upon in vitro stimulation. Similarly, CD4+ T cells from Cd109–/– mice showed in vitro Th1 and Th17 hyperactivation and caused increased EAE pathology in vivo. Unexpectedly, T cell CD109 fails to control TGF-b signaling but rather restrains IFN-g and/or IL-17 production by binding to and inhibiting the activity of a non-canonical costimulator linked to Th1/Th17 biology. Guided by modeling and atomic force microscopy data, we designed a CD109/costimulator inhibitory peptide, which fully recapitulated the phenotype observed in CD109-deficient T cells. Furthermore, inhibiting CD109’s restraint on cytokine release using this peptide boosted CAR T cell tumor-killing capacity in vitro. Together, these data suggest that CD109 is an unexpected and important brake on CD4 T cell co-stimulatory signals upstream of Th1/Th17 inflammatory pathways, and that modulating its function pharmacologically can be leveraged to improve CAR T cell responses against tumors. Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Signaling events mediated by the human-specific complement regulator and receptor CD46 are vital coordinators of the metabolic gene program underlying normal monocyte activation and Th1 immunity. Perturbations in CD46 signals therefore are associated with several disease states, including recurrent infections, autoimmunity, and cancer. The molecular mechanism by which CD46 controls cellular gene transcription is unknown. Here, we demonstrate that the cleaved, intracellular signaling domain of CD46 interacts with the transcription factor specificity protein 1 (Sp1) in human CD4 T cells to dynamically modulate Sp1–DNA interactions. Specifically, CD46–Sp1-controlled gene programs sustain naive CD4 T cell survival through tonic suppression of the death Caspase-3 machinery and, concurrently, enable initiation of the IFN-γ production pathways. Furthermore, the CD46–Sp1 axis also restrains transcription of integrated human deficiency virus type 1 (HIV) in human CD4 T cells, indicating an unexpected role for CD46 in HIV infection. Our findings add important insights into human-specific aspects of complement and T cell biology and provide a tangible opening to define novel physiological and pathophysiological roles of CD46 across cells and diseases. Immune Response Regulation: Molecular Mechanisms (IRM)

Systemic complement guards the vascular space, while cell intrinsic and/or intracellularly derived complement components C3 and C5 play pivotal roles in tissue immunity through the regulation of normal cell physiology and metabolism. In human Th cells, cell-autonomous C5a generation engages the intracellular C5aR1, which triggers Th1 program-supporting intrinsic NLRP3 activation. Here, we define a role for the alternative C5a receptor, C5aR2 (which is activated by C5a-desArg), in restraining T helper cell effector responses by assessing T cells from the first described patient with C5aR2 deficiency. This patient suffers from an autoinflammatory syndrome, with enhanced inflammatory Th responses, a profound loss of naïve CD4 T cells, and a preponderance of memory T cells. Furthermore, we identified T cell-intrinsic carboxypeptidase M (CPM) as the C5a-desArg/C5aR2 ligand generating enzyme. In consequence, CPM inhibition, or genetic deletion of CPM, resulted in enhanced inflammatory human T cell responses that were rescued by a C5aR2 agonist in vitro. Further, Cpm or C5ar2-deficient mouse CD4 T cells displayed largely overlapping gene signature perturbations. In line with these observations, Cpm–/– CD4 T cells transferred into mice caused increased pathology vs. WT CD4 T cells in a T cell transfer model of colitis. Overall, these findings highlight the importance of an unexpected, CPM-controlled, autoregulatory C5aR1 and C5aR2 signaling balance to limit pathological Th1 immunity. Immune Response Regulation: Cellular Mechanisms (IRC)

T helper 1 (Th1) cell initiation pathways are well characterized; however, those regulating their contraction are less understood. Here, we define a CD4+ T cell-autonomous pathway in which complement C5 orchestrated a shift from prostaglandin E2 (PGE2) dominance to enhanced prostacyclin (PGI2) production via activation of C5a receptor 2 (C5aR2). This pivot in lipid mediators induced autocrine signaling through the PGI2 receptor and expression of the interleukin-1 (IL-1) decoy IL-1 receptor type 2 (IL-1R2), which sequestered Th1 cell-driving intrinsic IL-1β, facilitating Th1 cell contraction. Disruption of this C5aR2-PGI2-R axis was a hallmark of pathologically persistent Th1 cell activity in inflammatory conditions, including cryopyrin-associated periodic syndromes (CAPS), Crohn's disease, and rheumatoid arthritis. Rebalancing this axis through selective PGE2 synthase inhibition rectified the hyperactive Th1 cell phenotype in vitro in T cells from individuals with CAPS. Therefore, complement is a key controller of prostanoid metabolism, and the latter is an intrinsic-and potentially druggable-checkpoint for the cessation of Th1 cell effector responses.

Malignant cells are part of a complex network within the tumor microenvironment, where their interaction with host cells and soluble mediators, including complement components, is pivotal. The complement system, known for its role in immune defense and homeostasis, exhibits a dual effect on cancer progression. This dichotomy arises from its antitumoral opsonophagocytosis and cytotoxicity versus its protumoral chronic inflammation mediated by the C5a/C5aR1 axis, influencing antitumor T-cell responses. Recent studies have revealed distinct co-expression patterns of complement genes in various cancer types, correlating with prognosis. Notably, some cancers exhibit co-regulated overexpression of complement genes associated with poor prognosis, while others show favorable outcomes. However, significant intra-patient heterogeneity further complicates this classification. Moreover, the involvement of locally produced and intracellular complement proteins adds complexity to the tumor microenvironment dynamics. This review highlights the unique interplay of complement components within different cancers and patient cohorts, showing that “one size does not fit all”, for complement in cancer. It summarizes the clinical trials for complement targeting in cancer, emphasizing the need for tailored therapeutic approaches. By elucidating the mechanistic basis of complement's context-dependent role, this review aims to facilitate the development of personalized cancer therapies, ultimately improving patient care and outcomes.

Natural killer (NK) cells play a pivotal role against cancer, both by direct killing of malignant cells and by promoting adaptive immune response though cytokine and chemokine secretion. In the lung tumor microenvironment (TME), NK cells are scarce and dysfunctional. By conducting single-cell transcriptomic analysis of lung tumors, and exploring pseudotime, we uncovered that the intratumoral maturation trajectory of NK cells is disrupted in a tumor stage-dependent manner, ultimately resulting in the selective exclusion of the cytotoxic subset. Using functional assays, we observed intratumoral NK cell death and a reduction in cytotoxic capacities depending on the tumor stage. Finally, our analyses of human public dataset on lung cancer corroborate these findings, revealing a parallel dysfunctional maturation process of NK cells during tumor progression. These results highlight additional mechanisms by which tumor cells escape from NK cell cytotoxicity, therefore paving the way for tailored therapeutic strategies.

Free heme released from hemoglobin contributes to exacerbated inflammation and tissue damage in hemolytic diseases. While a moderate level of free heme does not cause intravascular inflammation by itself, its presence during infection greatly enhances inflammation. Although specific serum proteins have been found to affect heme-induced inflammation, the selective contribution of serum proteins inhibiting macrophage activation by heme or, conversely, amplifying the production of cytokines by macrophages stimulated with heme and microbial molecules, is poorly defined. Here we identified a serum fraction containing proteins with > 50 KDa which was capable of inhibiting heme-stimulated TNF production and capable of enabling TNF production under conditions of a heme-LPS synergy. The inhibition of heme-induced TNF production was mimicked by Hemopexin (Hx), human serum albumin (HSA), serum from Hx-knockout mice, and less efficiently by serum from albumin-knockout mice, but not by serum LDL. Hx and HSA inhibited heme-induced ROS generation, MAPK/ Syk phosphorylation and cell death. However, Hx and HSA each also promoted the synergistic relationship between heme and LPS upon TNF production. Serum from Hx-knockout mice was fully capable of enabling this synergy, while serum from albumin-knockout mice was less efficient to promote TNF production under these conditions. Low concentrations of HSA mimicked the ability of serum to enable heme-stimulated IL-1 beta production after LPS priming, while high concentrations inhibited it. Together, our findings indicate how heme inflammatory effects are restrained in the blood upon sterile hemolysis, yet exacerbate inflammation in the presence of microbes. Moreover, it is interesting to note that opposing effects of serum proteins on heme-induced macrophage activation were selected through evolution, with both effects exerted by Hx and albumin.

Arginase 1 (ARG1), the enzyme catalyzing the conversion of arginine to ornithine and urea, is a hallmark of IL-10 producing immunosuppressive M2 macrophages, however ARG1 activity in T cells is disputed. Here we demonstrate that Arg1, but not Arg2, expression induction is a key feature of lung CD4 T cells during mouse in vivo influenza infection. Ablation of CD4 T cell-intrinsic Arg1 unexpectedly accelerated both the virus-specific Th1 effector response and its IL-10-associated contraction. Biologically, this led to efficient viral clearance, yet significantly reduced lung pathology. Surprisingly, loss of Arg1 in CD4 T cells did not result in disturbed intracellular ornithine or polyamine levels. Instead, by employing unbiased transcriptomic and metabolomic approaches, we found that Arg1 deficiency triggered altered glutamine metabolism, and rebalancing the glutamine flux normalized the Arg1 deficient T cell response. Further, the role of Arg1 in CD4 T cells was distinct from that of its’ isoenzyme Arg2, as ablation of CD4 T cell intrinsic Arg2 resulted in normal Th1 responses, and instead altered Th2 and Th17 responses. Finally, CD4 T cells from rare patients with a deficiency in ARG1, or from healthy donors with CRISPR-Cas9-mediated ARG1 deletion, recapitulated the mouse data, demonstrating that ARG1 also plays a CD4 T cell intrinsic role in human Th1 responses. Collectively, CD4 T cell-intrinsic ARG1, functions as an unexpected pace-keeper of human and mouse T helper 1 (Th1) responses with implications for Th1-associated tissue pathologies. Supported by grants from NIH (5K22HL125593 to M.K.) the Intramural Research Program of the NIH (NIDDK ZIA/DK075149 to B.A. and NHLBI ZIA/HL006223 to C.K.)

Arginase 1 (Arg1), the enzyme catalyzing the conversion of arginine to ornithine, is a hallmark of IL-10-producing immunoregulatory M2 macrophages. However, its expression in T cells is disputed. Here, we demonstrate that induction of Arg1 expression is a key feature of lung CD4+ T cells during mouse in vivo influenza infection. Conditional ablation of Arg1 in CD4+ T cells accelerated both virus-specific T helper 1 (Th1) effector responses and its resolution, resulting in efficient viral clearance and reduced lung pathology. Using unbiased transcriptomics and metabolomics, we found that Arg1-deficiency was distinct from Arg2-deficiency and caused altered glutamine metabolism. Rebalancing this perturbed glutamine flux normalized the cellular Th1 response. CD4+ T cells from rare ARG1-deficient patients or CRISPR-Cas9-mediated ARG1-deletion in healthy donor cells phenocopied the murine cellular phenotype. Collectively, CD4+ T cell-intrinsic Arg1 functions as an unexpected rheostat regulating the kinetics of the mammalian Th1 lifecycle with implications for Th1-associated tissue pathologies.

EDITORIAL article Front. Immunol., 12 July 2023Sec. Molecular Innate Immunity Volume 14 - 2023 | https://doi.org/10.3389/fimmu.2023.1248299