Host Immune Recognition Research Articles

The association between human leukocyte antigen (HLA)-G polymorphisms and human papillomavirus (HPV) infection in Inuit women of northern Quebec

BackgroundThe human leukocyte antigen (HLA)-G molecules act as negative regulators of the immune response. We analyzed the associations between HLA G polymorphisms and human papillomavirus (HPV) infection and squamous intraepithelial lesions (SIL) in Inuit women from Nunavik, northern Quebec. MethodsCervical specimens from a cohort study of 548 Inuit women were tested for HPV DNA. HPV genotypes were classified according to tissue-tropism groupings of alpha-papillomavirus species: alpha group 1 includes low risk (LR) cervical species, group 2 includes high risk (HR) cervical species, and group 3 includes LR vaginal species. HLA-G alleles were typed using direct DNA sequencing. ResultsHLA-G∗01:01:01 was associated with an increased risk of period prevalent alpha groups 1 (OR=2.23, 95% CI:1.08–4.59) and 3 (OR=1.70, 95% CI:1.09–2.65). The homozygous HLA-G∗01:04:01 genotype was associated with a decreased risk of alpha group 3 infection period prevalence (OR=1.69 95% CI=1.07–2.67). No HLA-G alleles were significantly associated with HPV persistence. HLA-G∗01:01:02, G∗01:04:01 and G∗01:06 were associated with high grade (HG)SIL, but the association did not reach statistical significance. ConclusionsThese results suggest that HLA-G polymorphisms play a role in the natural history of HPV infection, likely at the stage of host immune recognition. HLA-G polymorphisms interacted differently with the three alpha papillomavirus groups.

L03.05 - Immunomodulatory Activity of SGI-110, a Second Generation Hypomethylating Agent

Aberrant DNA hypermethylation down-regulates the expression of components of the “tumor recognition complex” (HLA class I, tumor-associated antigens (TAA) of the cancer testis antigens (CTA) family and accessory/co-stimulatory molecules) in neoplastic cells. This evidence strongly suggests that the extent of DNA methylation of cancer cells might allow tumor-escape from host's immune recognition, contributing to reduce the clinical efficacy of immunotherapeutic approaches. SGI-110 is a dinucleotide of decitabine (DAC) and deoxyguanosine formulated as a low volume SQ injection allowing extended DAC exposure compared to DAC IV.

Macrophages at work: phagocytosis of urinary fungi.

A 56-year-old woman who underwent a lung transplant followed 2 years later by a deceased donor kidney transplant presented with pyuria. Enterococcus bacteremia was previously treated with amoxicillin/sulbactam for 14 days. Subsequent to this therapy, urinalysis was carried out, which revealed the following: specific gravity 1.010; pH 7.5; nitrite negative; albumin 1+; hemoglobin 3+. Urine microscopy revealed one to two squamous epithelial cells/high power field (HPF); ∼20 white blood cells/HPF; >50 red blood cells/HPF; 0–2 hyaline casts/low power field; and blastoconidia and pseudomycelia (Candida sp.). Interestingly, numerous macrophages phagocytosing blastoconidia were noted throughout the specimen (Figure 1). Fig. 1. (A) Candida blastoconidia and pseudomycelia are seen on urine microscopy. Several macrophages have phagocytosed blastoconidia (arrows). (B) Several macrophages containing blastoconidia are noted (arrows). Candida sp. is a human fungal pathogen. The major host defense against systemic Candida infection is ingestion of fungi by cells of the immune system, in particular macrophages and neutrophils. The fungal cell membrane is the initial contact with the immune system and plays an important role in host immune cell recognition and phagocytosis [1]. It is a dynamic organelle that determines both the shape of the fungus and its viability. Depending on the environment, these organisms are able to undergo reversible morphological changes moving between yeast, pseudohyphal and hyphal forms [2]. This plasticity allows fungi to successfully infect different anatomical sites. Hyphae have invasive properties that allow tissue penetration and escape from immune cells [3], whereas yeast forms are suited to disseminate via the bloodstream [4]. Phagocytic clearance of fungal pathogens consists of four stages: (i) accumulation of phagocytes where fungi reside; (ii) recognition of fungal molecular patterns; (iii) ingestion of fungi attached to phagocyte cell membranes and (iv) phagocyte processing of engulfed fungi by fusion [5]. The urinary findings of macrophages ingesting fungi in our patient support the importance of this host defense against such invading microorganisms.

The Invisible Enemy – How Human Papillomaviruses Avoid Recognition and Clearance by the Host Immune System

Human papillomavirus (HPV) needs to persist in squamous epithelia for a certain amount of time to complete its reproductive cycle. Therefore, the virus has evolved multiple immune evasion strategies. The interplay of these immune evasion mechanisms with the host immune system decides whether a HPV infection is cleared or becomes persistent. Clearance of HPV-induced lesions is mediated by a cellular immune response, consisting of both cytotoxic T lymphocyte and T helper cell responses. Persistent HPV infection, on the other hand, is the single most important risk factor for the development of HPV-associated premalignant lesions and HPV-driven cancers. This article reviews the immune evasion mechanisms employed by high-risk HPVs to escape host immune recognition and attack.

Microbial recognition and evasion of host immunity

Plants are able to detect microbes by pattern recognition receptors in the host cells that, upon recognition of the enemy, activate effective immune responses in the invaded tissue. Recognition of microbes occurs by common conserved structures called microbe-associated molecular patterns (MAMPs). Plant pathogens and beneficial soil-borne microbes live in close contact with their host. Hence, prevention of the host's defence programme is essential for their survival. Active suppression of host defences by microbial effector proteins is a well-known strategy employed by many successful plant-associated microbes. Evasion of host immune recognition is less well studied but is emerging as another important strategy. Escape from recognition by the host's immune system can be caused by alterations in the structure of the recognized MAMPs, or by active intervention of ligand-receptor recognition. This paper reviews the structure and recognition of common MAMPs and the ways that plant-associated microbes have evolved to prevent detection by their host.

Abstract 3550: Differences in immune escape in HPV+ versus HPV- head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and has not seen an improvement in survival in decades. Current standard-of-care treatments including surgical resection, chemotherapy and radiation still lead to high rates of recurrence and loco-regional metastasis contributing to the poor prognosis in these patients. It is well established that HNSCC are highly immune-modulatory tumors and may therefore benefit from immunotherapy treatment regimens. Better methods are therefore needed to match patients with the appropriate immunotherapy. Recently, human papillomavirus (HPV) was implicated in the etiology of a subset of HNSCC arising in younger patients without a history of alcohol and tobacco use. Approximately 30% of HNSCC tumors are HPV+ and despite late stage presentation, often have a better prognosis and response to therapy. This study examines how host immune recognition and response varies between HPV+ and HPV- HNSCC, with the goal of identifying new targets for immunotherapy. The immune profile and HPV infection status of 41 HNSCC patient tumor samples obtained from surgical resection was characterized by qRT-PCR, PCR, and IHC techniques. Tumor samples were analyzed for the expression of 40 genes involved in immune activation and suppression in the tumor microenvironment to generate an immune profile. Gene expression levels were normalized to GAPDH and compared across HPV+, HPV- and normal tissue groups by ANOVA and post-test pairwise analysis. HPV infectivity of tumor biopsies was determined using HPV consensus primers (MY09/MY11 and GP5+/GP6+) and confirmed with p16 immunohistochemistry. From these studies, eight of 41 (20%) HNSCC biopsies were HPV+ by PCR and IHC analysis. HPV+ tumors demonstrated statistically significant increased expression of genes related to immunosuppression including regulatory T (CD4, FoxP3, CTLA-4, and FasL) and myeloid suppressor cell markers (CD11b and IL-6, an inducer of these suppressor cells), as well as negative-regulators (SOCS1) (p<0.05). These tumors also demonstrated an increase in markers of immune activation and infiltration, including evidence of T cells (CD4, Fas-L), antigen presenting cells (CD80, CD83, CD1c), and inflammatory cytokines and co-stimulatory molecules (IL-2, IL-12, IFN- γ, and OX40L) (p<0.05). There were no statistically significant differences in gene expression found between normal tissue and HPV- HNSCC tumors. These studies demonstrate a general pattern of increased immunogenicity and concurrent immunosuppression in patients with HPV+ versus HPV- HNSCC that could potentially be used to inform immunotherapy treatment of HNSCC patients. HPV+ tumors may be more effectively treated with a regimen that employs a combination of immune stimulation and reversal of immune tolerance mechanisms, while HPV- tumors may require greater immune stimulation to activate endogenous anti-tumor immune responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3550. doi:1538-7445.AM2012-3550

The Mycobacterium avium ESX-5 PPE protein, PPE25-MAV, interacts with an ESAT-6 family Protein, MAV_2921, and localizes to the bacterial surface

Previous research has demonstrated that inactivation of the Mycobacterium avium gene, PPE25-MAV (MAV_2928), leads to a significant attenuation of virulence in both in vitro and in vivo models. PPE25-MAV encodes for a PPE family protein, a family from which many members have been implicated in both bacterial virulence and host immune recognition. Recent research has shown that many PPE family proteins are exported by a specialized Type VII secretion system in mycobacteria. In this context, the mechanisms of PPE25-MAV in M. avium pathogenesis were investigated. A mycobacterial 2-hybrid system was used to perform a directed search for M. avium proteins that interact directly with PPE25-MAV. An interaction was observed between PPE25-MAV and the ESAT-6 family protein, MAV_2921, and was further defined by 2-hybrid analysis of truncated PPE25-MAV, and confirmed by co-immunoprecipitation. Localization of the PPE25-MAV protein was analyzed in Mycobacterium smegmatis expressing the recombinant protein and a significant percentage of PPE25-MAV was shown to be exposed at the bacterial surface by surface biotinylation and trypsin protection assays. Finally, transcriptional analysis of PPE25-MAV and its associated operon suggested that nutrient limitation, a condition which occurs in the phagosome, plays a role in regulating expression of the PPE25-MAV gene.

Mapping the Binding between the Tetraspanin Molecule (Sjc23) of Schistosoma japonicum and Human Non-Immune IgG

BackgroundSchistosomal parasites can establish parasitization in a human host for decades; evasion of host immunorecognition including surface masking by acquisition of host serum components is one of the strategies explored by the parasites. Parasite molecules anchored on the membrane are the main elements in the interaction. Sjc23, a member of the tetraspanin (TSP) family of Schistosoma japonicum, was previously found to be highly immunogenic and regarded as a vaccine candidate against schistosomiasis. However, studies indicated that immunization with Sjc23 generated rapid antibody responses which were less protective than that with other antigens. The biological function of this membrane-anchored molecule has not been defined after decades of vaccination studies.Methodology and Principal FindingsIn this study, we explored affinity pull-down and peptide competition assays to investigate the potential binding between Sjc23 molecule and human non-immune IgG. We determined that Sjc23 could bind human non-immune IgG and the binding was through the interaction of the large extra-cellular domain (LED) of Sjc23 (named Sjc23-LED) with the Fc domain of human IgG. Sjc23 had no affinity to other immunoglobulin types. Affinity precipitation (pull-down assay) in the presence of overlapping peptides further pinpointed to a 9-amino acid motif within Sjc23-LED that mediated the binding to human IgG.Conclusion and Significance S. japonicum parasites cloak themselves through interaction with human non-immune IgG, and a member of the tetraspanin family, Sjc23, mediated the acquisition of human IgG via the interaction of a motif of 9 amino acids with the Fc domain of the IgG molecule. The consequence of this interaction will likely benefit parasitism of S. japonicum by evasion of host immune recognition or immunoresponses. This is the first report that an epitope of schistosomal ligand and its immunoglobulin receptor are defined, which provides further evidence of immune evasion strategy adopted by S. japonicum.

Pathogenesis and Treatment of Human Immunodeficiency Virus-Associated Cytomegalovirus Retinitis

In the era of HAART, human cytomegalovirus (HCMV) retinitis remains the leading opportunistic ocular infection and the major cause of blindness in patients with AIDS. The virus has been subjected to selection and presented with the opportunity to occupy a niche to which it is highly adapted in order to escape from host immune recognition and establish persistent infection in the retina. The imbalance between host immune protection and viral immune evasion results in retinitis progression. Moreover, a synergistic interaction between HCMV and HIV in the pathogenesis of retinitis has been proposed. HAART has had a major beneficial impact on the prognosis for HIV-infected individuals. Both HAART and specific anti-HCMV treatment contribute to therapeutic success against HCMV retinitis in AIDS patients. The improved prognosis for AIDS patients with respect to the development of HCMV retinitis has been welcomed; however, we should bear in mind the occurrence of HIV drug resistance, relapse of retinitis and immune recovery uveitis after treatment, which mean that this complication of HIV infection remains a threat.

Conflicting selection pressures target the NS3 protein in hepatitis C virus genotypes 1a and 1b

Analysis of complete polyprotein-encoding sequences of the two most prevalent genotypes of hepatitis C virus (HCV-1a and HCV-1b) revealed evidence of abundant, slightly deleterious nonsynonymous variants subject to ongoing purifying selection. In the case of both HCV-1a and HCV-1b, the NS3 protein demonstrated a high incidence of forward-and-backward or parallel nonsynonymous changes in CTL epitopes as measured by the phylogenetic consistency index. These results imply that certain nonsynonymous mutations have occurred frequently throughout the HCV-1a and HCV-1b phylogenies in the codons encoding the epitopes in NS3. This pattern is best explained by the frequent re-occurrence of the same set of escape mutations in CTL epitopes of NS3, which are selectively favored within hosts presenting the class I major histocompatability complex molecule, but subject to purifying selection in the population at large. This pattern was more pronounced in HCV-1b than in HCV-1a, suggesting that there may be differences between the two genotypes with respect to NS3's interaction with host immune recognition.

Virus-Encoded Homologs of Cellular Interleukin-10 and Their Control of Host Immune Function

The capacity of viruses to successfully infect the immunocompetent host to cause disease argues in favor of virus-encoded functions that specifically target components of the immune system so as to orchestrate an environment that limits the capacity of the host immune response to clear infection. In this respect, many viruses have evolved to coexist with the host immune system by developing an arsenal of strategies to avoid immune surveillance and elimination from the host. These include viruses which have acquired homologs of cellular cytokines or cytokine receptors as a strategy to limit host immune recognition. Cellular interleukin-10 (IL-10) is a pleiotropic immunomodulatory cytokine produced by a wide variety of cells, including monocytes, macrophages, T and B lymphocytes, dendritic cells (DC), keratinocytes, epithelial cells, and mast cells. The properties of IL-10 have been comprehensively reviewed elsewhere (12, 69, 70, 72, 77) and so will not be covered in detail here, but the key features of this cytokine relate mainly to its capacity to exert potent immunosuppressive functions on the expression of a range of cytokines and chemokines (2, 16, 23), as well as the repression of major histocompatibility complex (MHC) molecules and costimulatory molecules (17, 104) and the maturation and function of DC (69). The immunosuppressive properties of IL-10 are primarily restricted to cells of the myeloid lineage (69). In contrast, IL-10 has been shown to exert a stimulatory effect on B lymphocytes (15, 28, 87), mast cells (99), thymocytes (64), and CD8+ T cells (31, 88, 90), highlighting the cell-type-dependent immunomodulatory properties of this cytokine. The immunomodulatory functions manifested by IL-10 require engagement of this cytokine with its cell surface bound receptor. The IL-10 receptor (IL-10R) consists of two different subunits (IL-10R1 and IL-10R2) (52, 60). IL-10 binds first to IL-10R1 with high affinity, and the resulting intermediate IL-10/IL-10R1 complex then binds with lower affinity to IL-10R2. The resulting active signaling complex induces the JAK/Stat signal transduction pathway (69, 72). In the context of viral pathogenesis, infections with a number of different viruses have been documented to upregulate the expression of IL-10, and in some cases, this upregulation has been shown to enhance infection by suppressing the immune function, suggesting that the far-reaching effects of this cytokine have many advantages for invading pathogens (3, 4, 18, 30, 45, 81, 82, 108, 111). IL-10-like open reading frames (ORF) have been identified by sequence homology in multiple members of the Herpesvirales and Poxviridae, all but one of which infect mammalian hosts (Table ​(Table1).1). The one exception, cyprinid herpesvirus 3, is a member of the Alloherpesviridae family of herpesviruses (order, Herpesvirales), which has the common carp (Cyprinus carpio) as its normal host (1). Otherwise, all of the herpesviruses identified to date as encoding IL-10-like ORF, including human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), are found only in the Beta- and Gammaherpesvirinae subfamilies of the Herpesviridae. No examples of the Alphaherpesvirinae that encode IL-10 homologs have been identified. Of the identified members of the Herpesviridae that encode IL-10 homologs, all but two, equid herpesvirus 2 and ovine herpesvirus 2 (OvHV2) (85, 96, 97), are confined to primate hosts (Table ​(Table1).1). All of the identified Poxviridae that encode IL-10 infect ruminants, including orf virus ([ORFV] sheep and goats), sheeppox virus ([SPPV] sheep), goatpox virus ([GPV] goats), and lumpy skin disease virus ([LSDV] cattle). The genomes of SPPV, GPV, and LSDV are 96 to 97% identical at the nucleic acid level (48, 101). Another member of the Poxviridae that has monkeys as its normal host, yaba-like disease virus, encodes an ORF (Y134R) that exhibits sequence homology to the IL-10-related cytokines IL-19, IL-20, and IL-24 (57). These viral IL-10 (vIL-10) homologs range in size from 139 to 191 amino acids (aa), bracketing the range in sizes for cellular IL-10 proteins (176 to 180 aa). TABLE 1. Viruses with IL-10-like or IL-10-related ORF The role of virus-encoded IL-10 homologs is likely to provide a tool to enable modulation of the local immune response so as to enhance the capacity to replicate, disseminate, and/or persist in an otherwise immunocompetent individual. In fact, there is emerging evidence that virus-encoded IL-10 homologs function in this capacity in a variety of settings. This review will cover those viruses which have thus far been identified as encoding homologs of IL-10. The similarities of each homolog to the IL-10 of the natural host species will be presented together with their biological functions (where known) and the role they may play in viral pathogenesis and evasion of the host immune response.

Functional PEG–peptide hydrogels to modulate local inflammation inducedby the pro-inflammatory cytokine TNFα

Hydrogels are an important class of biomaterials for cell encapsulation and delivery, providing a physical barrier or “immuno-isolation” between the host tissue and encapsulated cells. The semi-permeable gel protects the encapsulated cells from host immune cells and/or antibody recognition while allowing facile diffusion of nutrients. However, a previously un-addressed problem is that highly permissive hydrogels cannot exclude the infiltration of soluble immune-mediators, such as pro-inflammatory cytokines that are highly expressed in wounded environments in vivo. When encountered with pro-inflammatory cytokines, encapsulated cells fail to perform their desired functions. Here, we report the synthesis, characterization, and application of peptide-functionalized, cytokine-antagonizing poly(ethylene glycol) (PEG) hydrogels capable of sequestering the pro-inflammatory cytokine, tumor necrosis factor α (TNFα). Results demonstrate that the survival, function, and differentiation of encapsulated cells (e.g., rat adrenal pheochromocytoma cells – PC12s, mouse pancreatic islets, and human mesenchymal stem cells or hMSCs) are significantly hindered in un-modified PEG hydrogels under in vitro TNFα treatments. In contrast, cells encapsulated in TNFα-antagonizing hydrogels are un-affected by the infiltrated TNFα. This study demonstrates the importance of controlling the availability of pro-inflammatory cytokines in highly permissive hydrogels.

Viral sequence diversity: challenges for AIDS vaccine designs

Among the greatest challenges facing AIDS vaccine development is the intrinsic diversity among circulating populations of HIV-1 in various geographical locations and the need to develop vaccines that can elicit enduring protective immunity to variant HIV-1 strains. While variation is observed in all of the viral proteins, the greatest diversity is localized to the viral envelope glycoproteins, evidently reflecting the predominant role of these proteins in eliciting host immune recognition and responses that result in progressive evolution of the envelope proteins during persistent infection. Interestingly, while envelope glycoprotein variation is widely assumed to be a major obstacle to AIDS vaccine development, there is very little experimental data in animal or human lentivirus systems addressing this critical issue. In this review, the state of vaccine development to address envelope diversity will be presented, focusing on the use of centralized and polyvalent sequence design as mechanisms to elicit broadly reactive immune responses.

Extracellular Gelsolin Binds Lipoteichoic Acid and Modulates Cellular Response to Proinflammatory Bacterial Wall Components

The various functions of gelsolin in extracellular compartments are not yet clearly defined but include actin scavenging and antiinflammatory effects. Gelsolin was recently reported to bind endotoxin (LPS) from various Gram-negative bacteria with high affinity. In this study we investigate whether gelsolin also interacts with bacterial wall molecules of Gram-positive bacteria such as lipoteichoic acid (LTA) and whether gelsolin's interaction with bacterial lipids from Gram-negative or Gram-positive bacteria affects their cellular inflammatory responses. A peptide based on the PPI binding site of gelsolin (160-169) binds purified LTA at the same molecular ratio that it binds phosphatidylinositol 4,5-bisphosphate. The OD of recombinant human plasma gelsolin was found to decrease following the addition of purified LTA, and the binding of gelsolin to LTA inhibits F-actin depolymerization by gelsolin. Simultaneously, the ability of LTA to activate translocation of NF-kappaB, E-selectin expression, and adhesion of neutrophils to LTA-treated human aortic endothelial cells was compromised by gelsolin. Gelsolin was able to partially inhibit LPS- or LTA-induced release of IL-8 from human neutrophils but was unable to prevent Gram-positive Bacillus subtilis or Gram-negative Pseudomonas aeruginosa growth and had no effect on the antibacterial activity of the cathelicidin-derived antibacterial peptide LL37. These data suggest that extracellular gelsolin is involved in the host immune recognition of LTA or LPS following release of these molecules from the bacterial outer membrane during cell division or attack by drugs and immune components.

Association of antibodies to hepatitis C virus glycoproteins 1 and 2 (anti‐E1E2) with HCV disease

Hepatitis C virus (HCV) causes acute and chronic liver diseases in humans. Its two envelope glycoproteins, E1 and E2, provide a target for host immune recognition. HCV genotypes are classified into six genetic groups. To study the role of anti-HCV E1 and E2 (anti-E1E2) in HCV disease, the correlation between antibody level and viral load, genotype, disease severity and response to treatment was investigated. The levels of antibodies to HCV glycoproteins E1 and E2 antibodies were evaluated in 230 sera of patients with chronic hepatitis C by enzyme-linked immunosorbent assay. The antigens used were recombinant HCV glycoproteins derived from genotype 1 (H77c) and genotype 3 (UKN3A1.28). Seroreactivity was greater when sera were tested against antigen derived from their homologous genotype than against heterologous antigen. Reactivity against UKN3A1.28 in sera from patients infected with genotype 3 was significantly higher than corresponding reactivity between patients infected with genotype 1 and H77c. The seroreactivity was inversely proportional to the viral load and to the degree of liver fibrosis. The pre-treatment level of anti-E1E2 was higher in sustained responders to combination therapy. These results demonstrate that seroreactivity against E1E2 depends upon the genotypic origin of the E1E2 antigens and the infecting genotype, and suggest a possible protective effect of anti-E1E2 against disease progression.

Biochemical and Functional Characterization of Salp20, an Ixodes scapularis Tick Salivary Protein that Inhibits the Complement Pathway

Ixodes ticks are vectors of several pathogens including Borrelia burgdorferi. Tick saliva contains numerous molecules that facilitate blood feeding without host immune recognition and rejection. We have expressed, purified, and characterized Ixodes scapularis salivary protein 20 (Salp20), a potential inhibitor of the alternative complement pathway that shares homology with the Isac protein family. When analysed by SDS-PAGE and size exclusion chromatography, Salp20 was approximately 48 kDa, more than double its predicted mass, primarily due N- and O-linked glycosylations. Recombinant Salp20 inhibited the alternative complement pathway by dissociating the C3 convertase, and partially protected a serum sensitive species of Borrelia from lysis by normal human serum. We propose that Salp20 facilitates tick feeding and possibly protects tick-borne pathogens from complement components.

Murine Bone Marrow Stromal Progenitor Cells Elicit an In Vivo Cellular and Humoral Alloimmune Response

Stromal progenitor cells (SPC) exhibit immunosuppressive effects in vitro that have led to speculation regarding their capacity to evade host immune recognition and to treat autoimmune diseases and gravt-versus-host disease. However, there is little in vivo experimental data to support these immunologic claims. To assess immune recognition of SPC in vivo, we evaluated the immune response of animals transplanted with SPC. C57BL/6 (B6) or Balb/c adult, murine, bone marrow-derived SPC (AmSPC) were administered by intraperitoneal injection into B6 recipients. T cell proliferation and alloantibody response was assessed from spleens and peripheral blood harvested from transplanted animals and analyzed by cell proliferation assay and flow cytometry. To assess tolerance induction, transplanted animals also received allogeneic skin grafts. Animals injected with allogeneic AmSPC mounted an accelerated CD4 response to alloantigen compared to syngeneic AmSPC injected and uninjected controls. Allogeneic AmSPC-injected animals also demonstrated high titers (≥1:1000) of antibody directed against allogeneic AmSPC targets. Animals primed with donor or host-matched AmSPC also failed to induce tolerance, and all animals exhibited rejection of allogeneic skin grafts (n = 7, P < .0001). In contrast to their in vitro behavior, our data demonstrate that AmSPC are recognized by the host immune system in vivo, elicit a cellular and humoral immune response, and fail to induce tolerance. These findings have significant implications for all allogeneic SPC-based therapeutic strategies.

Preferential Activation of Toll-Like Receptor Nine by CD46-Utilizing Adenoviruses

Adenoviruses (Ads) are responsible for respiratory, ocular, and gastrointestinal illnesses in humans. While the majority of serotypes utilize coxsackievirus-adenovirus receptor (CAR) as their primary attachment receptor, subgroup B and subgroup D Ad37 serotypes use CD46. Given the propensity of Ad vectors to activate host immune responses, we sought to investigate their potential for type I interferon induction. We found that CD46 Ads were capable of alpha interferon (IFN-alpha) induction by peripheral blood mononuclear cells and that plasmacytoid dendritic cells (pDCs) were the principal producers of this cytokine. IFN-alpha induction correlated with the permissivity of pDCs to CD46- but not CAR-utilizing Ad serotypes. A role for Toll-like receptor 9 (TLR9) recognition of Ad was supported by the requirement for viral DNA and efficient endosomal acidification and by the ability of a TLR9-inhibitory oligonucleotide to attenuate IFN-alpha induction. Cell lines expressing TLR9 that are permissive to infection by both CAR- and CD46-utilizing serotypes showed a preferential induction of TLR9-mediated events by CD46-utilizing Ads. Specifically, the latter virus types induced higher levels of cytokine expression and NF-kappaB activation in HeLa cells than CAR-dependent Ad types, despite equivalent infection rates. Therefore, infectivity alone is not sufficient for TLR9 activation, but this activation instead is regulated by a specific receptor entry pathway. These data reveal a novel mode of host immune recognition of Ad with implications for Ad pathogenesis and for the use of unconventional Ad vectors for gene delivery and vaccine development.

Evidence of Host Immune Recognition of Allogeneic Mesenchymal Stem Cells.

The immunologic properties of Mesenchymal Stem Cells (MSCs) are of therapeutic interest. Previous work shows MSCs do not elicit an alloreactive T cell response in vitro due to suppressive mechanisms. These results suggested that allogeneic MSCs could be used in vivo without inducing an immune response. We further explored this hypothesis using a well-characterized population of Adult Murine Mesenchymal Stem Cells (AmMSCs). These AmMSC are free of hematopoietic contamination and have the following immunologic phenotype: Class I +, Class II-, CD40-, CD80+, and CD86−. Upon treatment with interferon gamma, upregulation of Class I, Class II, CD80, and CD86 was observed. T cell proliferation assays were performed using CFDA SE tracking dye and T cell subsets were analyzed using dual color flow cytometry. The AmMSCs were capable of suppressing CD4+ and CD8+ T cell proliferation in response to alloantigen or polyclonal mitogen, independent of MHC matching, when cultured in direct contact with the T cells. Intracellular cytokine staining of CD4+ and CD8+ T cells revealed that interferon gamma and IL-10 production increased in co-cultures with AmMSC. This suggested that AmMSC are recognized by T cells, but are suppressing proliferation due to other mechanisms in vitro.In order to determine if donor AmMSCs are detected by an immunocompetent host in vivo, we conducted the following study. C57/B6 mice received an intraperitoneal injection of either one million C57/B6 GFP AmMSC (congenic, n=5), one million Balb/c AmMSC (allogeneic, n=5) , or 5 million Balb/c splenocytes (allogeneic control, n=5) at time point zero, and then were given an additional injection of the same cells at 4 weeks. Mice were bled at 0,2,4,5, and 6 weeks after the first injection. Serum was collected and assayed for alloantibody production. Alloantibody results revealed IgG and IgM responses against donor alloantigen at titers of greater than 1:100 in all 5 animals injected with Balb/c MSC. This was significantly increased over titers observed in the 5 mice injected with congenic C57/B6 GFP AmMSC, and similar to the titers seen in mice injected with allogeneic splenocytes. At six weeks post injection animals were sacrificed and a mixed lymphocyte reaction (MLR) was performed using host splenocytes as responders and irradiated donor splenocytes as stimulators. Splenocytes were stained using CFDA SE tracking dye and stained for CD4+ and CD8+ positive T cells. Proliferation of CD4+ and CD8+ T cells was measured using dual color flow cytometry. No increase in proliferation compared to a naïve MLR was noted in the animals injected with the congenic C57/B6 GFP AmMSCs. However all animals injected with allogeneic AmMSCs or allogeneic splenocytes showed increased CD4+ and CD8+ proliferation.It has been suggested primarily based on their in vitro properties, that MSCs may evade the immune response or induce tolerance after allogeneic transplantation. Our results document immune recognition of AmMSCs in vitro and in vivo. While we observed suppression of T-cell proliferation in vitro, our results after in vivo administration support a specific allogeneic immunization response equivalent to that induced by allogeneic splenocytes. These results argue against the theory that allogeneic MSCs may induce tolerance after transplantation, or that they can escape immune surveillance.

Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors

Most viruses exploit a variety of host cellular proteins as primary cellular attachment receptors in the context of successful execution of infection. Furthermore, many viral agents have evolved precise mechanisms to subvert host immune recognition to achieve persistence. Herein we present data indicating that adenovirus (Ad) serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. CD80 and CD86 are co-stimulatory molecules that are present on mature dendritic cells and B lymphocytes and are involved in stimulating T-lymphocyte activation. To our knowledge, this is one of the first demonstrations of a virus utilizing immunologic accessory molecules as a primary means of cellular entry. This finding suggests a mechanism whereby viral exploitation of these proteins as receptors may achieve both goals of cellular entry and evading the immune system.