Intramural Research Program Research Articles

Dynamic chromatin accessibility licenses STAT5- and STAT6-dependent innate-like function of Th9 cells to promote allergic inflammation

Abstract Allergic diseases are a major global health issue, causing significant morbidity and mortality. Interleukin-9 (IL-9) producing T helper 9 (Th9) cells promote allergic inflammation, yet Th9 effector functions are incompletely understood because the heterogeneity and instability of Th9 cells makes them challenging to study. Although the significance and mechanisms of Th9 instability are unknown, T cell receptor (TCR) activation is hypothesized to play a key role. Conversely to this paradigm, we found that resting Th9 cells did not require TCR restimulation for IL-9 production, which was induced by STAT5- and STAT6-dependent paracrine cytokines. This mechanism was seen in circulating T cells from allergic patients and was restricted to recently activated cells. Analysis of IL-9 +T cells from allergic subjects revealed a transcriptional program that was enriched for activation pathways and unstable over time, with IL9 amongst the least stable genes. Prolonged resting of cells decreased the accessibility of STAT5-dependent IL9 enhancers, inactivating the locus. In vivo, Th9 cells induced airway inflammation via TCR-independent, STAT-dependent mechanisms. In allergic patients, Th9 expansion was associated with STAT activation and responsiveness to JAK inhibitors. These findings suggest that Th9 instability is a negative checkpoint on TCR-independent inflammation that breaks down in allergic disease, and that JAK inhibitors should be considered for allergic patients with a Th9 signature. Supported by the NIAID intramural research program (2018-2022), University of Pittsburgh start-up funds (2022-current), and the National Psoriasis Foundation (Robertson Fellowship, 2018-2021; Translational Grant, 2022-current)

CRISPR screening for cytotoxic granule degranulation pathway in CD8 T cell

Abstract Degranulation of perforin-containing cytotoxic granules is important for NK cells and CD8 cytotoxic T lymphocytes (CTLs) to kill infected cells or tumors. Defects in this pathway cause the primary form of hemophagocytic lymphohistiocytosis (HLH), which is known as familial HLH (FHL) syndrome. Most of reported FHL causative genes encode molecules that are involved in the intracellular trafficking of cytotoxic granules and their fusion to the plasma membrane, followed by the delivery of their contents into the target cell through immunological synapses. However, other components essential for cytotoxic granule degranulation for CTLs are not well understood. To identify indispensable molecules for cytotoxic granule degranulation, we established an efficient in vitro CD8 amplification and degranulation system to perform a CRISPR-screening. To test this system, we transduced OT-I CD8 cells with a gRNA library targeting PIP 3binding proteins, which are important for cell adhesion, subcellular trafficking and T cell activation. Cells were harvested at short (Day 8) and medium-timepoints (Day 15) to evaluate the frequency of gRNAs in the degranulated (CD107+) and non-degranulated fractions. Of the top 10 hits of genes whose deletion suppressed degranulation, five were already listed in the 2022 IUIS classification of inborn error of immunity. We will use individual knockouts to evaluate how these genes affect degranulation. To expand to genome-wide screening, we have also subcloned a full library of sgRNAs into our high efficiency retroviral vector. Thus, we established an efficient assay system to perform a CRISPR-screening for CD8 T cells and can detect genes affected by primary immunodeficiencies. This work was supported by the Intramural Research Program of NIAID, NIH.

Mucus sialylation determines intestinal host-commensal homeostasis

Abstract Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD. Supported by the Intramural Research Program of the NIAID, NIH, United States and by National Multiple Sclerosis Society Career Transition Award, United States (TA 3059-A-2 to C.W.).

Global inhibition of Ly-49/MHC-I interactions by anti-MHC-I augments innate and adaptive immunity against cancer metastasis.

Abstract We have identified an pan-anti-MHC-I mAb (M1/42), which blocks the interaction of NK cell inhibitory receptors (Ly-49 antigens) with MHC-I, but does not inhibit antigen presentation by MHC-I. Administration of M1/42 in vivo markedly activated IFNg-producing NK cells. NK cell-derived IFNg stimulated APC to produce IL-12/-15/-18 cytokine cascades that further drove the proliferation of NK cells and memory phenotype (MP) T cells. M1/42 treatments profoundly augmented innate and adaptive immunity against PD-1-sensitive and -resistant transplanted tumors. Given that NK cells are critical to control of cancer metastases, we evaluated the effects of M1/42 therapy in several metastatic cancer models. M1/42 treatments successfully constrained B16F10 lung metastasis by enhancing CD8 +T cell infiltration and inducing melanoma-specific MP CD8 +T cell expansion and activation. In contrast, treatment with conventional checkpoint inhibitors (anti-PD-1/PD-L1, anti-CTLA-4 and anti-NKG2A) had no therapeutic effect. scRNAseq analysis of B16F10 lung melanoma tumor infiltrating lymphocytes revealed that M1/42 treatment significantly enhanced the expression of multiple genes associated with NK cell proliferation and activation. M1/42 also enhanced Th1-type innate and adaptive immune responses, and greatly restricted liver and lung metastasis produced by pancreatic adenocarcinoma cell line via intrasplenic or intravenous injection. Thus, the global inhibition of Ly-49/MHC-I interactions by M1/42 results in activation of a potent innate and adaptive immune response that restrains the growth of both transplanted tumors and cancer metastasis produced by intrasplenic or intravenous injection of tumor cells. This work was supported by the Intramural Research Program of NIAID, NIH.

Targeted proteomics-driven computational modeling of the mouse macrophage toll-like receptor signaling pathway

Abstract The Toll-like receptor (TLR) signaling pathway is crucial for the initiation of effective immune responses. Computational modeling of biological pathways can test mechanistic hypotheses about how regulation is achieved and why it sometimes fails, causing disease (e.g., sepsis). In this investigation, mass spectrometry (LC-MS) and computational modeling are being integrated to develop a model of the TLR4 pathway. TLR4 pathway proteins within mouse macrophages were quantitated using targeted LC-MS and stable-isotope labeled internal (phospho)peptide standards (136 unmodified peptides and 29 phosphopeptides for 54 (phospho)proteins). AlphaFold-Multimer was used to predict protein complex structures, Rosetta was used to idealize and relax the structures, and Simulation of Diffusional Association (SDA) and TransComp were used to estimate protein-protein association rates. Simmune was used to perform rule-based pathway modeling, simulation, and training. LPS stimulation altered proteins known to be affected by TLR4 pathway activation (e.g., phosphorylated ERK1 increased from 30,000 to 250,000 copies per cell). Hundreds of protein-protein association rates were predicted. The (phospho)protein absolute abundance values and protein-protein association rates are being used as parameters for TLR4 pathway models. The parameter space is being explored to identify parameter sets that accurately reproduce the experimental data. Experimental and computational techniques are being integrated to generate a strongly data driven model of the TLR pathway. This work was supported by the Intramural Research Program of NIAID, NIH. This work was supported by the Intramural Research Program of NIAID, NIH.

Abstract B022: A non-conserved histidine residue on KRAS drives paralog selectivity of the KRAS G12D inhibitor MRTX1133

Abstract The discovery of small molecule inhibitors against mutant KRAS protein has been a breakthrough in targeted therapy. MRTX1133 is the first non-covalent inhibitor with selectivity for the mutant KRAS G12D protein. Using isogenic cell lines expressing single Ras allele to test the selectivity of this compound, we found that in addition to KRAS G12D, MRTX1133 shows significant activity against several other KRAS mutants as well as wildtype KRAS protein. In contrast, MRTX1133 exhibits no activity against both G12D and wildtype forms of HRAS and NRAS proteins. Functional analysis revealed that the selectivity of MRTX1133 towards KRAS is associated with its binding to histidine 95 on KRAS, a residue that is not conserved in HRAS and NRAS. Reciprocal mutation of amino acid 95 among the three Ras paralogs resulted in reciprocal change in their sensitivity towards MRTX1133. Thus, histidine 95 is an essential selectivity handle for MRTX1133 towards KRAS. This knowledge could aid the development of other KRAS-selective inhibitors. (Funding: this research was supported by the Intramural Research Program of the National Cancer Institute, NIH) Citation Format: Miles Keats, John Han, Yeon-Hwa Lee, Chih-Shia Lee, Ji Luo. A non-conserved histidine residue on KRAS drives paralog selectivity of the KRAS G12D inhibitor MRTX1133 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B022.

TCR/Chimeric Antigen Receptor (CAR) Cross-Antagonism to Fine-Tune CAR-T cell Immunotherapy

Abstract Chimeric antigen receptor (CAR) T cells, are created by extracting T cells from a cancer patient, engineering them to express a CAR targeting a tumor specific molecule, then reintroducing them back into the patient. A patient’s T cells contain their own endogenous T cell receptors (TCRs) however, which could potentially interact with the exogenous CAR inserted into the cell. In this study, we examine how TCR and CAR signals interact upon CAR-T activation. We show that weak TCR stimulation can reduce (antagonize) or increase overall CAR-T response, both in vitro and in vivo, across multiple tumor models, in both mouse and human T cells. We further show that the behavior of these TCR/CAR interactions can be manipulated by changing various characteristics of the TCR, CAR, and associated ligands. While this behavior is complex, we show that it can be described by a single mathematical model based on the adaptive kinetic proofreading scheme of ligand discrimination. We conclude by presenting potential applications for cancer immunotherapy. Intramural Research Program of the National Cancer Institute

Inhibition of Ly-49/LILRB-MHC-I interactions by anti-MHC-I augments innate and adaptive immunity.

Abstract Mouse NK cells recognize MHC-I antigens via stochastically expressed inhibitory Ly49 receptors. The loss of MHC-I expression on tumor cells (“missing self”) abrogates inhibitory signals, resulting in NK activation. We have identified an anti-mouse pan MHC-I mAb (M1/42), which blocks Ly49-MHC-I interactions but not TCR-MHC-I interactions. Administration of M1/42 in vivo markedly activated IFNg-producing NK cells that further drove the proliferation of NK cells and memory phenotype (MP) T cells and profoundly augmented adaptive immunity against viral infection and cancer metastasis. In contrast to mouse Ly-49 antigens, human killer cell inhibitory receptors bind to HLA at a site overlapping the TCR binding site and blocking of KIR binding to MHC-I would inhibit TCR recognition. However, two anti-pan-human MHC-I abs (DX17 and W6/32) markedly induced NK cell proliferation and IFNg production in cultures of human PBMC. DX17 potently blocked the interaction of leukocyte Ig-like inhibitory receptors LILRB1, B2, B3 and B5 with human MHC-I but had no effect on TCR-MHC-I interactions. The crystal structure of a DX17 Fab/MHC-I complex reveals that the footprint of DX17 overlaps the LILRB1 binding site on MHC-I. Administration of W6/32 Fab to humanized mice (PBMC reconstituted NSG or NK cell reconstituted NOG-IL-15) induced human NK and MP T cell proliferation. Marked reduction of tumor size was seen when NOG-hIL-15Tg mice were reconstituted with CD3 −cells and then treated with W6/32 Fab. DX17 Fc silenced mAb (DX17 LALAPG) activated human tumor infiltrating lymphocytes and monocytes. These results strongly suggest that inhibition of LILRB-MHC-I interactions by anti-MHC-I in humans may result in marked augmentation of anti-tumor immunity. This work was supported by the Intramural Research Program of NIAID, NIH.

GIMAP5 regulates a lymphocyte longevity assurance pathway

Abstract Elucidating the molecular mechanisms of immunodeficiency diseases is a powerful approach to discovering new immunoregulatory pathways in humans. One such previously unknown pathway is that of GIMAP5, an evolutionarily conserved GTPase expressed in T and NK lymphocytes, also known as the gene mutated in the Biobreeding diabetic rat and Sphinx lymphopenic mice. To better understand this disease association, we identified causative GIMAP5 mutations in 17 individuals of 9 kindreds with lymphopenia, liver disease, splenomegaly, thrombocytopenia, lymphadenopathy, lung infections, and lymphoma. As the mechanism of action, we now report that GIMAP5 controls the pathological accumulation of long chain ceramides. The ceramide synthase inhibitor, fumonisin B1, rescues GIMAP5-deficient T cells from ceramide overaccumulation and cell deterioration. Concordant with these findings, induction of ceramide accumulation and cell death by exogenous C6-ceramides in human T cell blasts was reduced by fumonisin B1 and GIMAP5 overexpression. Our work reveals the underlying long-chain ceramide mechanics of GIMAP5, opening new opportunities for further metabolomic studies of immunodeficiency diseases and targets for the development of therapeutics. This research was supported by NIAID by the Intramural Research Program of the NIH.

PI3Kδ shapes Th2 lineage restriction through coordination of IL-2 signaling, Foxo1 inactivation and epigenetic remodeling

Abstract Activated PI3K-delta syndrome (APDS) is a primary immunodeficiency caused by heterozygous activating mutations in PI3Kδ, resulting in recurrent infections as well as allergic diseases. Given that APDS patients show an inability to clear respiratory pathogens (Th1) and development of allergic diseases (Th2), we hypothesized that hyperactivated PI3Kδ (Pik3cd E1020K) may alter CD4 T cell differentiation. Using in vitro polarization of murine naïve CD4 T cells, we observed increased production of IFNγ from Pik3cd E1020Kmice under Th1 inducing conditions and marked increases in IL-4 and IL-13 production under Th2 conditions. However, Pik3cd E1020KTh2 cells also expressed high levels of Tbet and aberrantly produced IFNγ. Strikingly, this dysregulation was also observed in vivo; Pik3cd E1020KCD4 cells show pronounced alterations following house dust mite induced airway inflammation, with impaired Th2 and inappropriate polarization to Th1 responses as visualized by scRNAseq. We linked this unstable Th1 and Th2 differentiation in Pik3cd E1020KCD4 T cells in vitro to hyper-responsiveness to IL-2, associated with increased phosphorylation and inactivation of transcription factor Foxo1. CRISPR-mediated targeting of Foxo1 in naïve WT CD4 cells recapitulated the Pik3cd E1020Kphenotype. Transcriptomic analysis of Pik3cd E1020KCD4 T cells revealed differential expression of genes involved in histone methylation. Accordingly, Pik3cd E1020KCD4 exhibit altered chromatin modifications, including H3K27 and H3K4 trimethylation. Together these data suggest that balanced regulation of PI3Kδ plays a critical role in enforcing Th1 and Th2 lineage identity. This research was supported by the Intramural Research Program of NIAID, NIH

The 1000 mouse experiment: quantifying the variability of tumor response to CAR T cell therapy

Abstract Chimeric Antigen Receptor (CAR) T cell therapy is a type of cancer immunotherapy designed to target specific antigens on the surface of tumor cells using engineered T cells. While effective, there is often variability in patient responses and even within an experiment given the same experimental conditions. Analyzing this variability may help identify key modalities to successful CAR T therapy. We present a platform to explore this variability by quantifying the luminescence signal from in vivo tumor growth over time in a mouse model of human CAR T response. We developed a Python toolkit (https://github.com/soorajachar/radianceQuantifier) to automate labeling, cropping, quantification, and plotting of bioluminescent tumor image data. Applying this toolkit to digitize dozens of prior in vivo experiments conducted over several years allowed us to generate a dataset consisting of over 1000 mice. We then performed statistical analyses and mathematically modeled the time dynamics of tumor responses to CAR T cells in this dataset. We identified three distinct phases of tumor response: initial tumor growth, tumor decay due to killing by CAR T cells, and tumor relapse. These phases were fit to differential equations to extract the growth and decay rates of the tumor for each mouse. Upon fitting our model to the experimental data, we found that the growth rate of relapsed tumors was slower than the initial tumor growth rate within individual mice, suggesting a selection of edited tumor cells post CAR T cell therapy. Future work will leverage our quantitative framework to explore the alterations between relapsed tumor cells and those that are eradicated by CAR T cells. Supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute (Bethesda, MD)

Differential dengue antibody responses in recipients of a Zika DNA vaccine are governed by dengue immune history

Abstract Dengue virus (DENV 1–4) and Zika virus (ZIKV) are closely related flaviviruses that induce immunological cross-reactivity. The VRC5283 Zika virus wild type DNA vaccine is the first to complete a Phase 2b trial (NCT03110770). The objective of this study was to test if VRC5283 induced potent ZIKV-specific neutralizing antibodies (NAbs) while avoiding priming of cross-reactive antibodies that could potentially enhance dengue disease. Here, we measured NAbs to DENV1–4 and ZIKV in serum samples obtained from individuals living in dengue-endemic (Puerto Rico) and non-endemic areas (Texas) immunized with VRC5283 at weeks 0, 4, and 8. We performed plaque reduction neutralization tests (PRNT 50) with sera collected at weeks 0 and 16 after final vaccination. Sera were tested for ZIKV isolated in Brazil, and DENV1–4 isolated in Sri Lanka. Most individuals from dengue-endemic areas had NAbs >1:20 to DENV1–4 and ZIKV prior to vaccination, with the highest NAb titers to DENV2. VRC5283 boosted immunity across all flaviviruses in the dengue-endemic cohort, with an 8.47-fold increase in ZIKV, 2.76-fold increase in DENV1, 1.67-fold increase in DENV3 and 2.06-fold increase in DENV4 titers, but did not significantly boost DENV2 titers. Sera from non-endemic areas had no NAbs to DENV1–4 or ZIKV prior to vaccination, and a boost in NAbs following vaccination was observed only to ZIKV. Thus, VRC5283 only induces dengue cross-reactive NAbs in individuals with prior dengue immunity. Ongoing testing with binding assays will further evaluate whether vaccination induced beneficial or enhancing antibodies to DENV1–4. This work was supported by the Intramural Research Program of NIAID.

The role of MATRIN3 (MATR3) in innate immune response

Abstract MATR3, one of the most abundant inner nuclear matrix proteins, is implicated in amyotrophic lateral sclerosis (ALS) and distal myopathy. MATR3 has been reported to have both DNA- and RNA-binding abilities. However, it is not understood mechanistically how mutation of MATR3 leads to ALS pathogenesis. To understand its loss of function effects, we mutated MATR3 in the human HAP1 cell line using CRISPR/Cas9 technology. Using two different RNA sequencing methods, Oxford Nanopore Technologies and Illumina, we discovered the upregulation of many interferon-stimulated genes (ISGs) in MATR3-deficient HAP1 cells. Furthermore, we have validated these results by Nanostring technology. Next, we hypothesized that HAP1 cells may be responding to endogenous double-stranded RNAs. Interestingly, we observed similar amounts of dsRNA in both MATR3 knock-out (KO) and wildtype (WT) cells which suggests that indeed intracellular dsRNAs are present and could be stimulating HAP1 cells; however, it is not the case that MATR3 KO cells are stimulated because they are producing significantly more dsRNAs. Currently, we are investigating whether and which RNA sensors are activated in MATR3 KO cells. Also, we are investigating whether MATR3’s DNA- and/or RNA-binding abilities are responsible for the innate immune response. Accordingly, we plan to perform CUT & RUN and photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP) respectively, in HAP1 cells. Altogether, our results illustrate that MATR3 is a novel negative regulator of the innate immune response. This work was supported by the Intramural Research Program of NIAID, NIH. This work was supported by the Intramural Research Program of NIAID, NIH.

Homeostatic intestinal immunity is dominated by microbiota-independent cytotoxic Th1 cells

Abstract Barrier tissues such as the skin and gut form the first line of defense against pathogens and establish an immune dialogue with the resident microbiota. To that end, they harbor immune populations geared towards barrier function, tolerance, and repair. Of particular interest are the large numbers of T cells. Some are specific towards commensal microbes or recognize previously encountered pathogens. However, the origin, antigen specificity, and function of most barrier tissue-resident T cells remains elusive. We have identified a dominant CD4 T cell population in the small intestine lamina propria characterized by the expression of T-bet. Surprisingly, this population is present in both germ-free and conventionally reared (SPF) mice at homeostasis, and accounts for almost half of T cells at this site. Bulk and single-cell transcriptome analysis revealed that this population has cytotoxic potential. Furthermore, single-cell TCR sequencing showed parallel T cell repertoires in germ-free and SPF mice, suggesting shared antigen specificity. To explore the ontogeny of this population, we have identified clonally expanded TCRs and are generating low frequency retrogenic mice. Additionally, we have generated several T cell hybridomas from small intestine Th1 cells. Interestingly, preliminary results suggest that small intestinal Th1 TCRs exhibit some degree of self-reactivity, which may determine their development and differentiation in a foreign antigen-independent manner. Collectively, these studies will shed light on the function of a dominant intestinal T cell population and reveal novel insights into homeostatic tissue immune networks. This research was supported by the Intramural Research Program of NIAID, NIH.

Aberrant expansion of spontaneous splenic germinal centers induced by hallmark genetic lesions of aggressive lymphoma

Abstract Diffuse large B cell lymphoma (DLBCL) is an aggressive heterogeneous clinical entity that is thought to be derived from germinal center (GC) B cells or cells that have recently passed through the GC. Unique molecular vulnerabilities have been identified in the aggressive MCD/C5 genetic subclass of DLBCL. However, the pre-malignant cell-of-origin exhibiting MCD-like dependencies remains elusive. In this study, we examined animals carrying up to four hallmark genetic lesions found in MCD consisting of gain-of-function mutations in Myd88 and Cd79b, loss of Prdm1 and overexpression of BCL2. We discovered that expression of combinations of these alleles in vivo promoted a cell-intrinsic accumulation of B cells in spontaneous splenic GCs. Like MCD, these pre-malignant B cells were enriched for B-cell receptors (BCRs) with evidence of self-reactivity, showed a de novo dependence on Tlr9 and were more sensitive to inhibition of BTK. Mutant spontaneous splenic GC B cells (GCB) showed increased proliferation and IRF4 expression. Mice carrying all four genetic lesions showed a greater than 50-fold expansion of spontaneous splenic GCs exhibiting aberrant histologic features with a dark zone immunophenotype and went on to develop DLBCL in the spleen with age. Thus, by combining multiple hallmark genetic alterations associated with MCD, our study identifies aberrant spontaneous splenic GCB as a likely cell-of-origin for this aggressive genetic subtype of lymphoma. This research was supported by the Intramural Research Program of the NIH, CCR, NCI.

Mosquito saliva alters draining LN architecture and function

Abstract Current murine models of most arbovirus infections result in the development of viremia and antiviral adaptive immune responses. However, delivery of virus by injection via syringe does not fully encompass all aspects of the response that develops after viral delivery via mosquito bite. One reason for this is likely to be the lack of additional immunomodulatory responses specifically induced by mosquito saliva as previous studies have demonstrated profound proviral and immunosuppressive properties of saliva. While the adaptive antiviral response develops in the lymph node draining the site of infection, the impact of mosquito saliva on this critical lymphoid organ has not been extensively explored. Further, the lymphatic system acts both as centralized hubs for immune cell activation and as dissemination pathways to distal tissues, meaning that immunomodulation of the lymphatic system likely affects infection and immunity systemically. Therefore, we explored the effect of mosquito saliva on the structure and function of the draining lymph node in the presence of absence of the mosquito borne arbovirus Zika virus (ZIKV). Preliminary confocal microscopic analyses of lymph nodes after saliva injection into the skin revealed changes in nodal architecture and specific cellular populations. Future studies will explore the impact of saliva specifically on lymphatic endothelial cells within the lymph node. These studies should provide additional insight into the mechanisms of bite-induced immunomodulation in secondary lymphoid organs and the impact on the developing antiviral response. Supported by the Intramural Research Program of NIAID, NIH (H.D.H.)

Innate Immune Training Enhances the Reactivation of Latently Infected HIV-1 from Monocytic Cell lines

Abstract HIV virus can persist in a latent but activatable chronic state in resting CD4 +T-cells and macrophages that are more persistent, leading to the requirement of lifelong combination antiretroviral therapy (cART). The latency of HIV-1 in macrophage cells is associated with increased chromatin condensation induced by histone methylation at HIV-1 integration sites. Innate immune training of macrophages has been shown to relieve chromosome condensation through epigenetic rewiring. We hypothesized that training of macrophages could enhance the reactivation of HIV-1 by opening the chromatin to facilitate transcription in response to Latency Reversing Agents (LRAs). We used beta-glucan, Syk inhibitor and Rutaecarpine to train the THP89GFP cell line, which is an experimental model for latently infected HIV-1 monocytes. We found that these trained THP89GFP cells have higher transcription of HIV-1 specific genes in response to PMA stimulation. To check the epigenetic changes associated with training in the proviral HIV-1 DNA of THP89GFP, we performed a chromatin immunoprecipitation for the histone mark, H3K27Ac. Consistent with the higher induction of transcription of HIV-1 genes, we observed that multiple regions of the HIV-1 LTR had higher deposition of H3K27Ac in the trained macrophages. Our studies thus show that induction of trained innate immunity may be a viable approach to the reactivation of latently infected HIV-1. This finding supports the hypothesis that innate immune training stimuli could be developed as novel candidates for enhancing reactivation of latently infected HIV-1, which may facilitate elimination of macrophage reservoirs. This work was supported by the Intramural Research Program of NIAID, NIH This work was supported by the Intramural Research Program of NIAID, NIH and Office of AIDS research.

Dissecting the role of TGF-b in the skin

Abstract The skin is outermost barrier in the body. Its only breaches are represented by hair follicles, which represent the only point of entrance for all the skin-sitting pathogens and commensals. Therefore, they can be defined as the most immunologically active sites of the skin. As such, tight regulation is needed at these sites and it is achieved not only through immune cells, but also non-hematopoietic cells like keratinocytes and fibroblasts. Example of immune cells are regulatory T cells, which are not only activated by, but also produce large amounts of TGF-b, a multifunctional cytokine, present in the body in 3 isoforms (TGF-b1,2 and 3). These isoforms have been reported to be fundamental for skin development during organogenesis and additional findings report about their role in driving cell localization in the skin as well as involvement in tissue repair. Though, litte is known about their immunological role in the skin. Upon generating a working protocol to separate skin layers and isolate cells from them, we were able to start to define the cell composition of each layer both at steady state and during inflammation. Moreover, we could detect the three TGF-b isoforms in each layer at both gene and protein level further discriminating between hematopoietic and non-hematopoietic sources. Finally, the importance of TGF-b in the skin was confirmed by experiments on TGF-b receptor I depleted mice. For the future, we are planning on defining exactly which cells secrete and respond to TGF-band confirm these findings by generating conditional KO mice. Additionally, the role of TGF-b will be assessed in world-wide skin diseases. Intramural Research Program of NIDCR

PI3Kdelta coordinates transcriptional, epigenetic and metabolic changes to promote effector CD8 T cells differentiation.

Abstract Activated PI3K-delta syndrome (APDS) is a primary immunodeficiency caused by heterozygous activating mutations of PIK3CD, resulting in dysregulated immunity, recurrent infections and lymphoproliferation, yet underlying mechanisms behind these phenotypes remain unclear. Using patient samples and Pik3cd E1020K/+mouse model, we evaluated CD8 cell function both in vitro and in response to infectious agents, assessing cellular phenotypes, metabolism, gene expression and chromatin organization. Pik3cd E1020K/+CD8 cells exhibited accelerated differentiation to short-lived effectors, associated with increased mTORC1 and c-Myc pathways, as well as altered metabolic, transcriptional and epigenetic circuits characterized by a pronounced IL-2/STAT5 signature associated with heightened IL-2 responses that prevented IL-15 differentiation to memory-like cells. Moreover, Pik3cd E1020K/+CD8 cells failed to sustain expression of proteins critical for maintenance of long-lived memory cells, including TCF1, and mounted inadequate central memory responses in vivo with enhanced generation of long-lived effector populations. In response to chronic LCMV Clone 13 infection, Pik3cd E1020K/+mice rapidly die, yet the surviving mice recover more rapidly than WT counterparts. Pik3cd E1020K/+mice fail to sustain TCF1 +Ly108 +stem-like CD8 cells, with a simultaneous reduction in exhausted cells. However, Pik3cd E1020K/+mice also have an expansion of CX3CR1 +effector cells associated with increased viral clearance and increased immunopathology. Our data position PI3Kd as a central hub integrating multiple signaling nodes that promote an accelerated effector T cell program during both acute and chronic infections. This work was supported by the Intramural Research Program of NIAID and NHGRI, NIH.

Analyzing the immunological variability in T cell/tumor interactions to identify a subpopulation of CD8+ T cells (Spark T cells) that drives succesful cancer immunotherapy

Abstract Thousands of patients have benefited from the growing use of cancer immunotherapies. However, the success of these therapies can be highly variable. Strikingly, some murine tumor models show large variability in the outcome of cancer immunotherapies, even when the mice, tumor cells and anti-tumor immune cells injected into mice are all genetically identical. Here, we sought to analyze this variability in adoptive cell therapies, in order to identify the immune population driving this variability. To search for the occurrence of large variability ex vivo, we extracted mouse TCR-transgenic CD8+ T cells, and co-cultured them with antigen-expressing tumor cells. By using multiplexed in vitro assays and single-cell analysis of thousands of samples, we identified conditions (e.g. cell numbers, antigen quality, tumor cell types) where large variations in the immune activation against cancer cells is observed even between technical replicates. We then developed a quantitative framework that uses statistical modeling and machine learning to extract useful information from this immunotherapeutic variability. Our framework allowed the prediction and identification of a rare population of naïve CD8+ T cells (“Spark T cells”) that is necessary and sufficient to spark massive anti-tumor immune reactions. We are currently performing experiments to test the efficacy and functional significance of the identified immune population in vivo. We are also applying this framework in human TCR-engineered T cell blasts to identify the equivalent of the Spark T cells in humans. We envision this framework being applied to identify other relevant immune cell types that act as catalysts for successful cancer immunotherapies. Supported by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute