A novel anti-CTLA-4 nanobody-IL12 fusion protein in combination with a dendritic cell/tumour fusion cell vaccine enhances the antitumour activity of CD8+ T cells in solid tumours.

BACKGROUND: We previously developed a nanobody targeting CTLA-4 and demonstrated that it can boost antitumour T-cell responses in vitro; however, the resulting responses after the injection of T cells into cancer models are usually weak and transient. Here, we explored whether fusing our nanobody to IL-12 would enable it to induce stronger, longer-lasting T-cell immune responses after exposure to immature dendritic cell and tumour cell fusions. RESULTS: The fusion protein enhanced the response of CD8+ T cells to tumour antigens in vitro and led to stronger, more persistent immune responses after the T cells were injected into mice bearing different types of xenografts. CONCLUSION: Our in vitro and in vivo results suggest the anticancer potential of our nanobody-interleukin fusion system and support the clinical application of this fusion approach for various nanobodies.

Monocyte-regulated interleukin 12 production drives clearance of Staphylococcus aureus.

Staphylococcus aureus is a versatile bacterium responsible for conditions ranging from mild skin and soft-tissue infections to serious disorders such as pneumonia and sepsis. Monocytes play a role in protection against pathogens by migrating to inflamed tissues and differentiating into macrophages but their specific role in the context of S. aureus pulmonary infection has not been fully elucidated. Using a CCR2-DTR transgenic mouse model we demonstrate that over the course of infection monocyte depletion resulted in worse airway clearance of S. aureus. The bronchoalveolar lavage fluid (BALF) of CCR2-DTR mice after S. aureus infection displayed significant decreases in interleukin-12 (IL-12), IFN-γ, IP-10, MIG and RANTES, all IFN-γ regulated, compared to wild-type (WT) infected controls. NK cells were identified as the main producers of IFN-γ, but both NK cells and IFN-γ were dispensable for clearance. We demonstrated through cytokine production and RNA-seq analysis that IL-12 and IL-12 regulated genes are strongly induced in monocytes upon S. aureus infection. Administration of IL-12 during infection restored the bacterial burdens in the BALF and lungs of monocyte-depleted CCR2-DTR mice to the levels of WT mice, independent of IFN-γ. In the absence of monocytes, alveolar macrophages are the primary phagocytic cells, and IL-12 influences their capacity to produce reactive oxygen species and clear S. aureus. These results show that production of IL-12 contributes to the control of S. aureus via its influence on alveolar macrophage function.

TLR7-Induced Mitochondrial Reactive Oxygen Species Production in Monocyte-derived Dendritic Cells Drives IL-12-Dependent NK Cell Activation and Enhances Antitumor Immunity.

Dendritic cell (DC)-based vaccines are promising immunotherapies for cancer. Although DC-based therapies are known to activate tumor-specific T cells, the interplay between DCs and NK cells in this setting is not fully understood. In this study, we demonstrated a novel TLR7/ mitochondrial reactive oxygen species (mROS)/IL-12 axis that drives potent NK cell responses against tumors. We showed that TLR7 activation by imiquimod in peripheral blood monocyte-derived CD11c+ DCs triggered mROS production, leading to enhanced IL-12 secretion and subsequent NK cell activation, as evidenced by increased IFN-γ production and tumor cell cytotoxicity. Notably, mROS neutralization abrogates NK cell-mediated tumor cell lysis, and TLR7-mediated DC activation of NK cells occurs independently of MyD88, suggesting involvement of the noncanonical NF-κB pathway. Our findings provide a rationale for targeting the TLR7/mROS/IL-12 axis to enhance the efficacy of DC-based cancer immunotherapy.

DNA circuit-based immunoassay for ultrasensitive protein pattern classification.

Cytokines are important immune modulators, and pivotal biomarkers for the diagnostic of various diseases. In standard analytical procedure, each protein is detected individually, using for instance gold standard ELISA protocols or nucleic acid amplification-based immunoassays. In recent years, DNA nanotechnology has been employed for creating sophisticated biomolecular systems that perform neuromorphic computing on molecular inputs, opening the door to concentration pattern recognition for biomedical applications. This work introduces immuno-PUMA (i-PUMA), an isothermal amplification-based immunoassay for ultrasensitive protein detection. The assay couples the convenience of supported format of an ELISA protocol with the computing capabilities of a DNA/enzyme circuit. We demonstrate a limit of detection of 2.1 fM, 8.7 fM and 450 aM for IL12, IL4 and IFNγ cytokines, respectively, outperforming the traditional ELISA format. i-PUMA's versatility extends to molecular computation, allowing the creation of 2-input perceptron-like classifiers for IL12 and IL4, with tunable weight sign and amplitude. Overall, i-PUMA represents a sensitive, low-cost, and versatile immunoassay with potential applications in multimarker-based sample classification, complementing existing molecular profiling techniques.

Cytokine profiles and their roles in development of provoked vulvodynia.

Provoked, localized, vulvodynia (PVD) is the main subtype of vulvodynia. Although the etiology of PVD is still a topic of debate, inflammation caused by cytokines responding to a dysregulated microbiome is one of the leading proposed theories. Therefore, the purpose of our study is to further explore the cytokine profiles in the study group with PVD using multiplex immunoassays based on electrochemiluminescence. We compared a panel of 26 distinct cytokines levels in the study group with PVD (n = 23) to the control group (n = 18) and cytokine concentrations were measured using MESO QuickPlex SQ 120 instrument with 5 different multiplex assays. Statistical analysis used the Mann-Whitney U test, two-sided p-values, and a significance level of α = 0.05. Differences in cytokine concentrations are described as negligible, small, medium, or large based on Cliff's δ. Concentrations of three cytokines were significantly lower in the PVD group: a large difference in IP-10 (p = 0.029*) and medium differences in IL-1RA4 (p = 0.030*) and IL-12 (p = 0.034*). One cytokine level was significantly higher in the PVD group: a medium difference for IL-6 (p = 0.037*). Due to the lack of consistency in elevation of inflammatory profiles, it is not enough to support persistent inflammation as the etiology behind PVD. However, these findings may indicate there is a possible immune response deficiency in some patients who have PVD. The resemblance of cytokine profile in our study to cytokine profile of people with chronic yeast infection further support this proposed mechanism behind PVD. Future studies involving history and testing for yeast infection are necessary to explore this possibility further.

Anti-IL23/12 agents and JAK inhibitors for inflammatory bowel disease.

IBD (inflammatory bowel disease) is a chronic inflammatory disease of the gastrointestinal tract with increasing incidence worldwide. Multiple factors, such as genetic background, environmental and luminal factors, and mucosal immune dysregulation, have been implicated in the cause of IBD, although the cause of the disease remains unknown. IL-12 and IL-23 and their downstream signaling pathways participate in the pathogenesis of inflammatory bowel disease. Early and aggressive treatment with biologic therapies or novel small molecules is needed to decrease complications and the need for hospitalization and surgery. The landscape of inflammatory bowel disease (IBD) treatment has tremendously improved with the development of biologics and small molecule drugs. Several novel biologics and small molecule drugs targeting IL-12 and IL-23 and their downstream targets have shown positive efficacy and safety data in clinical trials, and several drugs have been approved for the treatment of IBD. In the future, numerous potential emerging therapeutic options for IBD treatment are believed to come to the fore, achieving disease cure.

Development of a targeted IL-12 immunotherapy platform for B-cell lymphomas.

Immunotherapy has emerged as a promising approach to cancer treatment that utilizes the potential of the immune system to precisely identify and eradicate cancerous cells. Despite significant progress in immunotherapy, innovative approaches are required to enhance the effectiveness and safety of these treatments. Interleukin-12 (IL-12), widely recognized for its essential function in immune responses, has been explored as a potential candidate for treating cancer. However, early attempts involving the systemic administration of IL-12 were ineffective, with significant adverse effects, thus underscoring the need for innovation. To address these challenges, we developed a therapeutic molecule that utilizes a single-chain IL-12 mutant (IL-12mut) linked to a tumor-targeting arm. Here, we describe the development of a highly effective IL-12-based TMEkine™ platform by employing a B-cell lymphoma model (termed CD20-IL-12mut). CD20-IL-12mut combined the attenuated activities of IL-12 with targeted delivery to the tumor, thereby maximizing therapeutic potential while minimizing off-target effects. Our results revealed that CD20-IL-12mut exhibited potent anticancer activity by inducing complete regression and generating immunological memory for tumor antigens. Collectively, our data provide a basis for additional research on CD20-IL-12mut as a potential treatment choice for patients with B-cell lymphomas such as non-Hodgkin's lymphoma.

A laponite-based immunologically active gel delivery system for long-acting tumor vaccine.

Traditional bolus vaccines typically require multiple doses, which complicates the vaccination process and may cause missed shots, leading to sub-optimal immunity and reduced vaccine effectiveness. Herein, a gel-based long-acting vaccine system with self-adjuvant properties based on laponite was constructed to simplify vaccination procedures and improve vaccine effectiveness. Firstly, the gel system could recruit multiple types of immune cells to form immune niches. Secondly, it could achieve sustained delivery of antigens to lymph nodes by active transport and passive drainage. Then, the gel system triggered the formation of a large number of germinal centers, which elicited enhanced and durable humoral immune responses, as well as strong cellular immune responses. As a result, it eventually showed good prophylactic and therapeutic effects in a variety of tumor models including melanoma, colorectal cancer and peritoneal metastasis models. By further combining the immunoadjuvant CpG ODN and cytokine IL-12, the effect of the gel-vaccine could be further enhanced. In a murine peritoneal metastasis model of colorectal carcinoma, a single administration of the gel-vaccine resulted in complete tumor eradication in 8/9 mice. In summary, this study developed an immunologically active gel-vaccine system. And as a robust and versatile vaccine platform, by loading different antigens and adjuvants, this gel-vaccine system is expected to realize its better therapeutic potential.

IL-12 drives the differentiation of human T follicular regulatory cells.

T follicular regulatory (Tfr) cells can counteract the B cell helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cytokines initiating the differentiation of human regulatory T (Treg) cells into Tfr cells is still missing. Herein, we report that low doses of the pro-Tfh cytokine interleukin-12 (IL-12) drive the induction of a Tfr cell program on activated human Treg cells while also preserving their regulatory function. Mechanistically, we found that IL-12 led to STAT4 (signal transducer and activator of transcription 4) phosphorylation and binding to IL-12-driven follicular signature genes. Patients with inborn errors of immunity in the IL12RB1 gene presented with a strong decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides an approach for the in vitro generation of human Tfr-like cells.

Innate acting memory Th1 cells modulate heterologous diseases.

Through immune memory, infections have a lasting effect on the host. While memory cells enable accelerated and enhanced responses upon rechallenge with the same pathogen, their impact on susceptibility to unrelated diseases is unclear. We identify a subset of memory T helper 1 (Th1) cells termed innate acting memory T (TIA) cells that originate from a viral infection and produce IFN-γ with innate kinetics upon heterologous challenge in vivo. Activation of memory TIA cells is induced in response to IL-12 in combination with IL-18 or IL-33 but is TCR independent. Rapid IFN-γ production by memory TIA cells is protective in subsequent heterologous challenge with the bacterial pathogen Legionella pneumophila. In contrast, antigen-independent reactivation of CD4+ memory TIA cells accelerates disease onset in an autoimmune model of multiple sclerosis. Our findings demonstrate that memory Th1 cells can acquire additional TCR-independent functionality to mount rapid, innate-like responses that modulate susceptibility to heterologous challenges.

Intestinal cDC1s provide cues required for CD4+ T cell-mediated resistance to Cryptosporidium.

Cryptosporidium is an enteric pathogen and a prominent cause of diarrheal disease worldwide. Control of Cryptosporidium requires CD4+ T cells, but how protective CD4+ T cell responses are generated is poorly understood. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to understand the basis for CD4+ T cell priming and effector function. These studies revealed that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node but differentiate into Th1 cells in the gut to provide local parasite control. Although type 1 conventional dendritic cells (cDC1s) were dispensable for CD4+ T cell priming, they were required for CD4+ T cell gut homing and were a source of IL-12 at the site of infection that promoted local production of IFN-γ. Thus, cDC1s have distinct roles in shaping CD4+ T cell responses to an enteric infection: first, to promote gut homing from the mesLN, and second, to drive effector responses in the intestine.

Anti-PD-1 cis-delivery of low-affinity IL-12 activates intratumoral CD8+T cells for systemic antitumor responses.

Immune checkpoint blockade (ICB) therapies function by alleviating immunosuppression on tumor-infiltrating lymphocytes (TILs) but are often insufficient to fully reactivate these dysfunctional TILs. Although interleukin 12 (IL-12) has been used in combination with ICB to improve efficacy, this remains limited by severe toxicity associated with systemic administration of this cytokine. Here, we engineer a fusion protein composed of an anti-PD-1 antibody and a mouse low-affinity IL-12 mutant-2 (αPD1-mIL12mut2). Systemic administration of αPD1-mIL12mut2 displays robust antitumor activities with undetectable toxicity. Mechanistically, αPD1-mIL12mut2 preferentially activates tumor-infiltrating PD-1+CD8+T cells via high-affinity αPD-1 mediated cis-binding of low-affinity IL-12. Additionally, αPD1-mIL12mut2 treatment exerts an abscopal effect to suppress distal tumors, as well as metastasis. Collectively, αPD1-mIL12mut2 treatment induces robust systemic antitumor responses with reduced side effects.

Fasting reshapes tissue-specific niches to improve NK cell-mediated anti-tumor immunity.

Fasting is associated with improved outcomes in cancer. Here, we investigated the impact of fasting on natural killer (NK) cell anti-tumor immunity. Cyclic fasting improved immunity against solid and metastatic tumors in an NK cell-dependent manner. During fasting, NK cells underwent redistribution from peripheral tissues to the bone marrow (BM). In humans, fasting also reduced circulating NK cell numbers. NK cells in the spleen of fasted mice were metabolically rewired by elevated concentrations of fatty acids and glucocorticoids, augmenting fatty acid metabolism via increased expression of the enzyme CPT1A, and Cpt1a deletion impaired NK cell survival and function in this setting. In parallel, redistribution of NK cells to the BM during fasting required the trafficking mediators S1PR5 and CXCR4. These cells were primed by an increased pool of interleukin (IL)-12-expressing BM myeloid cells, which improved IFN-γ production. Our findings identify a link between dietary restriction and optimized innate immune responses, with the potential to enhance immunotherapy strategies.

Differential detection of chicken heterodimeric cytokines, interleukin 12 and 23 using their subunit-specific mouse monoclonal antibodies.

Interleukin-23 (IL-23) is a recently identified member of the IL-12 family of heterodimeric cytokines that play a critical role in regulating T helper cell function. IL-12 and IL-23 share a common p40 subunit, but differ in their p35 and p19 subunits, respectively. This difference in subunit composition results in distinct signaling pathways and biological functions for IL-12 and IL-23. Here, we report the functional characterization and immunomodulatory properties of chicken IL-12 and IL-23 using the panels of newly developed mouse anti-IL-12p40, IL-12p35-α and IL-23p19 monoclonal antibodies (mAbs). Western blot and indirect ELISA analysis demonstrated that the anti-chicken IL-12p40 mAbs (chIL-12p40; #10G10F4 and #10D8G2) bound to both recombinant proteins (IL-12 and IL-23), the anti-chicken IL-12p35 mAb (chIL-12p35; #2F1) specifically recognized recombinant IL-12, and the anti-chicken IL-23p19 mAb (chIL-23p19; #15A3) exhibited specificity for recombinant IL-23, without any cross-reactivity. Two ELISAs detecting specific chicken IL-12 (#10G10F4 and #2F1) or IL-23 (#10D8G2 and #15A3) were developed using newly developed mAb combinations, #10G10F4/ #2F1 and #10D8G2/#15A3 for IL-12 and IL-23, respectively, identified through a pairing assay. The levels of IL-12 and IL-23 in Resiquimod-848 stimulated-HD11 chicken macrophage cells were monitored over time using antigen-capture sandwich ELISA developed in this study. Furthermore, the levels of chicken IL-12 and IL-23 in the circulation of Eimeria maxima (E. maxima) and Eimeria tenella (E. tenella)-infected chickens were determined. Notably, the anti-chIL-12p40 mAbs (#10G10F4 and #10D8G2) neutralized the function of both chIL-12 and chIL-23 proteins, which share the p40 subunit, while the anti-chIL-23p19 mAb (#15A3) specifically neutralized chIL-23 protein in HD11 cells in vitro. The anti-chIL-12p35 mAb (#2F1), which is specific to the p35 subunit of IL-12, showed a partial neutralizing effect on chIL-12 protein. Collectively, our study validates the specificity and significance of 2 newly developed antigen-capture immunoassays for chIL-12 and chIL-23 which will expand our understanding of the functional characteristics of IL-12 and IL-23 and their association in normal and diseased chickens. These mAbs for each subunit, anti-chIL-12p35, anti-chIL-12p40 and anti-chIL-23p19, will serve as valuable immune reagents to elucidate host immune responses against disease pathogenesis in both fundamental and applied studies of avian species.

IL-12 induces a B cell-intrinsic IL-12/IFNγ feed-forward loop promoting extrafollicular B cell responses.

While some infections elicit germinal centers, others produce only extrafollicular responses. The mechanisms controlling these dichotomous fates are poorly understood. We identify IL-12 as a cytokine switch, acting directly on B cells to promote extrafollicular and suppress germinal center responses. IL-12 initiates a B cell-intrinsic feed-forward loop between IL-12 and IFNγ, amplifying IFNγ production, which promotes proliferation and plasmablast differentiation from mouse and human B cells, in synergy with IL-12. IL-12 sustains the expression of a portion of IFNγ-inducible genes. Together, they also induce unique gene changes, reflecting both IFNγ amplification and cooperative effects between both cytokines. In vivo, cells lacking both IL-12 and IFNγ receptors are more impaired in plasmablast production than those lacking either receptor alone. Further, B cell-derived IL-12 enhances both plasmablast responses and T helper 1 cell commitment. Thus, B cell-derived IL-12, acting on T and B cells, determines the immune response mode, with implications for vaccines, pathogen protection and autoimmunity.

Association of Chlamydia trachomatis Infection With Breast Cancer Risk and the Modification Effect of IL-12.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) infection has been implicated in various cancers, yet its association with breast cancer remains unexplored. This infection triggers a cascade of immune responses primarily regulated by Interleukins-12 (IL-12). Thus, the objective of this case-control study was to investigate the link between C. trachomatis infection and breast cancer risk, as well as the modification effect of IL-12. METHODS: We assessed IgG levels against C. trachomatis in serum of 1,121 women with breast cancer (861 with estrogen receptor-positive (ER+) and 260 with estrogen receptor-negative (ER-) tumors) and 400 controls in Guangzhou, China. Logistic regression models were applied to estimate the odds ratios (ORs) and 95% confidence intervals (95% CIs) for breast cancer risk in association with C. trachomatis infection. The interaction between C. trachomatis infection and IL-12 on breast cancer risk was estimated by the product terms in the logistic regression models. RESULTS: Seropositivity of C. trachomatis IgG showed a slight association with an increased risk of breast cancer (OR = 1.20; 95% CI: 0.86∼1.78). This association was more pronounced among women with a higher (OR = 5.82; 95% CI: 1.31∼25.94) than a lower (OR = 0.73; 95% CI: 0.41∼1.30) level of IL-12, with a statistically significant interaction observed (Pinteraction = 0.013). In addition, C. trachomatis IgG seropositivity was related to an increased risk of breast cancer among PR+ patients (OR = 1.53; 95% CI: 1.04∼2.23). CONCLUSIONS: C. trachomatis infection may contribute to the development of hormone-responsive breast cancer in women with high levels of IL-12. Further studies are needed to uncover the underlying mechanisms.

The alteration of NK cells phenotypes related to the functions and dengue disease outcomes.

Natural killer cells (NK cells) are the front line of immune cells to combat pathogens and able to influence the subsequent adaptive immune responses. One of the factors contributing to pathogenesis in dengue hemorrhagic fever (DHF) disease is aberrant immune activation during early phase of infection. This study explored the profile of NK cells in dengue infected pediatric patients with different degrees of disease severity. DHF patients contained higher frequency of activated NK cells but lower ratio of CD56dim:CD56bright NK subsets. Activated NK cells exhibited alterations in several NK receptors. Interestingly, the frequencies of NKp30 expressing activated NK cells were more pronounced in dengue fever (DF) than in DHF pediatric patients. In vitro functional analysis indicated that degranulation of NK cells in responding to dengue infected dendritic cells (DCs) required cell-cell contact and type I IFNs. Meanwhile, Interferon gamma (IFN-γ) production initially required cell-cell contact and type I IFNs followed by Interleukin-12 (IL-12), Interleukin-15 (IL-15) and Interleukin-18 (IL-18) resulting in the amplification of IFN-γ producing NK cells over time. This study highlighted the complexity and the factors influencing NK cells responses to dengue virus. Degree of activation, phenotypes of activated cells and the crosstalk between NK cells and other immune cells, could modulate the outcome of NK cells function in the dengue disease.

IL-12 Mediates T-bet-Expressing Myeloid Cell-Dependent Host Resistance against Toxoplasma gondii.

To defend against intracellular pathogens such as Toxoplasma gondii, the host generates a robust type 1 immune response. Specifically, host defense against T. gondii is defined by an IL-12-dependent IFN-γ response that is critical for host resistance. Previously, we demonstrated that host resistance is mediated by T-bet-dependent ILC-derived IFN-γ by maintaining IRF8+ conventional type 1 dendritic cells during parasitic infection. Therefore, we hypothesized that innate lymphoid cells are indispensable for host survival. Surprisingly, we observed that T-bet-deficient mice succumb to infection quicker than do mice lacking lymphocytes, suggesting an unknown T-bet-dependent-mediated host defense pathway. Analysis of parasite-mediated inflammatory myeloid cells revealed a novel subpopulation of T-bet+ myeloid cells (TMCs). Our results reveal that TMCs have the largest intracellular parasite burden compared with other professional phagocytes, suggesting they are associated with active killing of T. gondii. Mechanistically, we established that IL-12 is necessary for the induction of inflammatory TMCs during infection and these cells are linked to a role in host survival.

Mesothelin CAR-T cells expressing tumor-targeted immunocytokine IL-12 yield durable efficacy and fewer side effects.

Chimeric antigen receptor (CAR)-modified T cell therapy has achieved remarkable efficacy in treating hematological malignancies, but it confronts many challenges in treating solid tumors, such as the immunosuppressive microenvironment of the solid tumors. These factors reduce the antitumor activity of CAR-T cells in clinical trials. Therefore, we used the immunocytokine interleukin-12 (IL-12) to enhance the efficacy of CAR-T cell therapy. In this study, we engineered CAR-IL12R54 T cells that targeted mesothelin (MSLN) and secreted a single-chain IL-12 fused to a scFv fragment R54 that recognized a different epitope on mesothelin. The evaluation of the anti-tumor activity of the CAR-IL12R54 T cells alone or in combination with anti-PD-1 antibody in vitro and in vivo was followed by the exploration of the functional mechanism by which the immunocytokine IL-12 enhanced the antitumor activity. CAR-IL12R54 T cells had potency to lyse mesothelin positive tumor cells in vitro. In vivo studies demonstrated that CAR-IL12R54 T cells were effective in controlling the growth of established tumors in a xenograft mouse model with fewer side effects than CAR-T cells that secreted naked IL-12. Furthermore, combination of PD-1 blockade antibody with CAR-IL12R54 T cells elicited durable anti-tumor responses. Mechanistic studies showed that IL12R54 enhanced Interferon-γ (IFN-γ) production and dampened the activity of regulatory T cells (Tregs). IL12R54 also upregulated CXCR6 expression in the T cells through the NF-κB pathway, which facilitated T cell infiltration and persistence in the tumor tissues. In summary, the studies provide a good therapeutic option for the clinical treatment of solid tumors.