Antibody-mediated Depletion Of CD8 Research Articles

Immune cells promote paralytic disease in mice infected with enterovirus D68.

Enterovirus D68 (EV-D68) is associated with acute flaccid myelitis (AFM), a poliomyelitis-like illness causing paralysis in young children. However, mechanisms of paralysis are unclear, and antiviral therapies are lacking. To better understand EV-D68 disease, we inoculated newborn mice intracranially to assess viral tropism, virulence, and immune responses. Wild-type (WT) mice inoculated intracranially with a neurovirulent strain of EV-D68 showed infection of spinal cord neurons and developed paralysis. Spinal tissue from infected mice revealed increased levels of chemokines, inflammatory monocytes, macrophages, and T cells relative to controls, suggesting that immune cell infiltration influences pathogenesis. To define the contribution of cytokine-mediated immune cell recruitment to disease, we inoculated mice lacking CCR2, a receptor for several EV-D68-upregulated cytokines, or RAG1, which is required for lymphocyte maturation. WT, Ccr2 -/- , and Rag1 -/- mice had comparable viral titers in spinal tissue. However, Ccr2 -/- and Rag1 -/- mice had significantly less paralysis relative to WT mice. Consistent with impaired T cell recruitment to sites of infection in Ccr2 -/- and Rag1 -/- mice, antibody-mediated depletion of CD4 + or CD8 + T cells from WT mice diminished paralysis. These results indicate that immune cell recruitment to the spinal cord promotes EV-D68-associated paralysis and illuminate new targets for therapeutic intervention.

Abstract A039: Silencing MICAL2 expression in pancreatic cancer cells reduces IL-1A expression and inhibits tumor growth through a CD8+ T cell dependent mechanism

Abstract Introduction: Pancreatic cancer is characterized by desmoplastic, fibroinflammatory stroma. The crosstalk between cancer cells and their surrounding tumor microenvironment (TME) promotes disease progression, metastasis, and chemoresistance. We identified MICAL2 as a super-enhancer-associated gene in pancreatic cancer. MICAL2 (molecule interacting with Cas-L) proteins are flavin monooxygenases that promote actin depolymerization and indirectly regulate SRF transcription. In other work, we have found that MICAL2 promotes pancreatic cancer growth and progression. This study evaluated how MICAL2 pancreatic cancer cell expression modulates the PDAC tumor microenvironment. Methods: RNA-Seq analysis performed on human pancreas cancer cells (AsPC-1) before and after MICAL2 KD revealed differential regulation of IL-1α. We validated the expression of IL-1α in multiple human and mouse pancreas cancer cell lines by q-PCR. We next exposed human (h) and mouse (m) PSCs to conditioned media (CM) from cancer cells before and after MICAL2 KD and checked the expression of a-SMA, fibronectin, and Col1A and IL-6 genes by qPCR. KrasG12D/+; Trp53R172H/+; Pdx1-cre (KPC) cells with and without MICAL2 were orthotopically injected to assess the in vivo tumor growth and metastasis. Sc-RNA seq and Immunophenotyping were performed on Shcontrol (ShCNT) and MICAL2 (M2KD) KD tumors. CD8+ T cell depletion was done using an antibody-mediated approach. The adoptive transfer of CD8+ T cells extracted from MICAL2 KD tumors was done by tail vein injection. Results: MICAL2 KD resulted in the downregulation of the IL-1α gene in both human and mouse pancreas cancer cell lines (50% reduction, p<0.05). Human and mouse pancreatic stellate cells (hPSCs and mPSCs) co-cultured with cancer cells CM showed significant downregulation of a-SMA, fibronectin, Col1A, and IL-6 upon MICAL2 KD as compared to hPSCs and mPSCs co-cultured with ShCNT cells. Orthotopic injections of KPC MICAL2 KD cells led to decreased tumor growth as compared to ShCNT (0.26 gm vs. 1 gm, p = 0.008). Immunofluorescence (IF) revealed less α-SMA, PDGF-α, and IL-6 expression and reduced deposition of collagen and fibronectin in MICAL2 KD tumors. Single-cell RNA seq and flow cytometry studies of tumors harvested from both groups showed significantly increased infiltration of activated cytotoxic CD8+ T cells in MICAL2 KD tumors which was further confirmed by IF. Antibody-mediated depletion of CD8+ T cells in MICAL2 KD tumors abrogated their reduced tumor growth in both immune-hot and immune-cold KPC cell lines. Adoptive transfer of CD8+ T cells extracted from M2KD tumors significantly reduced ShCNT flank tumor growth. Conclusion: These data reveal that MICAL2 expression mediates tumor-stromal crosstalk through IL1A and creates an immunosuppressive environment in PDAC and suggests MICAL2 may be an attractive immunomodulatory target for pancreatic cancer therapy. Ongoing work is focused on dissecting the role of MICAL2 in priming the metastatic niche through the remodeling of the TME. Citation Format: Bharti Garg, EvanGeline Mose, Edgar Esparza, Jay Patel, Sarah Sass, Alexei Martsinkovskiy, Dawn Jacquish, Herve Tiriac, Andrew M. Lowy. Silencing MICAL2 expression in pancreatic cancer cells reduces IL-1A expression and inhibits tumor growth through a CD8+ T cell dependent mechanism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A039.

Abstract 6190: Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers

Abstract Cancers deficient in homologous recombination (HR) repair secondary to mutations in genes such as BRCA1 or BRCA2, are dependent on alternative DNA damage response (DDR) pathways to maintain genomic integrity, rendering them susceptible to synthetic lethal targeting of these pathways. Recently, inhibitors of polymerase theta (POLθ, encoded by POLQ), the critical enzyme in microhomology-mediated end-joining (MMEJ), have been shown to be synthetic lethal with HR repair deficiency (Zhou et al. Nature Cancer 2021). Both HR and MMEJ require nucleolytic DNA end-resection to allow for DSB repair, and we have previously shown that MMEJ acts as a barrier to DNA end-resection at DSBs (Patterson-Fortin et al. Cancer Research 2022). Given the synthetic lethality between HR and MMEJ leads to unrestrained DNA end-resection generating chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, we hypothesized that POLθ inhibition in HR-deficient cancers would activate the cGAS/STING innate immune response pathway and facilitate immunotherapy. To investigate the interactions of POLθ inhibition with the immune microenvironment in HR-deficient cancers, we used human cell lines and genetically modified mouse models representative of BRCA1-deficient triple-negative breast cancer (TNBC) and BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). Genetic or pharmacological inhibition of POLθ using novobiocin, a first-in-class inhibitor of the POLθ ATPase domain, induced significantly increased cytosolic dsDNA contained in micronuclei. This free DNA was sensed by the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS), increasing synthesis of cGAMP, in HR-deficient tumor cells but not in HR-proficient tumor cells. Increased cGAMP bound to and activated stimulator of interferon genes (STING), triggering phosphorylation of TBK1 and ultimately of IRF3. Activation of the cGAS/STING pathway by POLθ inhibition drove the expression of type I interferon response elements, including PD-L1. Depletion of STING by siRNA or by CRISPR abrogated this pro-inflammatory signaling and abolished the anti-tumor efficacy of novobiocin-mediated POLθ inhibition. Pharmacologic inhibition of POLθ enhanced Granzyme B+ CD8+ T-cell tumor infiltration. Importantly, antibody-mediated depletion of CD8+ T-cell severely compromised the anti-tumor efficacy of novobiocin-mediated POLθ inhibition, whereas anti-tumor activity of POLθ inhibition was augmented with the addition of either anti-PD-1 or anti-CTLA-4 antibodies. These results demonstrate that POLθ inhibition in HR-deficient cancers mediates a pro-inflammatory response in HR-deficient TNBC or PDAC tumor microenvironments, and that immune checkpoint blockade inhibition enhances the therapeutic efficacy of POLθ inhibition. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Constantia Pantelidou, Tin Phan, Carter Grochala, Anita K. Mehta, Jennifer L. Guerriero, Gerburg M. Wulf, Brian M. Wolpin, Ben Z. Stanger, Andrew J. Aguirre, James M. Cleary, Alan D. D'Andrea, Geoffrey I. Shapiro. Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6190.

Systemic immune responses to irradiated tumours via the transport of antigens to the tumour periphery by injected flagellate bacteria.

Because the tumour microenvironment is typically immunosuppressive, the release of tumour antigens mediated by radiotherapy or chemotherapy does not sufficiently activate immune responses. Here we show that, following radiotherapy, the intratumoural injection of a genetically attenuated strain of Salmonella coated with antigen-adsorbing cationic polymer nanoparticles caused the accumulation of tumour antigens at the tumour's periphery. This enhanced the crosstalk between the antigens and dendritic cells, and resulted in large increases in activated ovalbumin-specific dendritic cells in vitro and in systemic antitumour effects, and extended survival in multiple tumour models in mice, including a model of metastasis and recurrence. The antitumour effects were abrogated by the antibody-mediated depletion of CD8+ T cells, indicating that systemic tumour regression was caused by adaptive immune responses. Leveraging flagellate bacteria to transport tumour antigens to the periphery of tumours to potentiate the activation of dendritic cells may open up new strategies for in situ cancer vaccination.

IL-10 Receptor Deficiency Aggravates Exhaustion of CD8+ T-Cells and Impedes Their Control of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is associated with substantial alteration in T-cell composition and function. However, the role of T-cells in CLL remains largely controversial. While T-cells can induce pro-survival signals in leukemic cells, there are several indications for existing, yet defective, anti-tumor immune responses in CLL patients. Here, we utilized the Eµ-TCL1 (TCL1) mouse model of CLL to delineate whether anti-tumoral immune responses exist in CLL, and characterize the phenotype and function of these potential anti-tumoral immune populations.First, we adoptively transferred TCL1 leukemic cells (TCL1 AT) into Rag2-/- mice, which lack B- and T-cells, and observed that these mice had significantly shorter survival compared to wild type (WT) recipients. Antibody-mediated depletion of CD8+, but not CD4+, T-cells (Figure 1A), or neutralization of IFNγ after TCL1 AT resulted in a substantial increase in CLL cell numbers in blood, bone marrow and secondary lymphoid organs, indicating that CD8+ T-cells play a crucial role in suppressing CLL progression in an IFNγ-dependent manner.Thus, we further studied the composition of CD8+ T-cells after TCL1 AT, and thereby identified a population of CD127low CD44int-hi effector cells that progressively increase along with disease. These cells presented the highest levels of activation markers (CD69, CD137 and GITR), degranulation capacity and Granzyme B (GzmB) production. Moreover, this population was composed of oligoclonal T-cells, as shown by T-cell receptor sequencing, indicating an enrichment of tumor-reactive T-cells. Of interest, expression of the inhibitory receptor, PD-1, was strictly confined to this effector CD8+ population, and we detected two distinct PD-1+ subsets, PD-1int and PD-1hi, that exhibited considerable phenotypical and functional differences. PD-1hi cells showed higher levels of activation, yet with increased expression of exhaustion markers (Lag3 and CD244). In contrast, PD-1int cells possessed superior functional capacity, as evident by higher cytokine (IFNγ, IL-2 and TNFα) release, stronger degranulation capacity and increased GzmB production. Gene expression profiling (GEP) revealed that PD-1int cells resemble effector T-cells in acute infections, while the PD-1hi subset is more similar to exhausted T-cells in chronic infections. Accordingly, PD-1hi cells expressed high levels of transcription factors mediating terminal differentiation, such as Batf and Blimp1, while PD-1int cells expressed memory transcription factors, like Tcf7.As GEP data showed that PD-1int cells expressed higher levels of Il10rb, we studied the role of the immunoregulatory cytokine IL-10 in controlling the balance between PD-1int and PD-1hi subsets. Using IL-10-GFP (tiger) mice, we observed IL-10 production by multiple cell types upon TCL1 AT. To assess the role of IL-10 in CLL, we treated CLL-bearing mice with IL-10R-blocking antibodies, and unexpectedly detected higher CLL load in all analyzed organs in anti-IL-10R-treated mice. Although effector CD8+ T-cells exhibited higher activation levels after IL-10R blockade, they were severely skewed towards PD-1hi cells with an almost complete loss of the PD-1int subset. This was accompanied by higher expression of inhibitory receptors and a substantial decrease in GzmB release and cytokine production. Moreover, GEP revealed a dramatic decrease in cell cycle and proliferation genes in PD-1hi cells following IL-10R blockade, which was further confirmed by Ki67 staining. To examine whether these effects are T-cell intrinsic, we transferred CD8+ T-cells from WT or Il10rb-/- mice into Rag2-/- mice followed by TCL1 AT. While WT T-cells substantially decreased the rate of CLL progression, Il10rb-/- cells were significantly inferior in controlling CLL in these mice (Figure 1B). Moreover, Il10rb-/- CD8+ T-cells were mostly PD-1hi cells with an increase in exhaustion markers and substantial drop in cytokine and GzmB production.Collectively, this study describes a novel role of IL-10 in maintaining immune responses in CLL by protecting anti-tumoral effector CD8+ T-cells from activation-induced exhaustion. Furthermore, these data suggest IL-10 as a therapeutic target in CLL, potentially in combination with immune checkpoint inhibitors. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD8+ T Cells in Atherosclerosis.

Atherosclerotic lesions are populated by cells of the innate and adaptive immune system, including CD8+ T cells. The CD8+ T cell infiltrate has recently been characterized in mouse and human atherosclerosis and revealed activated, cytotoxic, and possibly dysfunctional and exhausted cell phenotypes. In mouse models of atherosclerosis, antibody-mediated depletion of CD8+ T cells ameliorates atherosclerosis. CD8+ T cells control monopoiesis and macrophage accumulation in early atherosclerosis. In addition, CD8+ T cells exert cytotoxic functions in atherosclerotic plaques and contribute to macrophage cell death and necrotic core formation. CD8+ T cell activation may be antigen-specific, and epitopes of atherosclerosis-relevant antigens may be targets of CD8+ T cells and their cytotoxic activity. CD8+ T cell functions are tightly controlled by costimulatory and coinhibitory immune checkpoints. Subsets of regulatory CD25+CD8+ T cells with immunosuppressive functions can inhibit atherosclerosis. Importantly, local cytotoxic CD8+ T cell responses may trigger endothelial damage and plaque erosion in acute coronary syndromes. Understanding the complex role of CD8+ T cells in atherosclerosis may pave the way for defining novel treatment approaches in atherosclerosis. In this review article, we discuss these aspects, highlighting the emerging and critical role of CD8+ T cells in atherosclerosis.

Combination of PD-1 Inhibitor and OX40 Agonist Induces Tumor Rejection and Immune Memory in Mouse Models of Pancreatic Cancer

Advanced pancreatic ductal adenocarcinoma (PDAC) is resistant to therapy, including immune checkpoint inhibitors. We evaluated the effects of a neutralizing antibody against programmed cell death 1 (PD-1) and an agonist of OX40 (provides a survival signal to activated T cells) in mice with pancreatic tumors. We performed studies in C57BL/6 mice (controls), KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice, and mice with orthotopic tumors grown from Panc02 cells, KrasG12D;P53flox/flox;PDX-1-Cre;Luciferase (KPC-Luc) cells, or mT4 cells. After tumors developed, mice were given injections of control antibody or anti-OX40 and/or anti-PD-1 antibody. Some mice were then given injections of antibodies against CD8, CD4, or NK1.1 to deplete immune cells, and IL4 or IL7RA to block cytokine signaling. Bioluminescence imaging was used to monitor tumor growth. Tumor tissues collected and single-cell suspensions were analyzed by time of flight mass spectrometry analysis. Mice that were tumor-free 100 days after implantation of orthotopic tumors were rechallenged with PDAC cells (KPC-Luc or mT4) and survival was measured. Median levels of PD-1 and OX40 mRNAs in PDACs were determined from The Cancer Genome Atlas and compared with patient survival times. In mice with orthotopic tumors, all those given control antibody or anti-PD-1 died within 50 days, whereas 43% of mice given anti-OX40 survived for 225 days; almost 100% of mice given the combination of anti-PD-1 and anti-OX40 survived for 225 days, and tumors were no longer detected. KPC mice given control antibody, anti-PD-1, or anti-OX40 had median survival times of 50 days or less, whereas mice given the combination of anti-PD-1 and anti-OX40 survived for a median 88 days. Mice with orthotopic tumors that were given the combination of anti-PD-1 and anti-OX40 and survived 100 days were rechallenged with a second tumor; those rechallenged with mT4 cells survived an additional median 70 days and those rechallenged with KPC-Luc cells survived long term, tumor free. The combination of anti-PD-1 and anti-OX40 did not slow tumor growth in mice with antibody-mediated depletion of CD4+ T cells. Mice with orthotopic tumors given the combination of anti-PD-1 and anti-OX40 that survived after complete tumor rejection were rechallenged with KPC-Luc cells; those with depletion of CD4+ T cells before the rechallenge had uncontrolled tumor growth. Furthermore, KPC orthotopic tumors from mice given the combination contained an increased number of CD4+ T cells that expressed CD127 compared with mice given control antibody. The combination of agents reduced the proportion of T-regulatory and exhausted T cells and decreased T-cell expression of GATA3; tumor size was negatively associated with numbers of infiltrating CD4+ T cells, CD4+CD127+ T cells, and CD8+CD127+ T cells, and positively associated with numbers of CD4+PD-1+ T cells, CD4+CD25+ T cells, and CD8+PD-1+ T cells. PDACs with high levels of OX40 and low levels of PD-1 were associated with longer survival times of patients. Pancreatic tumors appear to evade the immune response by inducing development of immune-suppressive T cells. In mice, the combination of anti-PD-1 inhibitory and anti-OX40 agonist antibodies reduces the proportion of T-regulatory and exhausted T cells in pancreatic tumors and increases numbers of memory CD4+ and CD8+ T cells, eradicating all detectable tumor. This information can be used in development of immune-based combination therapies for PDAC.

Interferon regulatory factor 1 priming of tumour-derived exosomes enhances the antitumour immune response

Background:Tumour-derived exosomes (TEXs) have a potential for application in cancer vaccines. Whether TEXs after induction by interferon regulatory factor 1 (IRF-1) are capable of enhancing the antitumour response remains to be determined.Methods:Exosomes released by tumour cells infected with IRF-1-expressing adenovirus (IRF-1-Exo) or treated with interferon-γ (IFN-Exo) were isolated via ultracentrifugation. The IRF-1 target proteins IL-15Rα and MHC class I (MHC-I) were analysed by western blot. Exosomes along with CpG adjuvant were injected into tumour models to assess the antitumour effects. Tumours were harvested for immunofluorescence staining. Splenocytes from tumour-bearing mice were co-cultured with tumour cells. The IFNγ-positive and granzyme B-positive CD8α+ splenocyte cells were quantified by flow cytometry.Results:The IRF-1-Exo or IFN-Exo displayed increased IL-15Rα and MHC-I expression. Injection of IRF-1-Exo or IFN-Exo combined with CpG had improved antitumour effects in mice. This effect may be a result of increased infiltration of tumours by CD4+ and CD8α+ T cells. Antibody-mediated depletion of CD4+ or CD8+ T cells abrogated the antitumour effects. Splenocytes isolated from CpG+IRF-1-Exo-injected Hepa 1–6 tumour mice had increased IFNγ-positive and granzyme B-positive CD8+ cells after co-culturing with Hepa 1–6 cells as compared with MC38 cells.Conclusions:The IRF-1 priming of TEXs enhances antitumour immune response.

Abstract 1634: CDK4/6 inhibition directly enhances an anti-tumor immune response in breast cancer

Abstract The cyclin D:cyclin-dependent kinases 4 and 6 (CDK4/6) axis is one of the most frequently dysregulated pathways in human cancers, and CDK4/6 inhibitors have shown significant activity against a number of solid tumors, including breast cancer. Analogous to clinical experience, we found that the CDK4/6 inhibitor, abemaciclib, caused significant tumor regression in the MMTV-rtTA/tetO-HER2 mouse model of luminal breast cancer. However, as CDK4/6 inhibitors are known to block tumor cell proliferation, but not directly induce tumor cell apoptosis, as we confirmed in our study, the mechanisms by which CDK4/6 inhibition caused tumor regression were not clear. Notably, abemaciclib therapy increased total CD3+ T cells in these tumors, while decreasing the immunosuppressive CD4+ regulatory T cell population. Further investigation revealed that CDK4/6 inhibition directly suppresses regulatory T cells by inhibiting their proliferation without impacting natural regulatory T cell formation in the thymus, the differentiation of inducible regulatory T cells, or regulatory T cell apoptosis. We are currently exploring potential effects of CDK4/6 inhibition on regulatory T cell function and lineage stability. Analysis of CD8+ and CD4+ T cells in abemaciclib-treated tumors revealed a marked reduction in the expression of the inhibitory immune checkpoint receptors PD-1, Tim-3, CTLA-4, and LAG3. Specifically, the fraction of CD8+ T cells expressing known markers of T cell exhaustion (PD-1/Tim-3 double-positivity or high expression of PD-1) was decreased in abemaciclib-treated tumors. Importantly, antibody-mediated depletion of CD8+ T cells established that response of MMTV-rtTA/tetO-HER2 tumors to abemaciclib is dependent on CD8+ T cells. Finally, given the observed immunomodulatory effects of abemaciclib, we sought to determine if abemaciclib treatment would sensitize MMTV-rtTA/tetO-HER2 tumors to immune checkpoint blockade. Pretreatment with abemaciclib followed by combination abemaciclib and α-PDL1 significantly enhanced tumor regression compared to abemaciclib or α-PDL1 alone. Our studies reveal a novel mechanism by which CDK4/6 inhibitors directly elicit an anti-tumor immune response. These results provide strong rationale for further investigations into combining CDK4/6 inhibitors with immune checkpoint blockade in breast cancer. Citation Format: Molly J. DeCristo*, Shom Goel*, April C. Watt, Haley BrinJones, Jaclyn Sceneay, Ben Li, Jessalyn M. Ubellacker, Shaozhen Xie, Susanne Ramm, Hye-Jung Kim, Sandra S. McAllister, Jean J. Zhao. CDK4/6 inhibition directly enhances an anti-tumor immune response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1634. doi:10.1158/1538-7445.AM2017-1634

X-Box Binding Protein-1 Dependent Plasma Cell Responses Limit the Development of Atherosclerosis.

Diverse B cell responses and functions may be involved in atherosclerosis. Protective antibody responses, such as those against oxidized lipid epitopes, are thought to mainly derive from T cell-independent innate B cell subsets. In contrast, both pathogenic and protective roles have been associated with T cell-dependent antibodies, and their importance in both humans and mouse models is still unclear. To specifically target antibody production by plasma cells and determine the impact on atherosclerotic plaque development in mice with and without CD4+ T cells. We combined a model of specific antibody deficiency, B cell-specific CD79a-Cre x XBP1 (X-box binding protein-1) floxed mice (XBP1-conditional knockout), with antibody-mediated depletion of CD4+ T cells. Ldlr knockout mice transplanted with XBP1-conditional knockout (or wild-type control littermate) bone marrow were fed western diet for 8 weeks with or without anti-CD4 depletion. All groups had similar levels of serum cholesterol. In Ldlr/XBP1-conditional knockout mice, serum levels of IgG, IgE, and IgM were significantly attenuated, and local antibody deposition in atherosclerotic plaque was absent. Antibody deficiency significantly accelerated atherosclerosis at both the aortic root and aortic arch. T cell and monocyte responses were not modulated, but necrotic core size was greater, even when adjusting for plaque size, and collagen deposition significantly lower. Anti-CD4 depletion in Ldlr/wild-type mice led to a decrease of serum IgG1 and IgG2c but not IgG3, as well as decreased IgM, associated with increased atherosclerosis and necrotic cores, and a decrease in plaque collagen. The combination of antibody deficiency and anti-CD4 depletion has no additive effects on aortic root atherosclerosis. The endogenous T cell-dependent humoral response can be protective. This has important implications for novel vaccine strategies for atherosclerosis and in understanding the impacts of immunotherapies used in patients at high risk for cardiovascular disease.

Dual Role of the Adaptive Immune System in Liver Injury and Hepatocellular Carcinoma Development

Hepatocellular carcinoma (HCC) represents a classic example of inflammation-linked cancer. To characterize the role of the immune system in hepatic injury and tumor development, we comparatively studied the extent of liver disease and hepatocarcinogenesis in immunocompromised versus immunocompetent Fah-deficient mice. Strikingly, chronic liver injury and tumor development were markedly suppressed in alymphoid Fah(-/-) mice despite an overall increased mortality. Mechanistically, we show that CD8(+) Tcells and lymphotoxin βare central mediators of HCC formation. Antibody-mediated depletion of CD8(+) Tcells as well as pharmacological inhibition of the lymphotoxin-β receptor markedly delays tumor development in mice with chronic liver injury. Thus, our study unveils distinct functions of the immune system, which are required for liver regeneration, survival, and hepatocarcinogenesis.

CD8 T cells mediate polyomavirus-associated CNS demyelination

Abstract JC polyomavirus (JCV), a common member of the virome in healthy humans, can cause the frequently fatal brain demyelinating disease Progressive Multifocal Leukoencephalopathy (PML) in immunocompromised individuals. PML, a pre-HAART AIDS-defining disease, has reemerged and is increasing in incidence in patients receiving immunodulatory therapies for autoimmune and inflammatory disorders. Lack of an animal model has hampered understanding of PML pathogenesis. We have developed a novel mouse model using intracerebral (i.c.) inoculation of mouse polyomavirus (MPyV), which mimics JCV-associated demyelination and shows similar histological features to PML. By one month after MPyV inoculation, C57BL/6 mice develop diffuse subcortical white matter demyelination, predominantly in the corpus callosum. Diffusion tensor magnetic resonance imaging in infected mice reveals severe peri-ventricular edema and a loss of structural integrity predominately in the corpus callosum and caudate. Virus-specific CD8 T cells robustly infiltrate the CNS and are stably maintained after CNS entry. Antibody-mediated depletion of CD4 and CD8 T cells prior to infection abrogates demyelination in the corpus callosum, despite resulting in a 50–100 fold increase in virus levels. Additionally, CD8 T cell-depletion alone was sufficient to cause higher virus levels in the brain. MPyV was controlled in the absence of CD4 T cells; however, MPyV-specific CD8 T cells in CD4 T cell-deficient mice expressed lower levels of CD103 (aE integrin), which binds epithelial E-cadherin. These data demonstrate that CD8 T cells infiltrating the CNS of MPyV-infected mice play a major role in this demyelinating leukoencephalitis.

Intestinal inhibition of Atg7 prevents tumour initiation through a microbiome-influenced immune response and suppresses tumour growth.

Here, we show that autophagy is activated in the intestinal epithelium in murine and human colorectal cancer and that the conditional inactivation of Atg7 in intestinal epithelial cells inhibits the formation of pre-cancerous lesions in Apc(+/-) mice by enhancing anti-tumour responses. The antibody-mediated depletion of CD8(+) T cells showed that these cells are essential for the anti-tumoral responses mediated by the inhibition of autophagy. We show that Atg7 deficiency leads to intestinal dysbiosis and that the microbiota is required for anticancer responses. In addition, Atg7 deficiency resulted in a stress response accompanied by metabolic defects, AMPK activation and p53-mediated cell-cycle arrest in tumour cells but not in normal tissue. This study reveals that the inhibition of autophagy within the epithelium may prevent the development and progression of colorectal cancer in genetically predisposed patients.

Promotion of a subdominant CD8 T cell response during murine gammaherpesvirus 68 infection in the absence of CD4 T cell help.

CD8 and CD4 T cells are each critically important for immune control of murine gammaherpesvirus 68 (γHV68) infection. In immunocompetent mice, acute γHV68 infection results in lifelong latency, but in the absence of CD4 T cell help, mice succumb to viral recrudescence and disease. However, the requirements for CD4 T cell help in the generation and maintenance of antiviral CD8 T cell responses are incompletely understood, and it is unclear whether there are epitope-specific differences in the requirement of CD8 T cells for CD4 help. In this report, we characterized the CD8 T cell response to γHV68 in major histocompatibility complex (MHC) class II(-/-) mice, which lack CD4 T cells, or after antibody-mediated depletion of CD4 T cells. All antiviral CD8 T cells exhibited marked upregulation of surface expression of the inhibitory receptor programmed death-1 (PD-1), but surprisingly, while the immunodominant memory response appeared to be functionally impaired, helpless CD8 T cells of a subdominant specificity had increased numbers and enhanced functionality. Thus, we demonstrate differential requirements for CD4 help in the antiviral CD8 T cell response to a latent gammaherpesvirus. Importance: γHV68 is a mouse pathogen closely related to the oncogenic human γHVs, which infect a majority of the world's population. Reactivation of these viruses from latency can lead to complications, disease, and even death. CD4 T cells are required for complete immune control of long-term infection, in part by providing key signals to dendritic cells that in turn instruct optimal antiviral CD8 T cell responses. We have investigated multiple virus-specific CD8 T cell responses during infection and identified a subdominant CD8 T cell response that is numerically and functionally enhanced in the absence of CD4 T cell help. This occurs in spite of high surface expression of an inhibitory receptor and in contrast to the immunodominant response, which is impaired. Our data suggest that signals from CD4 T cells are important in maintaining the CD8 T cell hierarchy during γHV infections.

Myeloid Cell COX-2 deletion reduces mammary tumor growth through enhanced cytotoxic T-lymphocyte function

Cyclooxygenase-2 (COX-2) expression is associated with poor prognosis across a range of human cancers, including breast cancer. The contribution of tumor cell-derived COX-2 to tumorigenesis has been examined in numerous studies; however, the role of stromal-derived COX-2 is ill-defined. Here, we examined how COX-2 in myeloid cells, an immune cell subset that includes macrophages, influences mammary tumor progression. In mice engineered to selectively lack myeloid cell COX-2 [myeloid-COX-2 knockout (KO) mice], spontaneous neu oncogene-induced tumor onset was delayed, tumor burden reduced, and tumor growth slowed compared with wild-type (WT). Similarly, growth of neu-transformed mammary tumor cells as orthotopic tumors in immune competent syngeneic myeloid-COX-2 KO host mice was reduced compared with WT. By flow cytometric analysis, orthotopic myeloid-COX-2 KO tumors had lower tumor-associated macrophage (TAM) infiltration consistent with impaired colony stimulating factor-1-dependent chemotaxis by COX-2 deficient macrophages in vitro. Further, in both spontaneous and orthotopic tumors, COX-2-deficient TAM displayed lower immunosuppressive M2 markers and this was coincident with less suppression of CD8(+) cytotoxic T lymphocytes (CTLs) in myeloid-COX-2 KO tumors. These studies suggest that reduced tumor growth in myeloid-COX-2 KO mice resulted from disruption of M2-like TAM function, thereby enhancing T-cell survival and immune surveillance. Antibody-mediated depletion of CD8(+), but not CD4(+) cells, restored tumor growth in myeloid-COX-2 KO to WT levels, indicating that CD8(+) CTLs are dominant antitumor effectors in myeloid-COX-2 KO mice. Our studies suggest that inhibition of myeloid cell COX-2 can potentiate CTL-mediated tumor cytotoxicity and may provide a novel therapeutic approach in breast cancer therapy.

CD4+ helper T cells and immunity to therapeutic proteins in gene therapy (P4431)

Abstract Adeno-associated (AAV) vectors are being assessed for gene replacement therapy in humans. Proteins encoded by the vector transgene are potentially foreign. Strategies to temper destructive immune responses may be required to achieve a therapeutic effect, particularly in diseases like Duchenne Muscular Dystrophy where the defective gene to be replaced has a large frame-shifting deletion. Here we studied the impact of immunity on expression of enhanced green fluorescent protein (eGFP) in AAV-transduced skeletal muscle of rhesus macaques. Antibodies to eGFP developed rapidly after AAV vector delivery to the tibialis anterior muscle. Cellular immunity was variable. In some animals, strong CD4+ helper and CD8+ cytotoxic T cells were detected in blood 2 weeks after vector administration. In others, low frequency responses were not detected until week 5-6. Regardless of the pace of the response, eGFP was cleared or substantially diminished in muscle after week 6. Antibody-mediated depletion of CD4+ T cells before AAV vector treatment delayed antibody and CD8+ T cell immunity and facilitated robust expression of eGFP persisted through day 42. Some positive muscle fibers were still visible at month 10, an unexpected finding that suggested an inability to sustain destructive cellular immune responses initially primed without CD4+ T cell help. The capacity for recall of eGFP-CD8+ T cell responses upon administration of a serologically distinct AAV vector is now being assessed.

CD4 T Cell Depletion Exacerbates AcuteMycobacterium tuberculosisWhile Reactivation of Latent Infection Is Dependent on Severity of Tissue Depletion in Cynomolgus Macaques

CD4 T cells are believed to be important in protection against Mycobacterium tuberculosis, but the relative contribution to control of initial or latent infection is not known. Antibody-mediated depletion of CD4 T cells in M. tuberculosis-infected cynomolgus macaques was used to study the role of CD4 T cells during acute and latent infection. Anti-CD4 antibody severely reduced levels of CD4 T cells in blood, airways, and lymph nodes. Increased pathology and bacterial burden were observed in CD4-depleted monkeys during the first 8 weeks of infection compared to controls. CD4-depleted monkeys had greater interferon (IFN)-γ expression and altered expression of CD8 T cell activation markers. During latent infection, CD4 depletion resulted in clinical reactivation in only three of six monkeys. Reactivation was associated with lower CD4 T cells in the hilar lymph nodes. During both acute and latent infection, CD4 depletion was associated with reduced percentages of CXCR3(+) expressing CD8 T cells, reported to be involved in T cell recruitment, regulatory function, and effector and memory T cell maturation. CXCR3(+) CD8 T cells from hilar lymph nodes had more mycobacteria-specific cytokine expression and greater coexpression of multiple cytokines compared to CXCR3(-) CD8 T cells. CD4 T cells are required for protection against acute infection but reactivation from latent infection is dependent on the severity of depletion in the draining lymph nodes. CD4 depletion influences CD8 T cell function. This study has important implications for human HIV-M. tuberculosis coinfection.

The role of CD8+ T cells in a model of multiple sclerosis induced with recombinant myelin oligodendrocyte glycoprotein

Background and objectives: Since CD8+ T cells may be important in the pathogenesis of multiple sclerosis (MS), we examined their role in the DA rat experimental autoimmune encephalomyelitis (EAE) model induced by immunization with recombinant myelin oligodendrocyte glycoprotein (rMOG). Methods: The inflammatory infiltrate in the spinal cord of affected animals was assessed by histology, electrophysiology and flow cytometry during the course of the disease (the first peak, remission and the second peak). The proportions of activated/memory effector (CD8+CD44+) and putative suppressor (CD8+CD28-, CD8+CD25high) CD8+ T cells in the draining lymph nodes were determined. To explore the role of CD8+ T cells, similar experiments were performed in CD8+ T cell depleted rats, before, during and after the first peak of the disease. Results: Throughout the disease, both CD4+ T cells and macrophages/activated microglia outnumbered CD8+ T cells within the spinal cord. The number of putative suppressor CD8+ T cells increased significantly both during and after the first peak suggesting the induction of a regulatory CD8+ T-cell response. However, antibody-mediated depletion of CD8+ T cells before induction of the disease, or after the first peak, did not significantly alter the incidence, severity or course of rMOG-induced EAE. Conclusions: The findings suggest that CD8+ T cells do not play a significant role in the pathogenesis or regulation of EAE induced by rMOG in DA rats. In this respect, rMOG-induced EAE is not an appropriate model for studying the role of CD8+ T cells in MS.

A new dynamic model of three cell interactions by CD4+ T-licensed DCs and DC-primed CD4+ T cells (100.38)

Abstract CD8 cytotoxic T lymphocytes (CTLs) kill pathogen-infected cells. Recent in vivo imaging study suggested that presence of DC-CD4 T clusters during CTL priming are essential for T-cell memory, challenging independent roles for the DCs or the CD4 T-cells that dissociate following DC-CD4 T interactions. We have developed a novel in vivo experimental system based on the rapid inducible ablation of diphtheria toxin receptor (DTR) transgenic-DCs and antibody-mediated depletion of CD4 and CD8+ T cells in mice to create DC-CD4 T interactions in vivo first, followed by killing of either CD4 T-licensed DCs or DC-primed CD4 T cells. Later, adoptively transferring naive polyclonal CD8 T cells facilitated their interactions with either licensed-DCs or primed-CD4 T cells, thereby revealing their respective roles in CTL immunity. Our results provide direct in vivo evidence that CD4 T-cells and DCs, once activated by DC-CD4 T interactions, can stimulate CTL responses and programme memory on CTLs independent of DC-CD4 T clusters, uncovering the co-existence of direct and indirect mechanisms of T-cell help for CTLs by licensed DCs and primed CD4 T cells in non-inflammatory situations. In situations in which CTL immunity is heavily dependent on CD4 T-cell help, our findings suggest a new dynamic model of three cell interactions in CTL responses, that dissociated stage of primed CD4 T-cells and licensed DCs represent crucial “immune-intermediates” for CTL responses and memory development.

Inhibition of T Cells Provides Protection against Early Invasive Pneumococcal Disease

Infections caused by Streptococcus pneumoniae are major causes of morbidity and mortality, which are in part mediated by immune cell-dependent mechanisms. Yet, the specific contributions of individual cell types to immunopathology are only partially understood. T cells are well characterized with respect to their function in protective humoral immune responses; however, their roles during early stages of infection and invasive pneumococcal disease (IPD) are less well defined. Using a mouse model of pneumococcal sepsis, we found that CD4(+) T cells were recruited to the lung as early as 12 h after intranasal infection. Recruitment was accompanied by upregulation of CD69 and B7-H1, reflecting T-cell activation. Unexpectedly, major histocompatibility complex (MHC) class II-deficient mice, which lack CD4(+) T cells, displayed an increased survival despite comparable bacterial titers in the blood, spleen, and lung. The higher survival correlated with a lower cytokine and chemokine response upon S. pneumoniae challenge in MHC class II-deficient mice, suggesting that inflammation may contribute to the mortality of IPD. Comparable to the case for MHC class II-deficient mice, antibody-mediated depletion of CD4(+) T cells and drug-induced inhibition of T-cell function with cyclosporine, or interference with T-cell activation using CTLA4-immunoglobulin (Abatacept), led to significant increases in survival during IPD. Our results reveal an important and adverse role of CD4(+) T cells in the pathogenesis of IPD and suggest that modulation of T-cell activation during early phases of S. pneumoniae invasive infection may provide a therapeutic option.