Model Of Immune Response Research Articles

Abstract 655: Necroptosis promotes tumor growth through IL1-alpha release and recruitment of immunosuppressive myeloid cells

Abstract Programmed cell death (PCD) was originally limited to apoptosis, but now includes pyroptosis, ferroptosis, necroptosis, and paraptosis. Necroptosis has attracted particular interest as an approach for immunotherapy as activation of the RIPK1/RIPK3 necrosome phosphorylates MLKL to form membrane pores, leading to loss of integrity and enhanced ability to induce an antigen-specific immune response in models of vaccination. Here we sought to determine if necroptosis within tumor cells was an important regulatory of anti-tumor immunity. Deficiency in Ripk3 or Mlkl did not impact tumor incidence or growth in genetic models of mammary carcinoma or lung adenocarcinoma. Similarly, the growth of syngeneic cell lines was not altered by loss of Ripk3 or Mlkl in vitro or in vivo. We next examined the induction of apoptosis or necroptosis in syngeneic tumor models using genetic constructs with doxycycline-controlled expression. In line with published data, subcutaneous vaccination demonstrated that RIPK3-induced necroptosis promoted an antigen-specific CD8+ T cell response greater than that observed with caspase 8-induced apoptosis. Surprisingly however, when necroptosis was induced within established PyMT-B6 orthotopic mammary tumors, we observed significantly reduced survival, which correlated with higher neutrophil and macrophage recruitment, along with lower CD8+ T cell infiltration and IFN-γ expression. In vitro, necroptosis induced with either RIPK3 or MLKL greatly enhanced expression of Cxcl1 and Cxcl2, a property that could be recapitulated by transferring dead cell supernatant to live cells, suggesting the presence of an alarmin or damage-associated molecular pattern (DAMP). We therefore knocked out MyD88, which serves as a critical adapter protein to a number of surface receptors, including the TLR family, the IL1 receptor, and RAGE. The loss of MyD88 completely eliminated the ability of tumor cells to express Cxcl1 in response to supernatant from necrotic cells. Expression was not driven by release of HMGB1 or signaling through RAGE or TLRs. Instead, we found that blocking the interleukin (IL)1 receptor or IL1α, but not IL1β, prevented Cxcl1 expression. IL1α is a constitutively expressed by some tumors, suggesting a potential role for IL1α in regulating immune responses to therapies that induce necrotic-like forms of cell death. In support of this, Il1a expression was strongly predictive of progression free survival in triple-negative breast cancer. These findings suggest that IL1α has an unexplored role in regulating the tumor microenvironment. By understanding how IL1α regulates the immune response during cytotoxic therapy, our findings may explain why certain therapies are immunogenic (or not) in different cancers. Citation Format: Kay Hänggi, Aysenur Keske, Alycia Gardner, Jie Li, Olabisi Osunmakinde, Daiana Celias, Amer Beg, Brian Ruffell. Necroptosis promotes tumor growth through IL1-alpha release and recruitment of immunosuppressive myeloid cells [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 655.

Double stranded RNA drives anti-viral innate immune responses, sickness behavior and cognitive dysfunction dependent on dsRNA length, IFNAR1 expression and age

Double stranded RNA is generated during viral replication. The synthetic analogue poly I:C is frequently used to mimic anti-viral innate immune responses in models of psychiatric and neurodegenerative disorders including schizophrenia, autism, Parkinson’s disease and Alzheimer’s disease. Many studies perform limited analysis of innate immunity despite these responses potentially differing as a function of dsRNA molecular weight and age. Therefore fundamental questions relevant to impacts of systemic viral infection on brain function and integrity remain. Here, we studied innate immune-inducing properties of poly I:C preparations of different lengths and responses in adult and aged mice. High molecular weight (HMW) poly I:C (1–6 kb, 12 mg/kg) produced more robust sickness behavior and more robust IL-6, IFN-I and TNF-α responses than poly I:C of < 500 bases (low MW) preparations. This was partly overcome with higher doses of LMW (up to 80 mg/kg), but neither circulating IFNβ nor brain transcription of Irf7 were significantly induced by LMW poly I:C, despite brain Ifnb transcription, suggesting that brain IFN-dependent gene expression is predominantly triggered by circulating IFNβ binding of IFNAR1. In aged animals, poly I:C induced exaggerated IL-6, IL-1β and IFN-I in the plasma and similar exaggerated brain cytokine responses. This was associated with acute working memory deficits selectively in aged mice. Thus, we demonstrate dsRNA length-, IFNAR1- and age-dependent effects on anti-viral inflammation and cognitive function. The data have implications for CNS symptoms of acute systemic viral infection such as those with SARS-CoV-2 and for models of maternal immune activation.

Abstract PO056: The kinetics of the anti-glioblastoma immune response in immunocompetent mouse models is influenced by neglected factors

Abstract Due to the recent rise in immunotherapy research to treat Glioblastoma, immunocompetent mouse models have become increasingly crucial for the study of novel immunotherapies against brain tumors. However, the kinetics of the immune response against the most prevalent immunocompetent GBM models, GL261 and CT2A, have not been well studied, nor have they been compared adequately in the literature. In this study, we compared the immune response in these models using flow cytometry and immunohistochemistry. We investigated several factors that influence the immune response, such as kinetics, tumor size, and transfection status. We hypothesize that these factors influence the immune response enough to warrant consideration while studying new immunotherapeutic approaches for treating GBM. Our results will aid in the preclinical studies of immunotherapy for GBM by allowing scientists to make informed decisions about their chosen models. Citation Format: Breanna R. Noffsinger, Alexandra Witter, Natasha Shebani, Aizhen Xiao, Qing Zhong, Tajie Harris, Benjamin Purow. The kinetics of the anti-glioblastoma immune response in immunocompetent mouse models is influenced by neglected factors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO056.

Abstract 4140: Discovery and characterization of novel highly potent A2A adenosine receptor antagonists for cancerimmunotherapy

Abstract Introduction: Extracellular adenosine produced at high concentrations within the tumor micro-environment (TME) and suppresses immune function via inhibition of immune cell activation. Targeting adenosine receptors has emerged as a novel method to activate anti-tumor immunity. In particular, A2A adenosine receptor (A2AR), one of the G-protein-coupled-receptors, exhibits immunosuppressive functions. Herein, we suggest novel A2AR antagonist lead compounds as a highly potent antagonist of A2AR for immunotherapy. Materials and Methods: Novel A2AR antagonist lead compounds were designed using CADD and synthesized as the active biologic inhibitory compound. The protein preparation and molecular docking were performed using Glide (Schrödinger). A radioligand binding assay was performed to evaluate the affinities of A2AR Antagonists for the human adenosine A2AR. The potencie of A2AR antagonists were determined by cAMP assay in HEK293-hA2AR cells and cAMP-mediated pCREB assay in human CD8+ T cells from whole blood. In vivo CT26 and MC38 syngeneic tumor models were used to assess the therapeutic effect of A2AR antagonists. Results: A2AR antagonist lead compounds showed strong binding affinities toward human A2AR. They also potently inhibited the NECA-mediated production of intracellular cAMP in HEK293 cells expressing human A2AR. Elevated intracellular cAMP following A2AR activation results in the phosphorylation of CREB (cAMP response element-binding protein). A2AR antagonist lead compounds treatment inhibited pCREB in NECA-stimulated HEK293-hA2AR and human CD8+ T cells. A2AR antagonist lead compounds inhibited tumor growth of mouse syngeneic tumor models as a single agent and combination with anti-PD-L1. In combination with anti-PD-L1, A2AR antagonist lead compounds had remarkable antitumor activities in multiple mouse tumor models, including restoration of immune responses in models that incompletely responded to anti-PD-L1 monotherapy. Conclusion: These results showed the potencies of A2AR antagonist lead compounds with high capability of A2AR inhibition. Blockade of the adenosine signaling pathway may be vital for enhancing anti-tumor responses in solid tumors that show an incomplete response to anti-PD-L1 therapy. A2AR antagonist lead compounds demonstrate a novel approach to anti-cancer immunotherapy. Citation Format: Kyungik Lee, Seungah Jun, EunYoung Byun, Hosun Lee, Yongtaek Lee, MiJin Moon, Yu-Yon Kim, Hyun Jeong Kang, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. Discovery and characterization of novel highly potent A2A adenosine receptor antagonists for cancerimmunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4140.

A2AR Antagonism with CPI-444 Induces Antitumor Responses and Augments Efficacy to Anti-PD-(L)1 and Anti-CTLA-4 in Preclinical Models.

Adenosine signaling through A2A receptors (A2AR) expressed on immune cells suppresses antitumor immunity. CPI-444 is a potent, selective, oral A2AR antagonist. Blockade of A2AR with CPI-444 restored T-cell signaling, IL2, and IFNγ production that were suppressed by adenosine analogues in vitro CPI-444 treatment led to dose-dependent inhibition of tumor growth in multiple syngeneic mouse tumor models. Concentrations of extracellular adenosine in the tumor microenvironment, measured using microdialysis, were approximately 100-150 nmol/L and were higher than corresponding subcutaneous tissue. Combining CPI-444 with anti-PD-L1 or anti-CTLA-4 treatment eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that incompletely responded to anti-PD-L1 or anti-CTLA-4 monotherapy. Tumor growth was fully inhibited when mice with cleared tumors were later rechallenged, indicating that CPI-444 induced systemic antitumor immune memory. CD8+ T-cell depletion abrogated the efficacy of CPI-444 with and without anti-PD-L1 treatment, demonstrating a role for CD8+ T cells in mediating primary and secondary immune responses. The antitumor efficacy of CPI-444 with and without anti-PD-L1 was associated with increased T-cell activation, a compensatory increase in CD73 expression, and induction of a Th1 gene expression signature consistent with immune activation. These results suggest a broad role for adenosine-mediated immunosuppression in tumors and justify the further evaluation of CPI-444 as a therapeutic agent in patients with solid tumors. Cancer Immunol Res; 6(10); 1136-49. ©2018 AACR.

The Enigma of Heat Shock Proteins in Immune Tolerance.

The fundamental problem of autoimmune diseases is the failure of the immune system to downregulate its own potentially dangerous cells, which leads to destruction of tissue expressing the relevant autoantigens. Current immunosuppressive therapies offer relief but fail to restore the basic condition of self-tolerance. They do not induce long-term physiological regulation resulting in medication-free disease remissions. Heat shock proteins (HSPs) have shown to possess the capacity of inducing lasting protective immune responses in models of experimental autoimmune diseases. Especially mycobacterial HSP60 and HSP70 were shown to induce disease inhibitory IL-10-producing regulatory T cells in many different models. This in itself may seem enigmatic, since based on earlier studies, HSPs were also coined sometimes as pro-inflammatory damage-associated molecular patterns. First clinical trials with HSPs in rheumatoid arthritis and type I diabetes have also indicated their potential to restore tolerance in autoimmune diseases. Data obtained from the models have suggested three aspects of HSP as being critical for this tolerance promoting potential: 1. evolutionary conservation, 2. most frequent cytosolic/nuclear MHC class II natural ligand source, and 3. upregulation under (inflammatory) stress. The combination of these three aspects, which are each relatively unique for HSP, may provide an explanation for the enigmatic immune tolerance promoting potential of HSP.

Abstract 5593: Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies

Abstract Adenosine signaling via A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and limits the efficacy of immunotherapy, chemotherapy, CAR-T, and vaccines. CPI-444 is a potent and selective oral A2AR antagonist. Daily treatment of mice with CPI-444 led to dose-dependent inhibition of tumor growth in multiple syngeneic tumor models. Combining CPI-444 with anti-PD-L1 treatment synergistically eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that are poorly responsive to anti-PD-1 or anti-PD-L1 monotherapy. We have initiated a Phase 1/1b clinical trial to examine safety, tolerability, biomarkers, and preliminary efficacy of CPI-444 as a single agent and in combination with the anti-PD-L1 antibody, atezolizumab, in patients with non-small cell lung, melanoma, renal, triple negative breast, and other (bladder, prostate, head and neck, colorectal) tumors. Step 1 of the trial focused on determining the optimal dose and schedule for CPI-444; Step 2 is currently evaluating the efficacy of optimal CPI-444 dosing alone and with atezolizumab. In 48 patients treated in Step 1, CPI-444 was well tolerated with 1 Grade 3 or 4 treatment related adverse events. Preliminary evidence of clinical activity was observed in patients treated with single agent CPI-444, including patients who previously failed anti-PD-1 therapy. A pCREB-based pharmacodyamic assay showed that 100 mg, BID dose of CPI-444 resulted in a complete, sustained inhibition of A2AR on circulating immune cells. CPI-444 treatment alone or in combination with atezolizumab resulted in increased frequency of circulating CD8+PD-1+ cells and memory/effector subsets of CD4+ and CD8+ T cells. Substantial changes in TCR repertoire (Morisita Index &amp;lt;0.9) were observed in both anti-PD-1 native and refractory patients and correlated to patient responses. CD73 expression, CD8+ infiltration, TREG distribution, and gene expression signatures are currently being evaluated in serial tumor biopsy specimens and will be presented. In total, this shows that CPI-444 is well tolerated in cancer patients, exhibits functional inhibition of adenosine signaling, and treatment is associated with activation of anti-tumor immunity and clinical activity. Citation Format: Stephen Willingham, Andrew Hotson, Po Ho, Carmen Choy, Kim Walter, Erik Yusko, Sharon Benzeno, Ginna Laport, Richard Miller, Ian McCaffery. Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies [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 5593. doi:10.1158/1538-7445.AM2017-5593

The role of gut microbiota in shaping the immune response in relapse-remitting and chronic-progressive mouse models of multiple sclerosis

Abstract In this study, we used a mouse model of multiple sclerosis (MS), experimental autoimmune encephalitis (EAE), to evaluate the role of gut microbiota in modulating T helper cell function in chronic-progressive (CP) versus relapse-remitting (RR) forms of the disease. We hypothesized that encephalitogenic T cell function in RR-EAE and CP-EAE is differentially shaped by gut microbiota. Metagenomic sequencing of the variable V4 region of the prokaryotic 16S ribosomal RNA gene present in feces derived from naïve mice and mice exhibiting CP-EAE or RR-EAE revealed significantly diverse microbial populations, which may be responsible for differing disease courses. Specifically, the order Bacteroidales was dominantly represented by RR-EAE mice, with very little or no detection in naive or CP-EAE mice, suggesting a potential role for bacteria in this order in shaping tolerant or remittance-favoring conditions. Additionally, Akkermansia and rc4-4, of phyla Verrucomicrobia and Firmicutes, respectively, were identified specifically in CP-EAE, suggesting a potential pathogenic or pro-inflammatory role for bacteria belonging to these taxa, at least in the context of EAE. We performed qRT-PCR in order to test the validity of the sequencing findings, as well as used a gas chromatograph configured with flame-ionization detectors to evaluate short chain fatty acids from cecal flushes. The current study implies a role for gut microbiota in differentially shaping the immune response in models of RRMS and CPMS. Information regarding tolerance–promoting or –inhibiting conditions shaped by gut bacteria can be harnessed for the potential development of therapies for diseases possessing important inflammatory components.

Anti-Inflammatory and Neuroprotective Effects of PGE2 EP4 Signaling in Models of Parkinson's Disease.

Inflammation is a ubiquitous factor accompanying normal aging and neurodegeneration, and recent studies indicate a major contribution of inducible cyclooxygenase (COX-2) and its downstream prostaglandin signaling pathways in modulating neuroinflammatory responses and neuronal function. We have previously shown that the prostaglandin PGE2 receptor EP4 suppresses innate immune responses in models of systemic inflammation. Here we investigated the role of the EP4 receptor in models of Parkinson's disease (PD). Systemic co-administration of the EP4 agonist ONO-AE1-329 with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) prevented loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) without significant changes in glial activation, suggesting a potent neuroprotective effect of EP4 signaling in this acute model of DA neuronal loss. Cell-specific conditional ablation of EP4 in Cd11bCre;EP4lox/lox mice exacerbated MPTP-associated glial activation and T-cell infiltration in SNpc, consistent with anti-inflammatory functions of microglial EP4 signaling. In vitro, in primary microglia stimulated with oligomeric α-synuclein, EP4 receptor activation suppressed generation of pro-inflammatory and oxidative stress factors. Taken together, these findings suggest a dual neuroprotective and anti-inflammatory mechanism of action by the EP4 receptor in models of PD.

0095. Transvenous vagus nerve stimulation does not modulate the innate immune response in humans in vivo during experimental endotoxemia

In a variety of conditions excessive and/or persistent activation of the innate immune system has detrimental effects. In animals, electrical vagus nerve stimulation (VNS) inhibits the innate immune response in models of endotoxemia (administration of lipopolysaccharide [LPS]), sepsis, trauma, and hemorrhagic shock, via the so-called cholinergic anti-inflammatory pathway. However, human in vivo evidence is lacking. Up till now, VNS was possible through implantation of a cuff electrode wrapped around the nerve, which limits its use in acute inflammatory situations frequently encountered on the ICU. A novel, less invasive VNS method is transvenous VNS (tVNS).

YopP-Expressing Variant of Y. pestis Activates a Potent Innate Immune Response Affording Cross-Protection against Yersiniosis and Tularemia

Plague, initiated by Yersinia pestis infection, is a rapidly progressing disease with a high mortality rate if not quickly treated. The existence of antibiotic-resistant Y. pestis strains emphasizes the need for the development of novel countermeasures against plague. We previously reported the generation of a recombinant Y. pestis strain (Kim53ΔJ+P) that over-expresses Y. enterocolitica YopP. When this strain was administered subcutaneously to mice, it elicited a fast and effective protective immune response in models of bubonic, pneumonic and septicemic plague. In the present study, we further characterized the immune response induced by the Kim53ΔJ+P recombinant strain. Using a panel of mouse strains defective in specific immune functions, we observed the induction of a prompt protective innate immune response that was interferon-γ dependent. Moreover, inoculation of mice with Y. pestis Kim53ΔJ+P elicited a rapid protective response against secondary infection by other bacterial pathogens, including the enteropathogen Y. enterocolitica and the respiratory pathogen Francisella tularensis. Thus, the development of new therapies to enhance the innate immune response may provide an initial critical delay in disease progression following the exposure to highly virulent bacterial pathogens, extending the time window for successful treatment.

Deletion of tumor necrosis factor-α ameliorates neurodegeneration in Sandhoff disease mice

Sandhoff disease (SD) is a lysosomal storage disorder caused by a lack of a functional β-subunit of the β-hexosaminidase A and B enzymes, leading to the accumulation of gangliosides in the central nervous system (CNS). The Hexb-/- mouse model of SD shows a progressive neurodegenerative phenotype similar to the human equivalent. Previous studies have revealed that Hexb-/- mice suffer from chronic neuroinflammation characterized by microglial activation and expansion. Tumor necrosis factor-α (TNFα), a key modulator of the CNS immune response in models of neurodegeneration, is a hallmark of this activation. In this study, we explore the role of TNFα in the development and progression of SD in mice, by creating a Hexb-/- Tnfα-/- double-knockout mouse. Our results revealed that the double-knockout mice have an ameliorated disease course, with an extended lifespan, enhanced sensorimotor coordination and improved neurological function. TNFα-deficient SD mice also show decreased levels of astrogliosis and reduced neuronal cell death, with no alterations in neuronal storage of gangliosides. Interestingly, temporal microglia activation appears similar between the Hexb-/- Tnfα-/- and SD mice. Evidence is provided for the TNFα activation of the JAK2/STAT3 pathway as a mechanism for astrocyte activation in the disease. Bone marrow transplantation experiments reveal that both CNS-derived and bone marrow-derived TNFα have a pathological effect in SD mouse models, with CNS-derived TNFα playing a larger role. This study reveals TNFα as a neurodegenerative cytokine mediating astrogliosis and neuronal cell death in SD and points to TNFα as a potential therapeutic target to attenuate neuropathogenesis.

P001 Interleukin-21 exerts pathogenic and host-protective effects in experimental models of colitis

Background: Interleukin-21 is a T-cell derived cytokine implicated in the pathogenesis of chronic inflammatory diseases, including IBD. IL-21-receptor (IL-21R) is reported to be expressed on a diverse array of cells, including both haematopoetic and epithelial cells. In T-cells, IL-21 signaling may be a critical regulator of the balance of Th17 and regulatory (Treg) responses. However, the in vivo role of IL-21 in experimental IBD is not well understood. Methods: Using IL-21R-deficient mice, we examined the effects of IL-21 signals in complimentary models of murine IBD, including CD4+CD45RBhi T-cell transfer, Helicobacter hepaticus/anti-IL-10R, and Citrobacter rodentium infection. Results: In the Helicobacter hepaticus/anti-IL-10R model, IL-21 was pathogenic, with reduced Th17 populations and increased FOXP3+ Treg cell accumulation in the mucosa of IL-21R / animals. Similarly, in the T-cell transfer model of disease, IL-21R / T-cells were impaired in their ability to induce disease, associated with a cell-intrinsic requirement for IL-21 to regulate the balance of FOXP3+ Treg and IFN-g+ Th1 cells in the intestine. Further studies implicate a role for IL-21 in driving the expression of T-cell attracting chemokines in both these models, with studies in T-cell transfer suggesting this to be a direct T-cell dependent effect. In contrast, IL-21 was required for host protection in Citrobacter rodentium infection, with IL-21R / mice developing more severe intestinal disease, associated with greater bacterial burdens and systemic dissemination of infection. In this model, mice deficient in IL-21R were unimpaired in their Th17 responses, but developed robust Th1 responses, and an IFN-g driven immunopathology. Conclusions: IL-21 is a critical regulator of intestinal immune responses in models of IBD, exerting context specific pathogenic or host-protective effects. IL-21 may therefore be an important future therapeutic target in IBD, but its pleiotrophic effects must be considered in the development and use of drugs targeting this pathway. P002 Intestinal homeostasis is critically regulated by the transcription factor T-bet N. Powell1 *, E. Stolarczyk1, J. Lo1, J. Canavan1, E. Marks1, T. MacDonald2, G. Lord1. 1King’s College London, Experimental Immunobiology, London, 2Queen Mary University, Immunity and Infectious Diseases, London, United Kingdom

Effect of Probiotic Bacteria on Microbial Host Defense, Growth, and Immune Function in Human Immunodeficiency Virus Type-1 Infection

The hypothesis that probiotic administration protects the gut surface and could delay progression of Human Immunodeficiency Virus type1 (HIV-1) infection to the Acquired Immunodeficiency Syndrome (AIDS) was proposed in 1995. Over the last five years, new studies have clarified the significance of HIV-1 infection of the gut associated lymphoid tissue (GALT) for subsequent alterations in the microflora and breakdown of the gut mucosal barrier leading to pathogenesis and development of AIDS. Current studies show that loss of gut CD4+ Th17 cells, which differentiate in response to normal microflora, occurs early in HIV-1 disease. Microbial translocation and suppression of the T regulatory (Treg) cell response is associated with chronic immune activation and inflammation. Combinations of probiotic bacteria which upregulate Treg activation have shown promise in suppressing pro inflammatory immune response in models of autoimmunity including inflammatory bowel disease and provide a rationale for use of probiotics in HIV-1/AIDS. Disturbance of the microbiota early in HIV-1 infection leads to greater dominance of potential pathogens, reducing levels of bifidobacteria and lactobacillus species and increasing mucosal inflammation. The interaction of chronic or recurrent infections, and immune activation contributes to nutritional deficiencies that have lasting consequences especially in the HIV-1 infected child. While effective anti-retroviral therapy (ART) has enhanced survival, wasting is still an independent predictor of survival and a major presenting symptom. Congenital exposure to HIV-1 is a risk factor for growth delay in both infected and non-infected infants. Nutritional intervention after 6 months of age appears to be largely ineffective. A meta analysis of randomized, controlled clinical trials of infant formulae supplemented with Bifidobacterium lactis showed that weight gain was significantly greater in infants who received B. lactis compared to formula alone. Pilot studies have shown that probiotic bacteria given as a supplement have improved growth and protected against loss of CD4+ T cells. The recognition that normal bacterial flora prime neonatal immune response and that abnormal flora have a profound impact on metabolism has generated insight into potential mechanisms of gut dysfunction in many settings including HIV-1 infection. As discussed here, current and emerging studies support the concept that probiotic bacteria can provide specific benefit in HIV-1 infection. Probiotic bacteria have proven active against bacterial vaginosis in HIV-1 positive women and have enhanced growth in infants with congenital HIV-1 infection. Probiotic bacteria may stabilize CD4+ T cell numbers in HIV-1 infected children and are likely to have protective effects against inflammation and chronic immune activation of the gastrointestinal immune system.

EC144, a Synthetic Inhibitor of Heat Shock Protein 90, Blocks Innate and Adaptive Immune Responses in Models of Inflammation and Autoimmunity

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.

Monitoring FoxP3 expression in Tolerance to Factor VIII (128.4)

Abstract The FoxP3 transcription factor has emerged as a marker for regulatory T cells in the mouse. Our lab has identified a gene therapy based approach that allows us to abrogate the immune response in models for autoimmune diseases and the deleterious immune response in hemophilic patients being treated with factor VIII. Utilizing the FoxP3-GFP knock-in mouse that has been mated to our hemophilic fVIII−/− mouse, we are able to monitor fluctuations in the FoxP3 by flow cytometry. We have observed a relative increase in FoxP3 when tolerance is induced and a relative decrease when an immune response is initiated towards fVIII. We have also developed an in vitro model to follow FoxP3 as it changes in different populations with certain stimuli. Thus, we found that ~75% of CD4+CD25+ cells in a resting population are FoxP3 positive and 12% of all CD4+ cells are FoxP3+. After a single day of stimulation of splenocytes with soluble anti-CD3 antibody, nearly all the CD4+ cells become CD4+CD25+ while less than 6% remain FoxP3+. When presentation of anti-CD3 via Fc receptors is blocked, activation (monitored by CD25 levels) is abrogated, but FoxP3 levels also decrease. While this drop in the frequency of FoxP3 expression in CD4 T cells can be explained in part by the activation of the CD4+CD25− effector cells, we propose that anti-CD3 antibody is also actively diminishing FoxP3 levels in the regulatory T cell population. Supported by HL061883.

Chemokine scavenging by D6: a movable feast?

The atypical chemokine receptor, D6, is efficient at sequestering and scavenging inflammatory CC chemokines. The absence of D6 blocks the successful resolution of immune responses in models of inflammation, suggesting that CC-chemokine scavenging by D6 is an important component of the resolution phase of in vivo inflammatory responses. Most studies have suggested that lymphatic endothelial cells are the main vehicles for D6 function in vivo. Here, we propose that leukocytes, which also express D6, could be more-effective vehicles for D6 scavenging function. Thus, leukocytes might be the primary cell type that removes inflammatory chemokines from inflamed tissues. We also propose that lymphatic endothelial cell-expressed D6 might have a distinct but complementary role in restricting inflammatory leukocyte access to the lymphatic vasculature.

T-cell recognition of glycolipids presented by CD1 proteins

The most well-known molecular paradigm of antigen recognition by T cells involves partial digestion of proteins to generate small peptides, which bind to major histocompatibility complex (MHC) proteins. Recent studies of CD1, an MHC class I homolog encoded outside the MHC, have revealed that it presents diverse glycolipids to T cells. The molecular mechanism for lipid antigen recognition involves insertion of the lipid portion of antigens into a hydrophobic groove to form CD1-lipid complexes, which contact T-cell receptors (TCRs). Here, we examine the known antigen structures presented by CD1, the majority of which have sugar moieties that are capable of interacting with TCRs. Recognition of carbohydrate epitopes is precise, and lipid-reactive T cells alter systemic immune responses in models of infectious and autoimmune disease. These findings provide a previously unrecognized mechanism by which the cellular immune system can recognize alterations in many types of carbohydrate structures.

New Target Antigens for Cancer Immunoprevention

Prevention of cancer through the activation of the immune system has been explored in recent years in preclinical systems thanks to the availability of several new transgenic mouse models that closely mimic the natural history of human tumors. The most thoroughly investigated model of cancer immunoprevention is the mammary carcinoma of HER-2/neu transgenic mouse. In this system it has clearly been shown that the activation of immune defences in healthy individuals can effectively prevent the subsequent onset of highly aggressive mammary carcinomas. A complete prevention was obtained using a combination of three signals (the so called "triplex" vaccine) that included the specific antigen (p185, the product of HER-2/neu) and nonspecific signals like allogeneic histocompatibility antigens and interleukin 12. The analysis of protective immune responses in models of cancer immunoprevention revealed some unexpected features, in particular the central role of antibodies in immunoprevention, at variance with conventional immuno-therapy which is firmly based on cytotoxic T cells. In the HER-2/neu system anti-p185 antibodies, in addition to immunological functions leading to tumor cell lysis, inhibit p185 dimerization and induce its internalization, resulting in the inhibition of mitogenic signaling. Most current tumor antigens appear to be unsuitable targets for cancer immunoprevention. An ideal antigen should have a crucial pathogenetic role in tumor growth to avoid the selection of antigen loss variants. Downregulation of major histocompatibility complex (MHC) expression during tumor progression frequently limits antigen recognition by MHC-restricted T cells. Thus an ideal antigen for cancer immunoprevention should be recognized both by T cells and by antibodies. Antibody binding to cell surface oncogenic determinants, in addition to complement- and cell-mediated tumor cell lysis, can block mitogenic signaling and induce internalization, resulting in tumor growth arrest. A search for new tumor antigens should be conducted among molecules that are directly involved in neoplastic transformation and are recognizable by the immune response also in MHC loss variants. Novel tumor antigens fulfilling both conditions will be crucial for the development of cancer immunoprevention and will provide new targets also for cancer immunotherapy.

The IL-1 receptor 1 is critical for Th2 cell type airway immune responses in a mild but not in a more severe asthma model.

IL-1 alpha and IL-1 beta are potent pro-inflammatory cytokines that regulate many physiological systems by binding and signaling to the same receptor termed IL-1 receptor type 1 (IL-1R1). We have investigated the role of IL-1 for pulmonary immune responses in models of allergic asthma using IL-1R1-deficient (IL-1R1(-/-)) mice. In a model of mild asthma, based on repeated sensitization of mice with low doses of ovalbumin in the absence of any adjuvant and multiple intranasal challenges, the pulmonary eosinophilic inflammation and goblet cell hyperplasia were strongly reduced in IL-1R1(-/-) as compared to control BALB/c mice. Moreover, priming of CD4(+) T cells in bronchial lymph nodes and their recruitment to the lung was affected in IL-1R1(-/-) mice associated with impaired antibody responses including IgG, IgE, and IgA. In contrast, sensitization of mice in the presence of alum adjuvant, a more severe asthma model, rendered the IL-1 pathway dispensable for the development of pulmonary allergic Th2 responses, as eosinophilic inflammation, antibody responses, and CD4(+) T cell priming in lymph nodes were comparable between IL-1R1(-/-) and wild-type mice. These results suggest a critical role of IL-1/IL-1R1 for development of allergic Th2 responses, but its requirement can be overcome by using alum as adjuvant for sensitization.