CD8 T-cell Priming Research Articles

Cytomegalovirus inhibitors of programmed cell death restrict antigen cross-presentation in the priming of antiviral CD8 T cells.

CD8 T cells are the predominant effector cells of adaptive immunity in preventing cytomegalovirus (CMV) multiple-organ disease caused by cytopathogenic tissue infection. The mechanism by which CMV-specific, naïve CD8 T cells become primed and clonally expand is of fundamental importance for our understanding of CMV immune control. For CD8 T-cell priming, two pathways have been identified: direct antigen presentation by infected professional antigen-presenting cells (pAPCs) and antigen cross-presentation by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Studies in mouse models using murine CMV (mCMV) and precluding either pathway genetically or experimentally have shown that, in principle, both pathways can congruently generate the mouse MHC/H-2 class-I-determined epitope-specificity repertoire of the CD8 T-cell response. Recent studies, however, have shown that direct antigen presentation is the canonical pathway when both are accessible. This raised the question of why antigen cross-presentation is ineffective even under conditions of high virus replication thought to provide high amounts of antigenic material for feeding cross-presenting pAPCs. As delivery of antigenic material for cross-presentation is associated with programmed cell death, and as CMVs encode inhibitors of different cell death pathways, we pursued the idea that these inhibitors restrict antigen delivery and thus CD8 T-cell priming by cross-presentation. To test this hypothesis, we compared the CD8 T-cell responses to recombinant mCMVs lacking expression of the apoptosis-inhibiting protein M36 or the necroptosis-inhibiting protein M45 with responses to wild-type mCMV and revertant viruses expressing the respective cell death inhibitors. The data reveal that increased programmed cell death improves CD8 T-cell priming in mice capable of antigen cross-presentation but not in a mutant mouse strain unable to cross-present. These findings strongly support the conclusion that CMV cell death inhibitors restrict the priming of CD8 T cells by antigen cross-presentation.

Modulation of cytomegalovirus immune evasion identifies direct antigen presentation as the predominant mode of CD8 T-cell priming during immune reconstitution after hematopoietic cell transplantation.

Cytomegalovirus (CMV) infection is the most critical infectious complication in recipients of hematopoietic cell transplantation (HCT) in the period between a therapeutic hematoablative treatment and the hematopoietic reconstitution of the immune system. Clinical investigation as well as the mouse model of experimental HCT have consistently shown that timely reconstitution of antiviral CD8 T cells is critical for preventing CMV disease in HCT recipients. Reconstitution of cells of the T-cell lineage generates naïve CD8 T cells with random specificities among which CMV-specific cells need to be primed by presentation of viral antigen for antigen-specific clonal expansion and generation of protective antiviral effector CD8 T cells. For CD8 T-cell priming two pathways are discussed: "direct antigen presentation" by infected professional antigen-presenting cells (pAPCs) and "antigen cross-presentation" by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Current view in CMV immunology favors the cross-priming hypothesis with the argument that viral immune evasion proteins, known to interfere with the MHC class-I pathway of direct antigen presentation by infected cells, would inhibit the CD8 T-cell response. While the mode of antigen presentation in the mouse model of CMV infection has been studied in the immunocompetent host under genetic or experimental conditions excluding either pathway of antigen presentation, we are not aware of any study addressing the medically relevant question of how newly generated naïve CD8 T cells become primed in the phase of lympho-hematopoietic reconstitution after HCT. Here we used the well-established mouse model of experimental HCT and infection with murine CMV (mCMV) and pursued the recently described approach of up- or down-modulating direct antigen presentation by using recombinant viruses lacking or overexpressing the central immune evasion protein m152 of mCMV, respectively. Our data reveal that the magnitude of the CD8 T-cell response directly reflects the level of direct antigen presentation.

Direct antigen presentation is the canonical pathway of cytomegalovirus CD8 T-cell priming regulated by balanced immune evasion ensuring a strong antiviral response.

CD8 T cells are important antiviral effectors in the adaptive immune response to cytomegaloviruses (CMV). Naïve CD8 T cells can be primed by professional antigen-presenting cells (pAPCs) alternatively by "direct antigen presentation" or "antigen cross-presentation". In the case of direct antigen presentation, viral proteins are expressed in infected pAPCs and enter the classical MHC class-I (MHC-I) pathway of antigen processing and presentation of antigenic peptides. In the alternative pathway of antigen cross-presentation, viral antigenic material derived from infected cells of principally any cell type is taken up by uninfected pAPCs and eventually also fed into the MHC class-I pathway. A fundamental difference, which can be used to distinguish between these two mechanisms, is the fact that viral immune evasion proteins that interfere with the cell surface trafficking of peptide-loaded MHC-I (pMHC-I) complexes are absent in cross-presenting uninfected pAPCs. Murine cytomegalovirus (mCMV) models designed to disrupt either of the two presentation pathways revealed that both are possible in principle and can substitute each other. Overall, however, the majority of evidence has led to current opinion favoring cross-presentation as the canonical pathway. To study priming in the normal host genetically competent in both antigen presentation pathways, we took the novel approach of enhancing or inhibiting direct antigen presentation by using recombinant viruses lacking or overexpressing a key mCMV immune evasion protein. Against any prediction, the strongest CD8 T-cell response was elicited under the condition of intermediate direct antigen presentation, as it exists for wild-type virus, whereas the extremes of enhanced or inhibited direct antigen presentation resulted in an identical and weaker response. Our findings are explained by direct antigen presentation combined with a negative feedback regulation exerted by the newly primed antiviral effector CD8 T cells. This insight sheds a completely new light on the acquisition of viral immune evasion genes during virus-host co-evolution.

A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV-infected cells

A long-lived latent reservoir of HIV-1-infected CD4 Tcells persists with antiretroviral therapy and prevents cure. We report that the emergence of latently infected primary CD4 Tcells requires the activity of histone deacetylase enzymes HDAC1/2 and HDAC3. Data from targeted HDAC molecules, an HDAC3-directed PROTAC, and CRISPR-Cas9 knockout experiments converge on a model where either HDAC1/2 or HDAC3 targeting can prevent latency, whereas all three enzymes must be targeted to achieve latency reversal. Furthermore, HDACi treatment targets features of memory Tcells that are linked to proviral latency and persistence. Latency prevention is associated with increased H3K9ac at the proviral LTR promoter region and decreased H3K9me3, suggesting that this epigenetic switch is a key proviral silencing mechanism that depends on HDAC activity. These findings support further mechanistic work on latency initiation and eventual clinical studies of HDAC inhibitors to interfere with latency initiation.

The Influence of TET2 and/or DNMT3A Knockout on the 5hmC Levels and Growth Characteristics of Modified Human Primary CD4 T Cells after Activation

Angioimmunoblastic T-cell lymphoma (AITL) has a unique immunohistological profile featuring malignant CD4+ T follicular helper cells (Tfh) inter-mixed with an extensive infiltrate of multiple lineages of immune cells. The AITL tumor microenvironment (TME) classically features small or large B cells (variably Epstein-Barr Virus (EBV) positive), reactive CD8+ T-cells, plasma cells and proliferative high endothelial venules (HEVs). Common genetic mutations have been identified within the malignant Tfh clone in AITL including epigenetic modifiers TET2, DNMT3A, and IDH2. DNMT3A mutations in AITL occur at a frequency of 20-38.5%and most of the DNMT3A mutations occur together with TET2 mutations. Tet2/ Dnmt3a double-knockout mice show synergistic effects on the expression of lineage-specifying transcription factor genes via epigenetic mechanisms. However, it is still not clear how DNMT3A mutations enhance the effect of TET2 mutations on promoting AITL development. To investigate how TET2 and DNMT3A mutations interact in AITL development, we first generated TET2 and/or DNMT3A knockout human primary CD4-T cells with CRISPR-Cas9 mediated genome editing. TET proteins could oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmc) in replication- independent DNA demethylation. Unmethylated cytosine base can be methylated by the DNA methylation machinery (DNMT3A/B) to form 5mC, which in turn provides the substrate for TET enzymes to produce 5hmC. TET and DNMT family members are thus needed to be dynamically modulate the epigenome to maintain cellular homeostasis or alter cellular function or development. We found that in response to CD3/CD28 stimulation, WT CD4-T cells exhibited division mediated dilutional reduction in 5 hmC which was later reconstitution. However, in TET2 or DKO cells, this process was compromised, showing delayed global 5hmc levels recovery (Fig. 1A). We then investigated whether the changes in methylation dynamics would alter cellular function. When using CD3/CD28 beads stimulation (10 days/cycle), we found that after two cycles of stimulation, these CD4-T cells with different gene editing started showing distinct behaviors with proliferation and apoptosis when exposed to CD3/CD28 beads again. The TET2/ DNMT3A double knockout (DKO) CD4-T cells showed more rapid response in cell division and higher proliferation rate than the other three groups (Fig. 1B). Compared with WT cells, consistently higher apoptotic cells were found in DNMT3A-KO group, but not with either TET2-KO or DKO cells (Fig. 1C). Interestingly, withdraw of beads at the end of the 10 days cycle, DKO cells kept higher baseline proliferation level than WT cells (Fig. 1D). We then used autologous or allogeneic EBV transformed B-cell for stimulation of the CD4 T-cells after one or two cycles of CD3/28 beads stimulation, and different response kinetics were observed among these prior activated CD4-T cells with TET2 and/or DNMT3A mutations. DKO cells showed more rapid response in cell division and elevated percentages of replicated cells to either autologous or allogeneic B-cells compared with WT cells (Fig. 1E). An interesting aspect of this B/T interaction is that, even the autologous B-cells could activate a large percentage of the T-cell population, particularly in the DKO cells, so it is not likely due to specific antigen recognition but rather a non-specific stimulation by the B-cells, probably via one or more co-stimulatory molecules (ICOSL, CD86 or 4-1BBL). This may be relevant to in vivo evolution of the mutated T-cells with B-cells/activated B-cells providing support.

Structural Insights into the Mechanism of HIV-1 Tat Secretion from the Plasma Membrane

Human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (Tat) is a small, intrinsically disordered basic protein that plays diverse roles in the HIV-1 replication cycle, including promotion of efficient viral RNA transcription. Tat is released by infected cells and subsequently absorbed by healthy cells, thereby contributing to HIV-1 pathogenesis including HIV-associated neurocognitive disorder. It has been shown that, in HIV-1-infected primary CD4 T-cells, Tat accumulates at the plasma membrane (PM) for secretion, a mechanism mediated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural basis for Tat interaction with the PM and thereby secretion is lacking. Herein, we employed NMR and biophysical methods to characterize Tat86 (86 amino acids) interactions with PI(4,5)P2 and lipid nanodiscs (NDs). Our data revealed that Arg49, Lys50 and Lys51 (RKK motif) constitute the PI(4,5)P2 binding site, that Tat86 interaction with lipid NDs is dependent on PI(4,5)P2 and phosphatidylserine (PS), and that the arginine-rich motif (RRQRRR) preferentially interacts with PS. Furthermore, we show that Trp11, previously implicated in Tat secretion, penetrates deeply in the membrane; substitution of Trp11 severely reduced Tat86 interaction with membranes. Deletion of the entire highly basic region and Trp11 completely abolished Tat86 binding to lipid NDs. Our data support a mechanism by which HIV-1 Tat secretion from the PM is mediated by a tripartite signal consisting of binding of the RKK motif to PI(4,5)P2, arginine-rich motif to PS, and penetration of Trp11 in the membrane. Altogether, these findings provide new insights into the molecular requirements for Tat binding to membranes during secretion.

Ex vivo Platforms to Study the Primary and Recall Immune Responses to Intracellular Mycobacterial Pathogens and Peptide-Based Vaccines.

Progress in the study of the immune response to pathogens and candidate vaccines has been impeded by limitations in the methods to study the functional activity of T-cell subsets proliferating in response to antigens processed and presented by antigen presenting cells (APC). As described in this review, during our studies of the bovine immune response to a candidate peptide-based vaccine and candidate rel deletion mutants in Mycobacterium avium paratuberculosis (Map) and Mycbacterium bovis (BCG), we developed methods to study the primary and recall CD4 and CD8 T-cell responses using an ex vivo platform. An assay was developed to study intracellular killing of bacteria mediated by CD8 T cells using quantitative PCR to distinguish live bacteria from dead bacteria in a mixed population of live and dead bacteria. Through use of these assays, we were able to demonstrate vaccination with live rel Map and BCG deletion mutants and a Map peptide-based vaccine elicit development of CD8 cytotoxic T cells with the ability to kill intracellular bacteria using the perforin-granzyme B pathway. We also demonstrated tri-directional signaling between CD4 and CD8 T cells and antigen-primed APC is essential for eliciting CD8 cytotoxic T cells. Herein, we describe development of the assays and review progress made through their use in the study of the immune response to mycobacterial pathogens and candidate vaccines. The methods obviate some of the major difficulties encountered in characterizing the cell-mediated immune response to pathogens and development of attenuated and peptide-based vaccines.

In This Issue

In This Issue

Early events in hepatitis B infection: the role of inoculum dose.

The relationship between the inoculum dose and the ability of the pathogen to invade the host is poorly understood. Experimental studies in non-human primates infected with different inoculum doses of hepatitis B virus have shown a non-monotonic relationship between dose magnitude and infection outcome, with high and low doses leading to 100% liver infection and intermediate doses leading to less than 0.1% liver infection, corresponding to CD4 T-cell priming. Since hepatitis B clearance is CD8 T-cell mediated, the question of whether the inoculum dose influences CD8 T-cell dynamics arises. To help answer this question, we developed a mathematical model of virus-host interaction following hepatitis B virus infection. Our model explains the experimental data well, and predicts that the inoculum dose affects both the timing of the CD8 T-cell expansion and the quality of its response, especially the non-cytotoxic function. We find that a low-dose challenge leads to slow CD8 T-cell expansion, weak non-cytotoxic functions, and virus persistence; high- and medium-dose challenges lead to fast CD8 T-cell expansion, strong cytotoxic and non-cytotoxic function, and virus clearance; while a super-low-dose challenge leads to delayed CD8 T-cell expansion, strong cytotoxic and non-cytotoxic function, and virus clearance. These results are useful for designing immune cell-based interventions.

Early innate and adaptive immune perturbations determine long-term severity of chronic virus and Mycobacterium tuberculosis coinfection

Chronic viral infections increase severity of Mycobacterium tuberculosis (Mtb) coinfection. Here, we examined how chronic viral infections alter the pulmonary microenvironment to foster coinfection and worsen disease severity. We developed a coordinated system of chronic virus and Mtb infection that induced central clinical manifestations of coinfection, including increased Mtb burden, extra-pulmonary dissemination, and heightened mortality. These disease states were not due to chronic virus-induced immunosuppression or exhaustion; rather, increased amounts of the cytokine TNFα initially arrested pulmonary Mtb growth, impeding dendritic cell mediated antigen transportation to the lymph node and subverting immune-surveillance, allowing bacterial sanctuary. The cryptic Mtb replication delayed CD4 Tcell priming, redirecting T helper (Th) 1 toward Th17 differentiation and increasing pulmonary neutrophilia, which diminished long-term survival. Temporally restoring CD4 Tcell induction overcame these diverse disease sequelae to enhance Mtb control. Thus, Mtb co-opts TNFα from the chronic inflammatory environment to subvert immune-surveillance, avert early immune function, and foster long-term coinfection.

IL4I1 Accelerates the Expansion of Effector CD8+ T Cells at the Expense of Memory Precursors by Increasing the Threshold of T-Cell Activation.

IL4I1 is an immunoregulatory enzyme that inhibits CD8 T-cell proliferation in vitro and in the tumoral context. Here, we dissected the effect of IL4I1 on CD8 T-cell priming by studying the differentiation of a transgenic CD8 T-cell clone and the endogenous repertoire in a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. Unexpectedly, we show that IL4I1 accelerates the expansion of functional effector CD8 T cells during the first several days after infection and increases the average affinity of the elicited repertoire, supporting more efficient LCMV clearance in WT mice than IL4I1-deficient mice. Conversely, IL4I1 restrains the differentiation of CD8 T-cells into long-lived memory precursors and favors the memory response to the most immunodominant peptides. IL4I1 expression does not affect the phenotype or antigen-presenting functions of dendritic cells (DCs), but directly reduces the stability of T-DC immune synapses in vitro, thus dampening T-cell activation. Overall, our results support a model in which IL4I1 increases the threshold of T-cell activation, indirectly promoting the priming of high-affinity clones while limiting memory T-cell differentiation.

Single-Cell Transcriptomics of Human TET2 Knockout CD4 T-Cells and Their Clonal Evolution

Angioimmunoblastic T-cell lymphoma (AITL), the most frequent subtype of peripheral T-cell lymphoma (PTCL), is a neoplasm with characteristics of mature T follicular helper (TFH) cells. We and others have identified frequent (~75%) inactivating mutations in the TET2 (Ten-Eleven Translocation-2) gene in AITL. TET2 belongs to a 3 member family of TET dioxygenases that catalyze DNA demethylation by oxidation of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl-cytosine (5-hmC) and further oxidative cytosine products. Thus, loss of function (LOF) of TET2 will cause aberrant genome hypermethylation and reduction in 5-hmC. Studies of the variant allele fraction (VAF) of TET2 mutants suggest that this mutation is a founding abnormality in AITL. However, how TET2 loss promotes the development of AITL is still unclear. To study LOF of TET2 in CD4 T-cell lymphomagenesis without the noise generated by other mutations in an established lymphoma, we generated a human TET2 knock-out (KO) CD4 T-cell model using CRISPR/Cas9 technology, which allows us to perform functional genomic studies by directly editing genes at their genomic loci. Whole transcriptome sequencing and single-cell transcriptome sequencing were used to study the cell evolution after KO. We generated multiple TET2 KO primary CD4 T-cell models using two different CRISPR/Cas9 methods. The first approach used the plasmid PX458-a, which expresses green fluorescent protein (GFP) fused Cas9 and guide RNA-a targeting TET2 exon 6, to electroporate CD4 T-cell from healthy donor F25. The second approach used homologous DNA repair (HDR) mediated knock-in (KI) of tandem GFP gene and a SV40 transcription stop signal to terminate TET2 expression at exon 3. Cas9/sgRNA-e RNP complex, along with a long DNA template (about 1.6 kb), was electroporated into CD4 T-cells from two healthy donors, F25 and M40. GFP-positive cells were sorted by FACS after electroporation and were considered to be edited cells. Edited CD4 T-cells were cultured in vitro with 50 U/ml IL-2, and stimulated regularly (every 7~10 days) with 1:1 ratio of anti-CD3/CD28 T activator beads. TET2 KO in these cells was confirmed by qRT-PCR, Sanger sequencing and Western blotting. Compared with wild-type (WT) CD4 T-cells under the same culture conditions, a lower level of 5-hmC in TET2 KO cells was observed, indicating successful editing of TET2. Compared to WT cells, KO cells had a higher growth rate, due to a lower apoptosis rate and a higher proliferation rate, by Annexin V staining, EdU staining, and MTS experiments. The growth of KO cells or WT cells was still dependent on IL-2 and T activator beads stimulation. All batches of KO cells, generated by different guide RNAs or from different donors, showed a much longer life span than WT cells, which usually lived for 3~4 months, but KO cells can keep proliferating longer than one year. We also performed TCR analysis on these cell samples. Both WT and KO cells demonstrated oligoclonality when examined at Day 40 (40D, early stage) and TET2 KO cells showed a dominant clone by Day 90 (90D, late stage). We performed single-cell transcriptome analysis on M40 KO vs. WT cells, at 40D and 90D. KO90D cells had a low TCR diversity with the dominant population representing ~88% of cells (TRAV9-2,TRBV5-1). From single-cell transcriptome analysis, cell clustering profiles were very distinctive in these 4 cell populations analyzed (Figure 1A) and these clusters had unique gene expression profiles (Figure 1B). Cluster 6 was prominent in KO90D but almost absent in WT90D, whereas the reverse was true for clusters 1 and 5. From pathway analysis, KO90D cells showed a higher expression of signatures associated with proliferation, cell cycle and chemokine signaling and lower histidine and tryptophan metabolism signatures. Sanger sequencing showed a 79 bp indel in addition to the GFP KI allele in KO90D cells, demonstrated the homozygous deletion of TET2 on these cells. Similar results were observed in F25 TET2 KO cells by plasmid PX458-a. This indicated the selection of homozygously deleted TET2 cells in long-term culture. However, clonal evolution is highly dynamic and a minor clone in KO40D cells may become the dominant clone in KO90D cells. Comparison of the 5-mC and 5-hmC profiles between KO and WT cells are being conducted to elucidate epigenetic alterations that are associated with the functional alterations and predisposition to AITL lymphomagenesis. Figure Disclosures No relevant conflicts of interest to declare.

IFN-γ treatment protocol for MHC-Ilo/PD-L1+ pancreatic tumor cells selectively restores their TAP-mediated presentation competence and CD8 T-cell priming potential

BackgroundMany cancer cells express a major histocompatibility complex class I low/ programmed cell death 1 ligand 1 positive (MHC-Ilo/PD-L1+) cell surface profile. For immunotherapy, there is, thus, an urgent need...

Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells

Regulatory Tcells maintain immunological tolerance and dampen inflammatory responses. Administering regulatory Tcells can prevent the immune-mediated tissue destruction of graft-versus-host disease, which frequently accompanies hematopoietic stem cell transfer. Neutralizing the Tcell-specific kinase, protein kinase C theta, which promotes Tcell effector functions and represses regulatory Tcell differentiation, augments regulatory Tcell immunosuppression and stability. We used a synthetic, cell-penetrating peptide mimic to deliver antibodies recognizing protein kinase C theta into primary human CD4 Tcells. When differentiated exvivo into induced regulatory Tcells, treated cells expressed elevated levels of the regulatory Tcell transcriptional regulator forkhead box P3, the surface-bound immune checkpoint receptor programmed death receptor-1, and pro-inflammatory interferon gamma, previously ascribed to a specific population of stable, highly suppressive human induced regulatory Tcells. The invitro suppressive capacity of these induced regulatory Tcells was 10-fold greater than that of Tcells differentiated without antibody delivery. When administered at the time of graft-versus-host disease induction, using a humanized mouse model, antibody-treated regulatory Tcells were superior to non-treated Tcells in attenuating lethal outcomes. This antibody delivery approach may overcome obstacles currently encountered using patient-derived regulatory Tcells as a cell-based therapy for immune modulation.

Antibody, but not B-cell-dependent antigen presentation, plays an essential role in preventing Chlamydia systemic dissemination in mice.

The obligate intracellular bacterium Chlamydia trachomatis causes the most prevalent bacterial sexually transmitted infection worldwide. CD4 T cells play a central role in the protective immunity against Chlamydia female reproductive tract (FRT) infection, while B cells are thought to be dispensable for resolution of primary Chlamydia infection in mouse models. We recently reported an unexpected requirement of B cells in local Chlamydia-specific CD4 T-cell priming and bacterial containment within the FRT. Here, we sought to tackle the precise effector function of B cells during Chlamydia primary infection. Using mixed bone marrow chimeras that lack B-cell-dependent Ag presentation (MHCIIB ) or devoid of circulating antibodies (AID-/- × μS-/- ), we show that Chlamydia-specific CD4 T-cell expansion does not rely on Ag presentation by B cells. Importantly, we demonstrate that antibody, but not B-cell-dependent Ag presentation, is required for preventing systemic bacterial dissemination following Chlamydia FRT infection.

Resolving Molecular Mechanisms of Autoimmune Disease In Primary CD4 T Cells

Numerous genomic loci have been implicated in autoimmune disorders, but attempts to identify causal variants, and thereby disease mechanisms, have been hampered by strong linkage disequilibrium; leaving most loci unresolved and the potential of GWAS largely unfulfilled. To overcome this, we developed a massively-parallel reporter assay for use in primary CD4 T-cells, a key effector of many autoimmune diseases, and sought to resolve potential causal variants via their functional effects. This provided testable hypotheses into disease mechanisms, which we illustrate using a multi-disease-associated locus on chromosome 6. By investigating the lead expression-modulating SNP, we uncover an NF-κB-driven regulatory circuit which constrains T-cell activation through the dynamic formation of a super-enhancer that upregulates expression of TNFAIP3 (A20), a key NF-κB inhibitor. In activated T-cells, this circuit is disrupted – and super-enhancer formation prevented – by the risk variant at the lead SNP, thus revealing a molecular mechanism that predisposes to multiple autoimmune diseases.

Abstract B035: Enhancing antitumoral activity of cell-based immunotherapies by modulating the JAK-STAT axis

Abstract Mounting a T helper 1 (Th1) type of response is required for the successful priming of antigen specific CD8 T-cells which ultimately lead to effective antitumoral responses. Several cellular components of the immune system participate of the Th1 decision-making process. They include innate cells sensing the transformed cells, mainly dendritic cells (DCs), and the further involvement of antigen specific CD4 T-cells promoting direct activation of CD8 T-cells through cross-presentation (1). The correct stimulation of DCs by type I interferons and molecular pattern sensors as STING is required to drive their differentiation to conventional DC 1 (cDC1) which through the secretion of large amounts of IL-12 and the expression of CD40 skew the CD4 T-cells differentiation to Th1 (2). However, this system is sensitive to modifications of the tumor cytokine microenvironmen,t leading to failure. Adoptive cell transfer (ACT) cancer immunotherapies are promising treatments for advanced malignancies. These therapies attempt to supply key cellular actors missing and induce proper antitumoral immunity. They include mainly the in vitro modification of autologous cells, from DCs loaded with tumor antigens to the expression of chimeric antigen receptor (CAR) on T and NK cells. Constant development of these technologies has given successful results as clinical trials have already showed up to 90 % of complete remission in relapsed/refractory B cell acute lymphoid leukemia (B-ALL) (3), treatment now approved for commercial use. However, there is still lack of consistency in the responses obtained from different individuals and in different trials. Previously our laboratory has already shown that modulation of the JAK-STAT inhibitory protein tyrosine phosphatases (PTPs), PTPN1 and PTP-N2, enhance proinflammatory type I interferon signalling while decreases the effects of immunosuppressive cytokines acting through STAT3 signaling (4, 5). Specifically, we have demonstrated that partial inhibition of these phosphatases in immune cells enhances the secretion of IL-12p70 by DCs[6] and increases the cytotoxic activity of antigen specific CD8 T-cells [Pérez-Quintero LA, Tremblay ML, unpublished results]. Moreover, the partial inhibition of these phosphatases in CD8 T-cells enhance the acquisition of a central memory (Tcm) phenotype, which has been found to be associated with better remission in CAR-T-cells therapies. Nevertheless, alternative methods to enrich this phenotype, as cytokine cocktails, have proven expensive and ineffective. Having this in mind, we propose here the use of small-molecule inhibitors specific for PTPN1 and PTPN2 as a simple and cost-effective method to enhance antitumoral responses. By treating ex vivo the cellular products in animal models for DCs and CD8 ACT therapies we show, as a proof of concept, a marked improvement on the reduction of tumor burden and remission, with the late goal of translating these findings into a clinical setup.

Molecular architecture and regulation of BCL10-MALT1 filaments

The CARD11-BCL10-MALT1 (CBM) complex triggers the adaptive immune response in lymphocytes and lymphoma cells. CARD11/CARMA1 acts as a molecular seed inducing BCL10 filaments, but the integration of MALT1 and the assembly of a functional CBM complex has remained elusive. Using cryo-EM we solved the helical structure of the BCL10-MALT1 filament. The structural model of the filament core solved at 4.9 Å resolution identified the interface between the N-terminal MALT1 DD and the BCL10 caspase recruitment domain. The C-terminal MALT1 Ig and paracaspase domains protrude from this core to orchestrate binding of mediators and substrates at the filament periphery. Mutagenesis studies support the importance of the identified BCL10-MALT1 interface for CBM complex assembly, MALT1 protease activation and NF-κB signaling in Jurkat and primary CD4 T-cells. Collectively, we present a model for the assembly and architecture of the CBM signaling complex and how it functions as a signaling hub in T-lymphocytes.

Deciphering mediator's function and structure in mammalian cells

Introduction/Objectives Mediator is a multi-subunit complex organized into four modules (head, middle, tail and kinase module). It is involved in several steps of PolII-dependent transcription and enhancer–promoter (E–P) interactions. Mutations in different subunits (SU) are associated with cancer and developmental diseases. Mediator's function and structure have been mainly studied in yeast. Earlier studies suggest significant differences with the mammalian complex in terms of composition and structure. Additionally, the chromatin architecture between both species is different, particularly E–P interactions. We aimed to comprehensively characterize the mammalian complex. Materials/Patients and methods We screened for cell essentiality of Mediator subunits, in a murine B-lymphoma cell line, CH12, in embryonic stem cells and in primary CD4T cells, using CRISPR-Cas9 technology. We generated knockout clones (KO) whenever possible in CH12 cells, and a degron-based conditional KO of the essential Mediator's backbone, Med14. We analyzed the impact on gene expression (RNAseq), on PolII and Mediator recruitment at promoters and enhancers (ChIPseq), on chromatin accessibility (ATACseq), on histone modifications (MNase ChIPseq), on chromatin architecture (in situ HiC) and on structure (cryo-EM). Results Depletion of non-essential SUs induces transcriptional deregulation of only a subset of genes, whereas depleting the essential Med14 induces global decrease in transcription associated with a decrease in PolII recruitment. Interestingly, the ablation of the whole tail module is viable. With this tailless model and the Med14-degron we do not observe an impact on chromatin architecture. Preliminary cryo-EM results show a conserved location of common SUs between yeast and mammalian cells. We are able to locate mammalian specific SUs and describe the interactions between the different SUs in the mammalian complex. Conclusions Our study provides new insights into mammalian Mediator structure and a comprehensive view on how Mediator and its tail module are involved in transcription and chromatin architecture. This data is essential for a better understanding of transcriptional regulation and Mediator related diseases.

Overcoming Resistance of Human Non-Hodgkin's Lymphoma to CD19-CAR CTL Therapy by Celecoxib and Histone Deacetylase Inhibitors.

Patients with B-cell non-Hodgkin’s lymphoma (B-NHL) who fail to respond to first-line treatment regimens or develop resistance, exhibit poor prognosis. This signifies the need to develop alternative treatment strategies. CD19-chimeric antigen receptor (CAR) T cell-redirected immunotherapy is an attractive and novel option, which has shown encouraging outcomes in phase I clinical trials of relapsed/refractory NHL. However, the underlying mechanisms of, and approaches to overcome, acquired anti-CD19CAR CD8+ T cells (CTL)-resistance in NHL remain elusive. CD19CAR transduced primary human CTLs kill CD19+ human NHLs in a CD19- and caspase-dependent manner, mainly via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway. To understand the dynamics of the development of resistance, we analyzed several anti-CD19CAR CTL-resistant NHL sublines (R-NHL) derived by serial exposure of sensitive parental lines to excessive numbers of anti-CD19CAR CTLs followed by a limiting dilution analysis. The R-NHLs retained surface CD19 expression and were efficiently recognized by CD19CAR CTLs. However, R-NHLs developed cross-resistance to CD19CAR transduced human primary CTLs and the Jurkat human T cell line, activated Jurkat, and lymphokine activated killer (LAK) cells, suggesting the acquisition of resistance is independent of CD19-loss and might be due to aberrant apoptotic machinery. We hypothesize that the R-NHL refractoriness to CD19CAR CTL killing could be partially rescued by small molecule sensitizers with apoptotic-gene regulatory effects. Chromatin modifiers and Celecoxib partially reversed the resistance of R-NHL cells to the cytotoxic effects of anti-CD19CAR CTLs and rhTRAIL. These in vitro results, though they require further examination, may provide a rational biological basis for combination treatment in the management of CD19CAR CTL-based therapy of NHL.