A genetic screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation

Cytokine-mediated activation of host immunity is central to the control of pathogens. Interferon-gamma (IFNg) is a key cytokine in protective immunity that induces major histocompatibility complex class II molecules (MHCII) to amplify CD4+ T cell activation and effector function. Despite its central role, the dynamic regulation of IFNg-induced MHCII is not well understood. Using a genome-wide CRISPR-Cas9 screen in murine macrophages we identified genes that control MHCII surface expression. Mechanistic studies uncovered two parallel pathways of IFNg-mediated MHCII control that require the multifunctional glycogen synthase kinase 3 beta (GSK3b) or the mediator complex subunit MED16. Both pathways control distinct aspects of the IFNg response and are necessary for IFNg-mediated induction of the MHCII transactivator Ciita, MHCII expression, and CD4+ T cell activation. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4+ T cell responses.

KS01.3.A Tumoral MHC class II expression in gliomas drives T cell exhaustion

BACKGROUND Neoepitopes are presented on major histocompatibility class II (MHCII) molecules. In glioma, for instance, the recurrent driver mutation IDH1R132H was shown to bear an MHCII-restricted epitope in preclinical and clinical vaccine studies. The general relevance of MHCII expression in glioma for antitumor immunity, however, remains unknown. Here we evaluate stromal and tumoral MHCII expression, functionality, and its association with survival in gliomas. MATERIAL AND METHODS Immunostaining of human glioma tissues was used to identify tumoral, endothelial, and microglial MHCII expression and to enumerate T cell infiltrates. To gain insights into tumoral MHCII expression, bulk transcriptomic data from TCGA and single-cell transcriptomic data from publicly available datasets were analyzed. MHC ligandome analyses of an MHCII+ glioma cell line and human glioma tissues were used to determine the functionality of MHCII in vitro and ex vivo. Functional in vitro co-culture assays with an HLA-DR-matched tetanus toxoid (TT) epitope-overexpressing glioma cell line and in vitro-expanded TT-reactive T cells from healthy donors were used to examine direct target recognition by T helper cells. CRISPR-Cas9-mediated knockout of MHCII in preclinical hypermutant glioblastoma cell line GL261 was employed to further validate the consequences of tumoral MHCII expression and to probe potential clinical intervention with existing therapies. RESULTS MHCII is expressed in the majority of gliomas and associated with increased infiltration of T cells. In 10% of the analyzed glioma tissues and a subset of single cells, tumoral MHCII expression is detected. Clinical and transcriptomic data reveal that tumoral MHCII is associated with poor prognosis, cytokine responses, immune inhibition and T cell differentiation. Ligandome analyses evidence presentation of peptides by MHCII molecules on glioma cells. In in vitro assays, TT-reactive T helper cells specifically produce IFNg when co-cultured with MHCII+ glioma cells upon the presence of co-stimulation. In agreement with the clinical data, preclinical murine models demonstrate that tumoral MHCII expression leads to reduced survival. Co-culture assay shows that tumoral MHCII results in upregulation of PD-1 on T helper cells antigen-specifically. Concordantly, immune checkpoint blockade (ICB) therapy slows the disease progression of mice carrying MHCII+ tumors. CONCLUSION MHCII is expressed in gliomas by a subset of tumor cells. Although tumoral MHCII is functional, it is associated with poor survival in both clinical data and preclinical models. T cell exhaustion induced by tumoral MHCII expression can, in part, be overcome by ICB in vivo. Further experiments are required to decipher tumor cell intrinsic and microenvironmental consequences of tumoral MHCII expression.

Type II alveolar cell MHCII improves respiratory viral disease outcomes while exhibiting limited antigen presentation

Type II alveolar cells (AT2s) are critical for basic respiratory homeostasis and tissue repair after lung injury. Prior studies indicate that AT2s also express major histocompatibility complex class II (MHCII) molecules, but how MHCII expression by AT2s is regulated and how it contributes to host defense remain unclear. Here we show that AT2s express high levels of MHCII independent of conventional inflammatory stimuli, and that selective loss of MHCII from AT2s in mice results in modest worsening of respiratory virus disease following influenza and Sendai virus infections. We also find that AT2s exhibit MHCII presentation capacity that is substantially limited compared to professional antigen presenting cells. The combination of constitutive MHCII expression and restrained antigen presentation may position AT2s to contribute to lung adaptive immune responses in a measured fashion, without over-amplifying damaging inflammation.

Type II alveolar cells with constitutive expression of MHCII and limited antigen presentation capacity contribute to improved respiratory viral disease outcomes

Type II alveolar cells (AT2s) are critical for basic respiratory homeostasis and tissue repair after lung injury. Prior studies indicate that AT2s also express major histocompatibility complex II (MHCII) molecules, but how MHCII expression by AT2s is regulated and how it contributes to host defense remain unclear. Here we show that AT2s express high levels of MHCII independent of conventional inflammatory stimuli, and that selective loss of MHCII from AT2s in mice results in the worsening of respiratory virus disease following influenza and Sendai virus infections. We also find that AT2s exhibit MHCII presentation capacity that is substantially limited in comparison to professional antigen presenting cells. The combination of constitutive MHCII expression and restrained presentation may position AT2s to contribute to lung adaptive immune responses in a measured fashion, without over-amplifying damaging inflammation.

Smoking and SARS-CoV-2 Impair Dendritic Cells and Regulate DC-SIGN Expression in Tissues

SummaryThe current spreading novel coronavirus SARS-CoV-2 is highly infectious and pathogenic. In this study, we screened the gene expression of three SARS-CoV-2 host receptors (ACE2, DC-SIGN and L-SIGN) and DC status in bulk and single cell transcriptomic datasets of upper airway, lung or blood of smokers, non-smokers and COVID-19 patients. We found smoking increased DC-SIGN gene expression and inhibited DC maturation and its ability of T cell stimulation. In COVID-19, DC-SIGN gene expression was interestingly decreased in lung DCs but increased in blood DCs. Strikingly, DCs shifted from cDCs to pDCs in COVID-19, but the shift was trapped in an immature stage (CD22+ or ANXA1+ DC) with MHCII downregulation in severe cases. This observation indicates that DCs in severe cases stimulate innate immune responses but fail to specifically recognize SARS-CoV-2. Our study provides insights into smoking effect on COVID-19 risk and the profound modulation of DC function in severe COVID-19.Graphical AbstractHighlightsSmoking upregulates the expression of ACE2 and CD209 and inhibits DC maturation in lungs. SARS-CoV-2 modulates the DCs proportion and CD209 expression differently in lung and blood.Severe infection is characterized by DCs less capable of maturation, antigen presentation and MHCII expression.DCs shift from cDCs to pDCs with SARS-CoV-2 infection but are trapped in an immature stage in severe cases.

Podocyte-specific knockout of the neonatal Fc receptor (FcRn) results in differential protection depending on the model of glomerulonephritis

Podocytes have been proposed to be antigen presenting cells (APCs). In traditional APCs, the neonatal Fc receptor (FcRn) is required for antigen presentation and global knockout of FcRn protects against glomerulonephritis. Since podocytes express FcRn, we sought to determine whether the absence of podocyte FcRn ameliorates immune-mediated disease. We examined MHCII and costimulatory markers expression in cultured wild type (WT) and FcRn knockout (KO) podocytes. Interferon gamma (IFNγ) induced MHCII expression in both WT and KO podocytes but did not change CD80 expression. Neither WT nor KO expressed CD86 or inducible costimulatory ligand (ICOSL) at baseline or with IFNγ. Using an antigen presentation assay, WT podocytes but not KO treated with immune complexes induced a modest increase in IL-2. Induction of the anti-glomerular basement membrane (anti-GBM) model resulted in a significant decrease in glomerular crescents in podocyte-specific FcRn knockout mouse (podFcRn KO) versus controls but the overall percentage of crescents was low. To examine the effects of the podocyte-specific FcRn knockout in a model with a longer autologous phase, we used the nephrotoxic serum nephritis (NTS) model. We found that the podFcRn KO mice had significantly reduced crescent formation and glomerulosclerosis compared to control mice. This study demonstrates that lack of podocyte FcRn is protective in immune mediated kidney disease that is dependent on an autologous phase. This study also highlights the difference between the anti-GBM model and NTS model of disease.

A genome-wide screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation

Cytokine-mediated activation of host immunity is central to the control of pathogens. A key cytokine in protective immunity is interferon-gamma (IFNγ), which is a potent activator of antimicrobial and immunomodulatory effectors within the host. A major role of IFNγ is to induce major histocompatibility complex class II molecules (MHCII) on the surface of cells, which is required for CD4+ T cell activation. Despite its central role in host immunity, the complex and dynamic regulation of IFNγ-induced MHCII is not well understood. Here, we integrated functional genomics and transcriptomics to comprehensively define the genetic control of IFNγ-mediated MHCII surface expression in macrophages. Using a genome-wide CRISPR-Cas9 library we identified genes that control MHCII surface expression, many of which have yet to be associated with MHCII. Mechanistic studies uncovered two parallel pathways of IFNγ-mediated MHCII control that require the multifunctional glycogen synthase kinase 3 beta (GSK3β) or the mediator complex subunit MED16. Both pathways are necessary for IFNγ-mediated induction of the MHCII transactivator CIITA, MHCII expression, and CD4+ T cell activation. Using transcriptomic analysis, we defined the regulons controlled by GSK3β and MED16 in the presence and absence of IFNγ and identified unique networks of the IFNγ-mediated transcriptional landscape that are controlled by each gene. Our analysis suggests GSK3β and MED16 control distinct aspects of the IFNγ-response and are critical for macrophages to respond appropriately to IFNγ. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4+ T cell responses by macrophages. These discoveries will aid in our basic understanding of macrophage-mediated immunity and will shed light on mechanisms of failed adaptive responses pervasive in infectious disease, autoimmunity, and cancer.

Hormonally controlled ILC antigen presentation potential is reduced during pregnancy

Strategically located in mucosal barriers, innate lymphoid cells (ILCs) are relevant in local containment and tolerance of commensal microflora. ILCs have been recently described at the fetomaternal interface, where the development of a semi-allogeneic fetus can only succeed in a well-controlled immune environment. We postulate that ILCs adapt their antigen presentation capacity to protect pregnancy from excessive immune responses. Human ILCs were studied in deciduae of term pregnancies, peripheral blood and in in vitro generated ILCs. Fresh isolated lymphocytes or cells treated with pregnancy-related factors were investigated. The fetal antigen rejection-based CBA/J × DBA/2J mouse model (poor outcome pregnant mice; POPM) was used to characterize ILC antigen presentation potential in normal and immunologically disturbed pregnancies. ILC antigen presentation potential was characterized by flow cytometry and qPCR. We discovered that the distribution of ILC subsets changed during both human and murine pregnancy. Moreover, the pregnancy was accompanied by reduced MHCII expression in splenic ILCs during normal pregnancy (CBA/J × BALB/c; good outcome pregnant mice; GOPM) but increased in splenic and intestinal ILCs of CBA/J × DBA/2J mice. In vitro, splenic ILCs from pregnant mice increased MHCII expression after stimulation with IL-1β and IL-23. In contrast, uterine ILCs displayed lower MHCII expression, which remained unchanged after stimulation. Finally, pregnancy-related factors and hormones present in the uterine environment reduced antigen presentation potential of human ILCs in vitro. Together, these data indicate that, during pregnancy, peripheral and especially uterine ILCs adapt their antigen presenting potential to maintain a level of tolerance and support pregnancy.

Cancer Cell Intrinsic Expression of MHCII Regulates the Immune Microenvironment and Response to Anti-PD-1 Therapy in Lung Adenocarcinoma

MHC class II (MHCII) expression is usually restricted to antigen presenting cells, but can be expressed by cancer cells. We examined the effect of cancer cell-intrinsic MHC class II (csMHCII) expression in lung adenocarcinoma on T cell recruitment to tumors and response to anti-PD-1 therapy. The functional significance of altering csMHCII expression was explored using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis Lung Carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, while LLC tumors are resistant. RNA-seq analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, while resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss- and gain-of-function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII, and converted tumors from anti-PD-1-sensitive to anti-PD-1-resistant. This was associated with decreased T cell infiltration, lower levels of Th1 cytokines, increased B cell number and decreased macrophage recruitment. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, provided a survival benefit and positively correlated with T cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T cell infiltration and sensitivity to anti-PD-1.