IL-10 Responses Research Articles

REL and BHLHE40 Variants Are Associated with IL-12 and IL-10 Responses and Tuberculosis Risk.

The major human genes regulating Mycobacterium tuberculosis-induced immune responses and tuberculosis (TB) susceptibility are poorly understood. Although IL-12 and IL-10 are critical for TB pathogenesis, the genetic factors that regulate their expression in humans are unknown. CNBP, REL, and BHLHE40 are master regulators of IL-12 and IL-10 signaling. We hypothesized that common variants in CNBP, REL, and BHLHE40 were associated with IL-12 and IL-10 production from dendritic cells, and that these variants also influence adaptive immune responses to bacillus Calmette-Guérin (BCG) vaccination and TB susceptibility. We characterized the association between common variants in CNBP, REL, and BHLHE40, innate immune responses in dendritic cells and monocyte-derived macrophages, BCG-specific T cell responses, and susceptibility to pediatric and adult TB in human populations. BHLHE40 single-nucleotide polymorphism (SNP) rs4496464 was associated with increased BHLHE40 expression in monocyte-derived macrophages and increased IL-10 from peripheral blood dendritic cells and monocyte-derived macrophages after LPS and TB whole-cell lysate stimulation. SNP BHLHE40 rs11130215, in linkage disequilibrium with rs4496464, was associated with increased BCG-specific IL-2+CD4+ T cell responses and decreased risk for pediatric TB in South Africa. SNPs REL rs842634 and rs842618 were associated with increased IL-12 production from dendritic cells, and SNP REL rs842618 was associated with increased risk for TB meningitis. In summary, we found that genetic variations in REL and BHLHE40 are associated with IL-12 and IL-10 cytokine responses and TB clinical outcomes. Common human genetic regulation of well-defined intermediate cellular traits provides insights into mechanisms of TB pathogenesis.

Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology.

BackgroundCellular immune memory responses post coronavirus disease 2019 (COVID‐19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large‐scale long‐term follow‐up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)‐specific peptide pool that contains no overlapping peptides with endemic human coronaviruses.MethodsPeptide stimulated memory T cell responses were assessed with dual interferon‐gamma (IFNγ) and interleukin (IL)‐2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array.ResultsOur work demonstrates that long‐term SARS‐CoV‐2‐specific memory T cell responses feature dual IFNγ and IL‐2 responses, whereas cross‐reactive memory T cell responses primarily generate IFNγ in response to SARS‐CoV‐2 peptide stimulation. T cell responses correlated to long‐term humoral immune responses. Disease severity as well as specific COVID‐19 symptoms correlated with the magnitude of the SARS‐CoV‐2‐specific memory T cell response four to five months post seroconversion.ConclusionUsing a large cohort and a SARS‐CoV‐2‐specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS‐CoV‐2‐specific memory T cell responses.

Tumor MHC Class I Expression Associates with Intralesional IL2 Response in Melanoma

Cancer immunotherapy can result in lasting tumor regression, but predictive biomarkers of treatment response remain ill-defined. Here, we performed single-cell proteomics, transcriptomics, and genomics on matched untreated and IL2 injected metastases from patients with melanoma. Lesions that completely regressed following intralesional IL2 harbored increased fractions and densities of nonproliferating CD8+ T cells lacking expression of PD-1, LAG-3, and TIM-3 (PD-1-LAG-3-TIM-3-). Untreated lesions from patients who subsequently responded with complete eradication of all tumor cells in all injected lesions (individuals referred to herein as "extreme responders") were characterized by proliferating CD8+ T cells with an exhausted phenotype (PD-1+LAG-3+TIM-3+), stromal B-cell aggregates, and expression of IFNγ and IL2 response genes. Loss of membranous MHC class I expression in tumor cells of untreated lesions was associated with resistance to IL2 therapy. We validated this finding in an independent cohort of metastatic melanoma patients treated with intralesional or systemic IL2. Our study suggests that intact tumor-cell antigen presentation is required for melanoma response to IL2 and describes a multidimensional and spatial approach to develop immuno-oncology biomarker hypotheses using routinely collected clinical biospecimens.

Caspase-1 inhibition by YVAD generates tregs pivoting IL-17 to IL-22 response in β-glucan induced airway inflammation

Background During Aspergillus fumigatus mediated lung inflammation, NLRP3 inflammasome is rapidly activated that aggravates IL-1β production contributing to lung inflammation. Previously, we have shown the protective role of SYK-1 inhibition in inhibiting inflammasome activation during lung inflammation. In the current manuscript, we explored the protective role of direct caspase-1 inhibition during β-glucan-induced lung inflammation. Methods We have mimicked the lung inflammation by administering intranasal β-glucan in mice model. YVAD was used for caspase-1 inhibition. Results We have shown that caspase-1 inhibition by YVAD did not alter inflammasome independent inflammatory cytokines, while it significantly reduced inflammasome activation and IL-1β secretion. Caspase-1 inhibited bone marrow derived dendritic cells (BMDCs), co-cultured with T cells showed decreased T-cell proliferation and direct them to secrete high TGF-β and IL-10 compared to the T cells co-cultured with β-glucan primed dendritic cells. Caspase-1 inhibition in BMDCs also induced IL-22 secretion from CD4+T cells. Caspase-1 inhibition in intranasal β-glucan administered mice showed decreased tissue damage, immune cell infiltration and IgA secretion compared to control mice. Further, splenocytes challenged with β-glucan show high IL-10 secretion and increased FOXp3 and Ahr indicating an increase in regulatory T cells on caspase-1 inhibition. Conclusion Caspase-1 inhibition can thus be an attractive target to prevent inflammation mediated tissue damage during Aspergillus fumigatus mouse model and can be explored as an attractive therapeutic strategy. HIGHLIGHTS Caspase-1 inhibition protects lung damage from inflammation during β-glucan exposure Caspase-1 inhibition in dendritic cells decreases IL-1β production resulting in decreased pathogenic Th17 Caspase-1 inhibition promotes regulatory T cells thereby inhibiting lung inflammation

Viral PB1-F2 and host IFN-γ guide ILC2 and T cell activity during influenza virus infection

Functional plasticity of innate lymphoid cells (ILCs) and T cells is regulated by host environmental cues, but the influence of pathogen-derived virulence factors has not been described. We now report the interplay between host interferon (IFN)-γ and viral PB1-F2 virulence protein in regulating the functions of ILC2s and T cells that lead to recovery from influenza virus infection of mice. In the absence of IFN-γ, lung ILC2s from mice challenged with the A/California/04/2009 (CA04) H1N1 virus, containing nonfunctional viral PB1-F2, initiated a robust IL-5 response, which also led to improved tissue integrity and increased survival. Conversely, challenge with Puerto Rico/8/1934 (PR8) H1N1 virus expressing fully functional PB1-F2, suppressed IL-5+ ILC2 responses, and induced a dominant IL-13+ CD8 T cell response, regardless of host IFN-γ expression. IFN-γ-deficient mice had increased survival and improved tissue integrity following challenge with lethal doses of CA04, but not PR8 virus, and increased resistance was dependent on the presence of IFN-γR+ ILC2s. Reverse-engineered influenza viruses differing in functional PB1-F2 activity induced ILC2 and T cell phenotypes similar to the PB1-F2 donor strains, demonstrating the potent role of viral PB1-F2 in host resistance. These results show the ability of a pathogen virulence factor together with host IFN-γ to regulate protective pulmonary immunity during influenza infection.

Contribution of the STAT Family of Transcription Factors to the Expression of the Serotonin 2B (HTR2B) Receptor in Human Uveal Melanoma.

Uveal melanoma (UM) remains the most common intraocular malignancy among diseases affecting the adult eye. The primary tumor disseminates to the liver in half of patients and leads to a 6 to 12-month survival rate, making UM a particularly aggressive type of cancer. Genomic analyses have led to the development of gene-expression profiles that can efficiently predict metastatic progression. Among these genes, that encoding the serotonin receptor 2B (HTR2B) represents the most discriminant from this molecular signature, its aberrant expression being the hallmark of UM metastatic progression. Recent evidence suggests that expression of HTR2B might be regulated through the Janus kinase/Signal Transducer and Activator of Transcription proteins (JAK/STAT) intracellular signalization pathway. However, little is actually known about the molecular mechanisms involved in the abnormally elevated expression of the HTR2B gene in metastatic UM and whether activated STAT proteins participates to this mechanism. In this study, we determined the pattern of STAT family members expressed in both primary tumors and UM cell-lines, and evaluated their contribution to HTR2B gene expression. Examination of the HTR2B promoter sequence revealed the presence of a STAT putative target site (5′-TTC (N)3 GAA3′) located 280 bp upstream of the mRNA start site that is completely identical to the high affinity binding site recognized by these TFs. Gene profiling on microarrays provided evidence that metastatic UM cell lines with high levels of HTR2B also express high levels of STAT proteins whereas low levels of these TFs are observed in non-metastatic UM cells with low levels of HTR2B, suggesting that STAT proteins contribute to HTR2B gene expression in UM cells. All UM cell lines tested were found to express their own pattern of STAT proteins in Western blot analyses. Furthermore, T142 and T143 UM cells responded to interleukins IL-4 and IL-6 by increasing the phosphorylation status of STAT1. Most of all, expression of HTR2B also considerably increased in response to both IL-4 and IL-6 therefore providing evidence that HTR2B gene expression is modulated by STAT proteins in UM cells. The binding of STAT proteins to the −280 HTR2B/STAT site was also demonstrated by electrophoretic mobility shift assay (EMSA) analyses and site-directed mutation of that STAT site also abolished both IL-4 and IL-6 responsiveness in in vitro transfection analyses. The results of this study therefore demonstrate that members from the STAT family of TFs positively contribute to the expression of HTR2B in uveal melanoma.

Characterization of the cell-mediated immune response to Takeda’s live-attenuated tetravalent dengue vaccine in adolescents participating in a phase 2 randomized controlled trial conducted in a dengue-endemic setting

BackgroundAs robust dengue-specific CD4+ and CD8+ T cell responses are essential for protective immunity, we assessed cell-mediated immune (CMI) responses to a DENV-2-based dengue tetravalent vaccine candidate (TAK-003) in adolescents living in Panama, a dengue-endemic country. MethodsPeripheral blood mononuclear cells were collected from a subset of 67 participants ≥ 10 years old included in a phase 2 clinical trial of TAK-003 (Clinicaltrials.gov: NCT02302066). Following stimulation with dengue peptides, the frequency, magnitude, and cross-reactivity of the CD8+ and CD4+ T cell IFN-γ, TNF-α and IL-2 responses were assessed by flow cytometry. ResultsIntracellular cytokine staining identified NS1, NS3, and NS5 as the most common non-structural (NS) targets of the CD4+ T-cell response (IFN-γ+); NS3 and NS5 were the main NS targets of the CD8+ T cell response (IFN-γ+). Both CD4+ and CD8+ T-cell responses were multi-functional (IFN-γ + TNF-α + IL-2+) and cross-reactive against DENV-1, -3, and -4 serotypes. Similar responses were seen in all CMI assessments irrespective of participant baseline status for dengue neutralizing antibodies and T cells. ConclusionsTAK-003 elicited cross-reactive, multi-functional CD4+ and CD8+ T-cell responses, irrespective of dengue pre-exposure.

The Type 2 Asthma Mediator IL-13 Inhibits Severe Acute Respiratory Syndrome Coronavirus 2 Infection of Bronchial Epithelium.

Asthma is associated with chronic changes in the airway epithelium, a key target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many epithelial changes, including goblet cell metaplasia, are driven by the type 2 cytokine IL-13, but the effects of IL-13 on SARS-CoV-2 infection are unknown. We found that IL-13 stimulation of differentiated human bronchial epithelial cells (HBECs) cultured at air-liquid interface reduced viral RNA recovered from SARS-CoV-2-infected cells and decreased double-stranded RNA, a marker of viral replication, to below the limit of detection in our assay. An intact mucus gel reduced SARS-CoV-2 infection of unstimulated cells, but neither a mucus gel nor SPDEF, which is required for goblet cell metaplasia, were required for the antiviral effects of IL-13. Bulk RNA sequencing revealed that IL-13 regulated 41 of 332 (12%) mRNAs encoding SARS-CoV-2-associated proteins that were detected in HBECs (>1.5-fold change; false discovery rate < 0.05). Although both IL-13 and IFN-α each inhibit SARS-CoV-2 infection, their transcriptional effects differed markedly. Single-cell RNA sequencing revealed cell type-specific differences in SARS-CoV-2-associated gene expression and IL-13 responses. Many IL-13-induced gene expression changes were seen in airway epithelium from individuals with type 2 asthma and chronic obstructive pulmonary disease. IL-13 effects on airway epithelial cells may protect individuals with type 2 asthma from COVID-19 and could lead to identification of novel strategies for reducing SARS-CoV-2 infection.

T-cells of invasive candidiasis patients show patterns of T-cell-exhaustion suggesting checkpoint blockade as treatment option

Recent data imply that strengthening host immunity by checkpoint inhibition improves outcome in invasive fungal infections (IFI), particularly in candidiasis. To assess T-cell exhaustion in this context, we compared peripheral blood mononuclear cells (PBMCs) and serum samples of patients with invasive Candida albicans infection (IC, n=21) to PBMCs or tumor-infiltrating lymphocytes (TILs) from cancer patients (n=14) and PBMCs of healthy controls (n=20). Type and differentiation of lymphocytes and expression of 29 immune-regulatory molecules were analyzed by flow cytometry. C. albicans specific responses were assessed by FluoroSpot (n=8) and antibody measurement (n=14). Fractions and phenotypes of lymphocyte subsets in PBMCs of IC patients were similar compared to PBMCs of controls, while they were different in TILs. PBMCs of patients with IC showed increased expression of immune-checkpoint molecules. The pattern of upregulated molecules was similar to TILs, but not present in PBMCs of control cancer patients. Fractions of T-cells expressing PD-1 and TIGIT were higher in IC patients that died. FluoroSpot analysis showed a Candida-specific IFN-y or IL-2 response in 5/8 patients, enhanced by addition of nivolumab in vitro. Together with preclinical data and preliminary evidence of clinical efficacy in mucormycosis, our results support clinical evaluation of immune-checkpoint inhibition in IFI treatment. NCT04533087; retrospectively registered on August 31, 2020.

A Theory on the Impact of Copper and Micronutrients Against COVID-19 in Humans

Copper (Cu) has a strong impact on the function of the immune system through several different pathways. These impacts include helping the function of monocytes, neutrophils, and macrophages, and enhancing Natural Killer cells’ activities. Cu also has a role in antimicrobial properties and inflammatory response. It is also important for IL-2 production and response, which is a component of adaptive immune cells. Additionally, Cu has multiple roles in both proliferation and differentiation of T cells and is involved in the production of antibodies. Cu deficiency can even lead to "increased viral virulence"1. Copper has a long history of use in medicine, and has continued to be used for purification of water, including use in hospitals to prevent legionnaires disease. The CDC pre released information on a study completed in March 2020 on the lifespan of COVID-19 on different surfaces which included its lifespan on copper, where it was completely dead within 4 hours. In addition, "Several reports demonstrated that Cu deficiency weakens the human immune response" 2. Given the multiple avenues of impact, it has been suggested that Cu supplementation, within recommended levels, be given to individuals who are low in Cu to help them fight off COVID-19. It is also possible that Cu supplementation, within recommended levels, may help prevent COVID-19 infection, or help individuals who are not low in Cu to fight off COVID-19 infection. However, dosage would have to be carefully managed, as excess levels of Cu can lead to both neurodegenerative and neurodevelopmental diseases.

Engineering T cell memory for antitumor immunity

Cancer therapy with the T cell growth factor interleukin (IL)-2 is limited by low response rates and toxicity. Multiple protein engineering strategies have attempted to improve IL-2 therapy, typically through enhanced IL-2 receptor (IL-2R) binding. Intriguingly, Mo et al. show that an IL-2R partial agonist may dramatically improve IL-2 responses by altering T cell differentiation.

Loss of aryl hydrocarbon receptor suppresses the response of colonic epithelial cells to IL22 signaling by upregulating SOCS3.

IL22 signaling plays an important role in maintaining gastrointestinal epithelial barrier function, cell proliferation, and protection of intestinal stem cells from genotoxicants. Emerging studies indicate that the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, promotes production of IL22 in gut immune cells. However, it remains to be determined if AhR signaling can also affect the responsiveness of colonic epithelial cells to IL22. Here, we show that IL22 treatment induces the phosphorylation of STAT3, inhibits colonic organoid growth, and promotes colonic cell proliferation in vivo. Notably, intestinal cell-specific AhR knockout (KO) reduces responsiveness to IL22 and compromises DNA damage response after exposure to carcinogen, in part due to the enhancement of suppressor of cytokine signaling 3 (SOCS3) expression. Deletion of SOCS3 increases levels of pSTAT3 in AhR KO organoids, and phenocopies the effects of IL22 treatment on wild-type (WT) organoid growth. In addition, pSTAT3 levels are inversely associated with increased azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon tumorigenesis in AhR KO mice. These findings indicate that AhR function is required for optimal IL22 signaling in colonic epithelial cells and provide rationale for targeting AhR as a means of reducing colon cancer risk.NEW & NOTEWORTHY AhR is a key transcription factor controlling expression of IL22 in gut immune cells. In this study, we show for the first time that AhR signaling also regulates IL22 response in colonic epithelial cells by modulating SOCS3 expression.

Citrullinated Epitopes Identified on Tumour MHC Class II by Peptide Elution Stimulate Both Regulatory and Th1 Responses and Require Careful Selection for Optimal Anti-Tumour Responses

BackgroundSomatic mutations or post-translational modifications of proteins result in changes that enable immune recognition. One such post-translational modification is citrullination, the conversion of arginine residues to citrulline. Citrullinated peptides are presented on MHC class II (MHCII) via autophagy which is upregulated by cellular stresses such as tumourigenesis.MethodsPeptides were eluted from B16 melanoma expressing HLA-DP4 and analysed by mass spectrometry to profile the presented citrullinated repertoire. Initially, seven of the identified citrullinated peptides were used in combination to vaccinate HLA-DP4 transgenic mice. Immune responses were characterised from the combination and individual vaccines by ex vivo cytokine ELISpot assay and assessed for tumour therapy.ResultsThe combination vaccine induced only weak anti-tumour therapy in the B16cDP4 melanoma model. Immune phenotyping revealed a dominant IFNγ response to citrullinated matrix metalloproteinase-21 peptide (citMMP21) and an IL-10 response to cytochrome p450 peptide (citCp450). Exclusion of the IL-10 inducing citCp450 peptide from the combined vaccine failed to recover a strong anti-tumour response. Single peptide immunisation confirmed the IFNγ response from citMMP21 and the IL-10 response from citCp450 but also showed that citrullinated Glutamate receptor ionotropic (citGRI) peptide stimulated a low avidity IFNγ response. Interestingly, both citMMP21 and citGRI peptides individually, stimulated strong anti-tumour responses that were significantly better than the combined vaccine. In line with the citGRI T cell avidity, it required high dose immunisation to induce an anti-tumour response. This suggests that as the peptides within the combined vaccine had similar binding affinities to MHC-II the combination vaccine may have resulted in lower presentation of each epitope and weak anti-tumour immunity.ConclusionWe demonstrate that tumours present citrullinated peptides that can stimulate Th1 and regulatory responses and that competition likely exists between similar affinity peptides. Characterisation of responses from epitopes identified by peptide elution are necessary to optimise selection for tumour therapy.

An allergen-fused dendritic cell-binding peptide enhances in vitro proliferation of equine T-cells and cytokine production

Allergen-specific immunotherapy (AIT) constitutes the only curative approach for allergy treatment. There is need for improvement of AIT in veterinary medicine, such as in horses suffering from insect bite hypersensitivity, an IgE-mediated dermatitis to Culicoides. Dendritic cell (DC)-targeting represents an efficient method to increase antigen immunogenicity. It is studied primarily for its use in improvement of cancer therapy and vaccines, but may also be useful for improving AIT efficacy. Immunomodulators, like the Toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid-A (MPLA) has been shown to enhance the IL-10 response in horses, while CpG-rich oligonucleotides (CpG-ODN), acting as TLR-9 agonists, have been shown to induce Th1 or regulatory responses in horses with equine asthma. Our aim was to evaluate in vitro effects of antigen-targeting to equine DC with an antigen-fused peptide known to target human and mouse DC and investigate whether addition of MPLA or CpG-ODN would further improve the induced immune response with regard to finding optimal conditions for equine AIT. For this purpose, DC-binding peptides were fused to the model antigen ovalbumin (OVA) and to the recombinant Culicoides allergen Cul o3. Effects of DC-binding peptides on cellular antigen uptake and induction of T cell proliferation were assessed. Polarity of the immune response was analysed by quantifying IFN-γ, IL-4, IL-10, IL-17 and IFN-α in supernatants of antigen-stimulated peripheral blood mononuclear cells (PBMC) in presence or absence of adjuvants. Fusion of DC-binding peptides to OVA significantly enhanced antigen-uptake by equine DC. DC primed with DC-binding peptides coupled to OVA or Cul o3 induced a significantly higher T-cell proliferation compared to the corresponding control antigens. PBMC stimulation with DC-binding peptides coupled to Cul o3 elicited a significant increase in the pro-inflammatory cytokines IFN-γ, IL-4, IL-17, as well as the anti-inflammatory IL-10, but not of IFN-α. Adjuvant addition further enhanced the effect of the DC-binding peptides by significantly increasing the production of IFN-γ, IL-4, IL-10 and IFN-α (CpG-ODN) and IL-10 (MPLA), while simultaneously suppressing IFN-γ, IL-4 and IL-17 production (MPLA). Targeting equine DC with allergens fused to DC-binding peptides enhances antigen-uptake and T-cell activation and may be useful in increasing the equine immune response against recombinant antigens. Combination of DC-binding peptide protein fusions with adjuvants is necessary to appropriately skew the resulting immune response, depending on intended use. Combination with MPLA is a promising option for improvement of AIT efficacy in horses, while combination with CpG-ODN increases the effector immune response to recombinant antigens.

The Impact of T Cell Immunity on Chemotherapy Response in Childhood Acute Lymphoblastic Leukemia

While most childhood acute lymphoblastic leukemia (ALL) can be cured with risk-adapted combination chemotherapy, a significant proportion of patients experience relapse and eventually succumb to leukemia. In fact, ~50% of ALL relapses occur in patients who have excellent early treatment response (i.e., negative minimal residual disease [MRD] at the end of induction therapy), suggesting that long-term remission is determined by factors beyond leukemia cell drug sensitivity. Inherited genetic variants in genes related to host immune functions have been linked to ALL prognosis, pointing to a possible role of immune vigilance against residual leukemia. However, little is known about how and which host immune factors influence the long-term-remission of this cancer.To this end, we first compared leukemia-free survival of murine BCR-ABL1 B-ALL in response to chemotherapy, in immunocompetent mice (C57BL/6J, “B6”) or mice with various T cell deficiencies. Complete loss of all T cells in the host (Tcra KO) led to a drastic increase in leukemia relapse after treatment with ABL inhibitor dasatinib (p=0.0014) or cytotoxic chemotherapy (e.g., dexamethasone [p=0.0002], and mercaptopurine [p=0.0106]). Similar results were observed when we specifically depleted CD4 or CD8 T cells in host mice (p=0.0302 and 0.0010 for CD4 and CD8 depletion, respectively). ABL1 mutation analysis showed that the immunocompetent mice were much more likely to relapse with wild-type BCR-ABL1 B-ALL after dasatinib withdrawal (71.4%). By contrast, most immunocompromised mice (60.0%, 75%, and 80% of TcraKO, CD4 T cell depletion, and CD8 T cell depletion) relapsed with ABL1-T315I B-ALL. Deep sequencing of BCR-ABL1 mutations during dasatinib therapy indicated that drug resistance mutations emerged early in leukemia regardless of immunocompetency of the host, but the lack of T cell immunity strongly predisposed mice to subsequently develop drug resistance and thus relapse. Furthermore, immunocompetent mice that achieved long-term remission also rejected re-engraftment of BCR-ABL1 ALL, suggesting immune memory against leukemia established during dasatinib treatment.To explore the molecular basis of T-cell immunity against ALL, we collected peripheral T cells during dasatinib therapy from mice which were eventually cured vs. those experienced relapse and compared their global gene expression pattern. Through expression array-based profiling of bulk T cells, we noted upregulation of memory/effector T cell signature genes in mice achieving long-term cure (especially in CD8 T cells), along with genes associated with TCR signaling and cytokine response (IL12 and IFNG). Similarly, scRNA-seq profiling also confirmed the association of TCR signaling, IL12 and IFNG signaling activation in T cells with the cure of ALL in mice. Single-cell RNA-seq analyses of T cell subpopulation specifically identified differences in memory-like T cells from relapse mice vs. those cured with the same chemotherapy, with the latter showing more robust activation of TCR and cytokine signalings. We thus hypothesized that IL12 and IFNG response signaling are key determinants of successful immune surveillance against ALL. Indeed, our in vivo experiments confirmed that 1) IfngKO mice relapsed much more quickly during dasatinib treatment with drug resistance BCR-ABL1 mutations than immunocompetent mice (p<0.0001), and 2) the addition of IL12 to dasatinib therapy significantly improved leukemia-free survival in immunocompetent mice with BCR-ABL1 B-ALL (p=0.0028).Finally, we validated these findings by evaluating the immune cell composition of peripheral blood samples collected during remission from 105 children with ALL treated on the St. Jude Total Therapy Study XV clinical trial. Using the gene expression-based deconvolution method, we quantified the frequency of 9 types of immune cells. There was a significant association of higher level of total T cell (CD8, CD4 naïve, CD4 memory, follicular helper, regulatory, and gamma delta T cells) with superior event-free survival (p=0.031), whereas other immune cells had no impact on ALL treatment outcomes. Even after adjusting for ALL molecular subtypes and MRD levels, T cell frequency remained prognostic (p=0.034).In conclusion, our results firmly established the role of T cell immunity in the cure of ALL and also pointed to T cell activation as a means of improving treatment outcomes of ALL. DisclosuresPui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee.

PRDM1 Is Sufficient for Inducing Human Primary T Cell Hyporesponsiveness and Implicates Low Gvhd Occurrence after Allo-HSCT

T cell hyporesponsiveness is crucial for functional immune system and prevent the damage induced by alloreactive T cells in autoimmune pathology and transplantation. As one of the most important regulators during B cell development, PRDM1 (also known as BLIMP-1) has been demonstrated its essential role for maintaining T cell hyporesponsiveness and homeostasis, evidenced by Prdm1 deficient mice accumulating activated T cells and developing multiorgan inflammatory disease. However, the mechanism of PRDM1 regulating T cell hyporesponsiveness is still ambiguous.In this study, we took advantage of multiomics technologies and systemic report the central role of PRDM1 in inducing human primary T cell hyporesponsiveness. Firstly, we overexpressed PRDM1 in human primary T cells and found increased ratio of CD4 +CD25 +FOXP3 + Treg cell subsets and increased IL-4 secretion. In parallel, inhibited PRDM1 expression level in human T cells decreased ratio of Treg cells and secretion of IL-4. Meanwhile, transcriptome analyses revealed that overexpressed PRDM1 enriched negative regulation of cell proliferation signaling pathway and resulted in a global reduction in IL-2 and inflammatory response signaling pathways. Furthermore, overexpressed PRDM1 in primary T cells upregulated several negative regulators of T cell function like EOMES, KLF2, LILRB1, KLRB1 and CD244, indicating a pioneer role of PRDM1 in inducing T cell hyporesponsive. To further investigate the regulation role of T cell hyporesponsiveness of PRDM1, we performed CUT&Tag and ATAC-seq in PRDM1 overexpressed primary T cells. CUT&Tag analysis showed PRDM1 could directly upregulated T cell hyporesponsiveness related gene such as KLF2, CD244 and KLRD1. Importantly, we observed consistent changes of IL-2, central regulator of T cell activation, in PRDM1 overexpressed T cell from ATAC-seq, CUT&Tag and RNA-seq data. We found PRDM1 could binding to IL-2 locus and decreased the chromatin accessibility of IL-2, consequently downregulated the expression level of IL-2 in human primary T cells. Moreover, altered open chromatin regions (OCRs) in PRDM1 overexpressed T cells enriched the similar transcription factors (TFs) with PRDM1 binding sites, indicating PRDM1 might be a pioneer TF in T cell hyporesponsiveness. These results demonstrated PRDM1 is sufficient for inducing T cell hyporesponsiveness in human primary T cells.To further validate the coexpression relationship between PRDM1 and Treg cell central TF FOXP3, we upregulated PRDM1 expression level on Jurkat T cells lines. The results also showed that elevated FOXP3 both in mRNA and protein level accompanied with upregulated PRDM1 expression level. To analyze the mechanism of PRDM1 regulating FOXP3 expression level, CUT&Tag data analyses showed that PRDM1 might upregulated FOXP3 by directly binding to the enhancer region of upstream of FOXP3 locus. Meanwhile, PRDM1 indirectly upregulated FOXP3 by upregulated KLF2, evidenced by inhibiting KLF2 in PRDM1 overexpressed primary T cells downregulated FOXP3 expression level. To further investigate the clinical implication of PRDM1 inducing T cell hyporesponsiveness, we detected the relationship of PRDM1 expression level and acute graft-versus-host disease(aGVHD) occurrence after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our results showed that patients with aGVHD (n=7) exhibited lower PRDM1 expression level than those without aGVHD in the same period after HSCT (n=7). Furthermore, we detected the expression level of PRDM1 in CD4 + T cell and CD8 + T cells from bone marrow allografts (BM) or peripheral blood allografts (PB) and followed up the occurrence of GVHD after HSCT for 2 years (n=18). There are low expression levels of PRDM1 in CD4 + T cells both from BM or PB grafts corelated with aGVHD occurrence in patients after allo-HSCT compared with those without aGVHD occurrence. In conclusion, our study provides the global regulatory model of PRDM1 in human primary T cell. We introduced PRDM1 as a sufficient regulator in T cell hyporesponsiveness induction, which is altering the chromatin accessibility and directly upregulated T cell inhibitory signals and downregulated T cell activated signals. The negative relationship between PRDM1 expression level with GVHD occurrence indicated it might be a potential biomarker for indicating HSCT prognosis. DisclosuresNo relevant conflicts of interest to declare.

Genomic and Immune Signatures Predict Sustained MRD Negativity in Newly Diagnosed Multiple Myeloma Patients Treated with Daratumumab, Carfilzomib, Lenalidomide, and Dexamethasone (D-KRd)

Genomic and Immune Signatures Predict Sustained MRD Negativity in Newly Diagnosed Multiple Myeloma Patients Treated with Daratumumab, Carfilzomib, Lenalidomide, and Dexamethasone (D-KRd)

Targeting IFN activity to both B cells and plasmacytoid dendritic cells induces a robust tolerogenic response and protection against EAE

Type I Interferon (IFN) was the very first drug approved for the treatment of Multiple Sclerosis (MS), and is still frequently used as a first line therapy. However, systemic IFN also causes considerable side effects, affecting therapy adherence and dose escalation. In addition, the mechanism of action of IFN in MS is multifactorial and still not completely understood. Using AcTaferons (Activity-on-Target IFNs, AFNs), optimized IFN-based immunocytokines that allow cell-specific targeting, we have previously demonstrated that specific targeting of IFN activity to dendritic cells (DCs) can protect against experimental autoimmune encephalitis (EAE), inducing in vivo tolerogenic protective effects, evidenced by increased indoleamine-2,3-dioxygenase (IDO) and transforming growth factor β (TGFβ) release by plasmacytoid (p) DCs and improved immunosuppressive capacity of regulatory T and B cells. We here report that targeting type I IFN activity specifically towards B cells also provides strong protection against EAE, and that targeting pDCs using SiglecH-AFN can significantly add to this protective effect. The superior protection achieved by simultaneous targeting of both B lymphocytes and pDCs correlated with improved IL-10 responses in B cells and conventional cDCs, and with a previously unseen very robust IDO response in several cells, including all B and T lymphocytes, cDC1 and cDC2.

ESAT-6 impedes IL-18 mediated phagosome lysosome fusion via microRNA-30a upon Calcimycin treatment in mycobacteria infected macrophages

The weaponry possessed by Mycobacterium tuberculosis (M. tb) in the form of immunodominant antigens hijack the host immune system to give a survival advantage to this intracellular fiend, but the mechanism of this control is not entirely known. Since we have previously reported the mechanism of autophagy inhibition by early secreted antigenic target 6 kDa (ESAT-6) through microRNA (miR)-30a-3p in Calcimycin treated differentiated THP-1 (dTHP-1) cells, the present study was undertaken to deduce the effect of miR-30a on the immunomodulatory profile of ESAT-6 treated cells and the mechanism involved thereof, if any. Initially, the effect of recombinant ESAT-6 (rESAT-6) on the immunomodulatory profile in Calcimycin-treated phorbol 12-myristate 13-acetate (PMA) dTHP-1 cells was checked. Later, transfection studies using miR-30a-3p inhibitor or -5p mimic highlighted the contrary roles of different arms of the same miRNA in regulating IL-18 response by ESAT-6 in dTHP-1 cells after Calcimycin treatment. By using either IL-18 neutralizing antibody or inhibitors of phosphoinositide 3-kinase (PI3K)/NF-κB/phagosome-lysosome fusion in the miRNA-30a transfected background, IL-18 mediated signaling and intracellular killing of mycobacteria was reversed in the presence of ESAT-6. Overall, the results of this study conclusively prove the contrary roles of miR-30a-3p and miR-30a-5p in regulating IL-18 signaling by ESAT-6 in dTHP-1 cells upon Calcimycin treatment that affected phagosome-lysosome fusion and intracellular survival of mycobacteria.

Lymphocyte Activation Gene-3 Regulates Dendritic Cell Metabolic Programing and T Cell Priming Function.

Deficiency of lymphocyte activation gene-3 (LAG3) is significantly associated with increased cardiovascular disease risk with in vitro results demonstrating increased TNF-α and decreased IL-10 secretion from LAG3-deficient human B lymphoblasts. The hypothesis tested in this study was that Lag3 deficiency in dendritic cells (DCs) would significantly affect cytokine expression, alter cellular metabolism, and prime naive T cells to greater effector differentiation. Experimental approaches used included differentiation of murine bone marrow-derived DCs (BMDCs) to measure secreted cytokines, cellular metabolism, RNA sequencing, whole cell proteomics, adoptive OT-II CD4+Lag3 +/+ donor cells into wild-type (WT) C57BL/6 and Lag3 -/- recipient mice, and ex vivo measurements of IFN-γ from cultured splenocytes. Results showed that Lag3 -/- BMDCs secreted more TNF-α, were more glycolytic, used fewer fatty acids for mitochondrial respiration, and glycolysis was significantly reduced by exogenous IL-10 treatment. Under basal conditions, RNA sequencing revealed increased expression of CD40 and CD86 and other cytokine-signaling targets as compared with WT. Whole cell proteomics identified a significant number of proteins up- and downregulated in Lag3 -/- BMDCs, with significant differences noted in exogenous IL-10 responsiveness compared with WT cells. Ex vivo, IFN-γ expression was significantly higher in Lag3 -/- mice as compared with WT. With in vivo adoptive T cell and in vitro BMDC:T coculture experiments, Lag3 -/- BMDCs showed greater T cell effector differentiation and proliferation, respectively, compared with WT BMDCs. In conclusion, Lag3 deficiency in DCs is associated with an inflammatory phenotype that provides a plausible mechanism for increased cardiovascular disease risk in humans with LAG3 deficiency.