Immune response to regressor and progressor tumor variants

Most skin cancers induced in mice by Ultraviolet (UV) radiation express highly immunogenic Tumor specific transplantation antigens (TSTAs) and thus exhibit a regressor phenotype. In this study, I have used cloned genes encoding tumor antigens and oncogenes in conjunction with DNA transfection technique to isolate and characterize regressor variants from progressor tumors and vice versa. The purpose of this study was (1) to determine whether the product of a cloned gene (216) from UV-1591 tumor, which encodes a novel MHC class I antigen can function as a tumor rejection antigen when expressed on unrelated, nonantigenic, murine tumor cells or whether its function is restricted to UV-induced tumors, and (2) to determine the processes by which progressor variants derived from a regressor UV-2240 cell line by transfection with an activated Ha-ras oncogene escape the immune defenses of the normal immunocompetent host.^ To answer the first question, a spontaneously transformed, nonimmunogenic cell line (10T-1) was cotransfected with DNA from p216 and pSV2-neo plasmids. Results demonstrate that the product of a cloned TSTA gene from a UV-induced murine tumor is capable of functioning as a tumor rejection antigen when expressed on unrelated, nonantigenic tumor cells. In addition, these results indicate that this approach could be used to augment the immune response against poorly antigenic tumors.^ To answer the second question, progressor variants were isolated from a highly antigenic UV radiation-induced C3H mouse regressor fibrosarcoma cell line, UV-2240, by transfection with an activated Ha-ras oncogene. Subcutaneous injection of Ha-ras-transfected UV-2240 cells into immunocompetent C3H mice produced tumors in 4 of 36 animals. In addition, the Ha-ras-induced progressor variants produced experimental lung metastasis in both normal C3H and nude mice, although they induced more lung nodules in nude mice than in normal C3H mice. Results indicate that the progressor phenotype of the Ha-ras-induced tumor variants is not due to loss of TSTAs or MHC class I antigens. This implies that some tumors can escape the immune defenses of the normal immunocompetent host by mechanisms other than the loss of TSTAs and MHC class I antigens. (Abstract shortened with permission of author.) ^

The immune response to the VlsE antigenic variation protein of Borrelia burgdorferi

The spirochete Borrelia burgdorferi (Bb) is the causative agent of Lyme disease. During infection, a strong immune response is elicited towards Bb by its host; however, the organism is able to persist and to disseminate to many different tissues. The vls locus is located on the linear plasmid lp28-1, a plasmid shown to be important for virulence in the mouse model. During infection, vlsE undergoes antigenic variation through a series of gene conversions, which results in the insertion of sequences from the silent, unexpressed cassettes into the vlsE cassette. We hypothesize that this antigenic variation is important in the spirochete's ability to persist within mammals by allowing it to evade the immune system. To define the role of vls in immune evasion, the immune response against VlsE was determined by using a recombinant form of VlsE (VlsE1-His) as an antigen to screen patient sera. Lyme patients produce antibodies that recognize VlsE, and these antibodies are present throughout the course of disease. Immunization with the VlsE1-His protein provided protection against infection with Bb expressing the same variant of VlsE (VlsE1), but was only partially protective when mice were infected with organisms expressing VlsE variants; however, subsequent VlsE immunization studies yielded inconsistent protection. Successful immunizations produced different antibody reactivities to VlsE epitopes than non-protective immunizations, but the reason for this variable response is unclear. In the process of developing genetic approaches to transform infectious Bb, it was determined that the transformation barrier posed by plasmids lp25 and lp56 could be circumvented by replacing the required lp25 gene pncA. To characterize the role of vlsE in infectivity, Bb lacking lp28-1 were complemented with a shuttle plasmid containing the lp25 encoded virulence determinant pncA and vlsE. Complemented spirochetes express VlsE, but the gene does not undergo antigenic variation and infectivity in the mouse model was not restored, indicating that either antigenic variation of vlsE is necessary for survival in the mouse model or that other genes on lp28-1 are important for virulence. ^

Comparison of compliance and immune response of drug users on the standard and accelerated hepatitis B vaccine schedules - Houston, Texas

Despite the availability of hepatitis B vaccine for over two decades, drug users and other high-risk adult populations have experienced low vaccine coverage. Poor compliance has limited efforts to reduce transmission of hepatitis B infection in this population. Evidence suggests that immunological response in drug users is impaired compared to the general population, both in terms of lower seroprotection rates and antibodies levels.^ The current study investigated the effectiveness of the multi-dose hepatitis B vaccine and compared the effect of the standard and accelerated vaccine schedules in a not-in-treatment, drug-using adult population in the city of Houston, USA.^ A population of drug-users from two communities in Houston, susceptible to hepatitis B, was sampled by outreach workers and referral methodology. Subjects were randomized either to the standard hepatitis vaccine schedule (0, 1-, 6-month) or to an accelerated schedule (0, 1-, 2-month). Antibody levels were detected through laboratory analyses at various time-points. The participants were followed for two years and seroconversion rates were calculated to determine immune response.^ A four percent difference in the overall compliance rate was observed between the standard (73%) and accelerated schedules (77%). Logistic regression analyses showed that drug users living on the streets were twice as likely to not complete all three vaccine doses (p=0.028), and current speedball use was also associated with non-completion (p=0.002). Completion of all three vaccinations in the multivariate analysis was also correlated with older age. Drug users on the accelerated schedule were 26% more likely to achieve completion, although this factor was marginally significant (p=0.085).^ Cumulative adequate protective response was gained by 65% of the HBV susceptible subgroup by 12-months and was identical for both the standard and accelerated schedules. Excess protective response (>=100 mIU/mL) occurred with greater frequency at the later period for the standard schedule (36% at 12-months compared to 14% at six months), while the greater proportion of excess protective response for the accelerated schedule occurred earlier (34% at 6 months compared to 18% at 12-months). Seroconversion at the adequate protective response level of 10 mIU/mL was reached by the accelerated schedule group at a quicker rate (62% vs. 49%), and with a higher mean titer (104.8 vs. 64.3 mIU/mL), when measured at six months. Multivariate analyses indicated a 63% increased risk of non-response for older age and confirmed the existence of an accelerating decline in immune response to vaccination manifesting after 40 years (p=0.001). Injecting more than daily was also highly associated with the risk of non-response (p=0.016).^ The substantial increase in the seroprotection rate at six months may be worth the trade-off against the faster antibody titer decrease and is recommended for enhancing compliance and seroconversion. Utilization of the accelerated schedule with the primary objective of increasing compliance and seroconversion rates during the six months after the first dose may confer early protective immunity and reduce the HBV vulnerability of drug users who continue, or have recently initiated, increased high risk drug use and sexual behaviors.^

Immune response to hepatitis B vaccination in drug using populations: A systematic review and meta-regression analysis

Hepatitis B virus (HBV) is a significant cause of liver diseases and related complications worldwide. Both injecting and non-injecting drug users are at increased risk of contracting HBV infection. Scientific evidence suggests that drug users have subnormal response to HBV vaccination and the seroprotection rates are lower than that in the general population; potentially due to vaccine factors, host factors, or both. The purpose of this systematic review is to examine the rates of seroprotection following HBV vaccination in drug using populations and to conduct a meta-analysis to identify the factors associated with varying seroprotection rates. Seroprotection is defined as developing an anti-HBs antibody level of ≥ 10 mIU/ml after receiving the HBV vaccine. Original research articles were searched using online databases and reference lists of shortlisted articles. HBV vaccine intervention studies reporting seroprotection rates in drug users and published in English language during or after 1989 were eligible. Out of 235 citations reviewed, 11 studies were included in this review. The reported seroprotection rates ranged from 54.5 – 97.1%. Combination vaccine (HAV and HBV) (Risk ratio 12.91, 95% CI 2.98-55.86, p = 0.003), measurement of anti-HBs with microparticle immunoassay (Risk ratio 3.46, 95% CI 1.11-10.81, p = 0.035) and anti-HBs antibody measurement at 2 months after the last HBV vaccine dose (RR 4.11, 95% CI 1.55-10.89, p = 0.009) were significantly associated with higher seroprotection rates. Although statistically nonsignificant, the variables mean age>30 years, higher prevalence of anti-HBc antibody and anti-HIV antibody in the sample population, and current drug use (not in drug rehabilitation treatment) were strongly associated with decreased seroprotection rates. Proportion of injecting drug users, vaccine dose and accelerated vaccine schedule were not predictors of heterogeneity across studies. Studies examined in this review were significantly heterogeneous (Q = 180.850, p = 0.000) and factors identified should be considered when comparing immune response across studies. The combination vaccine showed promising results; however, its effectiveness compared to standard HBV vaccine needs to be examined systematically. Immune response in DUs can possibly be improved by the use of bivalent vaccines, booster doses, and improving vaccine completion rates through integrated public programs and incentives.^

Antibody chemical reactivity: Beneficial and pathogenic roles

Antibodies (Abs) to autoantigens and foreign antigens (Ags) mediate, respectively, various pathogenic and beneficial effects. Abs express enzyme-like nucleophiles that react covalently with electrophiles. A subpopulation of nucleophilic Abs expresses proteolytic activity, which can inactivate the Ag permanently. This thesis shows how the nucleophilicity can be exploited to inhibit harmful Abs or potentially protect against a virus. ^ Inactivation of pathogenic Abs from Hemophilia A (HA) patients by means of nucleophile-electrophile pairing was studied. Deficient factor VIII (FVIII) in HA subjects impairs blood coagulation. FVIII replacement therapy fails in 20-30% of HA patients due to production of anti-FVIII Abs. FVIII analogs containing electrophilic phosphonate group (E-FVIII and E-C2) were hypothesized to inactivate the Abs by reacting specifically and covalently with nucleophilic sites. Anti-FVIII IgGs from HA patients formed immune complexes with E-FVIII and E-C2 that remained irreversibly associated under conditions that disrupt noncovalent Ab-Ag complexes. The reaction induced irreversible loss of Ab anti-coagulant activity. E-FVIII alone displayed limited interference with coagulation. E-FVIII is a prototype reagent suitable for further development as a selective inactivator of pathogenic anti-FVIII Abs. ^ The beneficial function of Abs to human immunodeficiency virus type 1 (HIV-1) was analyzed. HIV-1 eludes the immune system by rapidly changing its coat protein structure. IgAs from noninfected subjects hydrolyzed gp120 and neutralized HIV-1 with modest potency by recognizing the gp120 421-433 epitope, a conserved B cell superantigenic region that is also essential for HIV-1 attachment to host cell CD4 receptors. An adaptive immune response to superantigens is generally prohibited due to their ability to downregulate B cells. IgAs from subjects with prolonged HIV-1 infection displayed improved catalytic hydrolysis of gp120 and exceptionally potent and broad neutralization of diverse CCR5-dependent primary HIV isolates attributable to recognition of the 421-433 epitope. This indicates that slow immunological bypass of the superantigenic character of gp120 is possible, opening the path to effective HIV vaccination. ^ My research reveals a novel route to inactivate pathogenic nucleophilic Abs using electrophilic antigens. Conversely, naturally occurring nucleophilic Abs may help impede HIV infection, and the Abs could be developed for passive immunotherapy of HIV infected subjects. ^

The function of the murine complement *C3a and *C5a anaphylatoxin receptors in endotoxemia, bacteremia and septic shock

The complement system functions as a major effector for both the innate and adaptive immune response. Activation of the complement cascade by either the classical, alternative, or lectin pathway promotes the proteolysis of C3 and C5 thereby generating C3a and C5a. Referred to as anaphylatoxins, the C3a and C5a peptides mediate biological effects upon binding to their respective receptors; C3a binds to the C3a receptor (C3aR) while C5a binds to the C5a receptor (C5aR, CD88). Both C3a and C5a are known for their broad proinflammatory effects. Elevated levels of both peptides have been isolated from patients with a variety of inflammatory diseases such as COPD, asthma, RA, SLE, and sepsis. Recent studies suggest that C5a is a critical component in the acquired neutrophil dysfunction, coagulopathy, and progressive multi-organ dysfunction characteristic of sepsis. The primary hypothesis of this dissertation was that preventing C3a-C3aR and C5a-C5aR mediated pro-inflammatory effects would improve survival in endotoxic, bacteremic and septic shock. To test this hypothesis, the murine C3aR and C5aR genes were disrupted. Following disruption of both the C3aR and C5aR genes, no abnormalities were identified other than the absence of their respective mRNA and protein. In models of both endotoxic and bacteremic shock, C3aR deficient mice suffered increased mortality when compared to their wild type littermates. C3aR deficient mice also had elevated circulating IL-1β levels. Using a model of sepsis, C3aR deficient mice had a higher circulating concentration of IL-6 and decreased peritoneal inflammatory infiltration. While these results were unexpected, they support an emerging role for C3a in immunomodulation. In contrast, following endotoxic or bacteremic shock, C5aR deficient mice experienced increased survival, less hemoconcentration and less thrombocytopenia. It was later determined that C5a mediated histamine release significantly contributes to host morbidity and mortality in bacteremic shock. These studies provide evidence that C5a functions primarily as a proinflammatory molecule in models of endotoxic and bacteremic shock. In the same models, C3a-C3aR interactions suppress the inflammatory response and protect the host. Collectively, these results present in vivo evidence that C3a and C5a have divergent biological functions. ^

IMMUNE MODULATION OF THE MYCOBACTERIUM TUBERCULOSIS GRANULOMATOUS RESPONSE

Tuberculosis (TB) remains a major public health burden. The immunocompetant host responds to Mycobacterium tuberculosis (MTB) infection by the formation of granulomas, which initially prevent uncontrolled bacterial proliferation and dissemination. However, increasing evidence suggests that granuloma formation promotes persistence of the organism by physically separating infected cells from effector lymphocytes and by inducing a state of non-replicating persistence in the bacilli, making them resistant to the action of antibiotics. Additionally, immune-mediated tissue destruction likely facilitates disease transmission. The granulomatous response is in part due to mycobacterial glycolipid antigens. Therefore, studies were first undertaken to determine the innate mechanisms of mycobacterial cord factor trehalose-6’6-dimycolate (TDM) on granuloma formation. Investigations using knock-out mice suggest that TNF-a is involved in the initiation of the granulomatous response, complement factor C5a generates granuloma cohesiveness, and IL-6 is necessary for maintenance of an established granulomatous responses. Studies were next performed to determine the ability of lactoferrin to modulate the immune response and pathology to mycobacterial cord factor. Lactoferrin is an iron-binding glycoprotein with immunomodulatory properties that decrease tissue damage and promote Th1 responses. Mice challenged with TDM and treated with lactoferrin had decreased size and numbers of granulomas at the peak of the granulomatous response, accompanied by increased IL-10 and TGF-b production. Finally, the ability of lactoferrin to serve as a novel therapeutic for the treatment of TB was performed by aerosol challenging mice with MTB and treating them with lactoferrin added to the drinking water. Mice given tap water had lung log10 CFUs of 7.5 ± 0.3 at week 3 post-infection. Lung CFUs were significantly decreased in mice given lactoferrin starting the day of infection (6.4 ± 0.7) and mice started therapeutically on lactoferrin at day 7 after established infection (6.5 ± 0.4). Total lung inflammation in lactoferrin treated mice was significantly decreased, with fewer areas of macrophages, increased total lymphocytes, and increased numbers of CD4+ and CD8+ cells. The lungs of lactoferrin treated mice had increased CD4+ IFN-g+ cells and IL-17 producing cells on ELISpot analysis. It is hypothesized that lactoferrin decreases bacterial burden during MTB infection by early induction of Th1 responses.

Simultaneous induction of NKG2D and NKG2D ligand for promoting immune surveillance by IL-12 plus doxorubicin treatment

NKG2D (natural killer group 2, member D) and its ligands interaction in tumor microenvironment directs tumor infiltrating immune cells to recognize tumor cells, stimulate cytotoxic effector immune cells, and therefore eradicate tumor cells. IL-12, a cytokine produced by antigen presenting cells, has remarkable antitumor effect by activating innate and adaptive immunity. Doxorubicin, a commonly used chemotherapeutic agent also boosts the host antitumor immune response to cause tumor cell death. Our previous publication suggests that IL-12 plus doxorubicin enhances NKG2D function-dependent inhibition of tumor progression and promotes CD8+T cells infiltrating into tumors. The purpose of this study is to determine the underlying mechanism. Our study reveals a novel function of doxorubicin, which is to augment IL-12–induced NKG2D expression in CD8+T cells but not in NK or CD4+T cells. This observation was further validated by NK and CD8+T cell-depletion studies, in which only depletion of CD8+T cells abolished the expression of NKG2D in lymphocytes. The induced NKG2D expression in CD8+T cells is tightly associated with tumor-specific localization of CD8+T cells and improved antitumor efficacy. The IL-12 plus doxorubicin treatment-induced antitumor efficacy is also due to NKG2D ligand Rae-1 induction in tumors. Rae-1 induction in tumors is a long term effect in multiple tumor models, but not in normal tissues. A novel CD8+T cell direct contact dependent mechanism accounts for Rae-1 induction in vivo and in vitro, and CD80 is the receptor through which CD8+T cells interplay with tumor cells to upregulate Rae-1 on tumor cells. In summary, increased NKG2D expression in CD8+T cells in response to IL-12 plus doxorubicin was closely associated with tumor-specific localization of CD8+T cells and greater antitumor efficacy of the combined regimen than either agent alone. NKG2D ligand Rae-1 induction is triggered by the interaction of CD80 on tumor cells with tumor infiltrating CD+8 T cells.

Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

Early stages of experimental colon carcinogenesis inhibit mast cell-dependent mucosal immune function of the distal colon

Inhibition of local host immune reactions is one mechanism contributing to tumor progression. To determine if alterations in local immune functioning occur during colon carcinogenesis, a model mucosal immune response, type I hypersensitivity against the intestinal parasite Trichinella spiralis, was first characterized in normal mice and then examined during experimental colon carcinogenesis. Segments of sensitized colon mounted in Ussing chambers and challenged with T. spiralis-derived antigen resulted in a rise in short-circuit current ($\rm\Delta I\sb{sc}$) that was antigen-specific and inhibited by furosemide, implicating epithelial Cl$\sp-$ secretion as the ionic mechanism. The immune-regulated Cl$\sp-$ secretion by colonic epithelial cells required the presence of mast cells with surface IgE. Inhibition of potential anaphylactic mediators with various pharmacological agents in vitro implicated prostaglandins and leukotrienes as the principal mediators of the antigen-induced $\rm\Delta I\sb{sc}$, with 5-hydroxytryptamine also playing a role. Distal colon from immune mice fed an aspirin-containing diet (800 mg/kg powdered diet) ad libitum for 6 wk had a decreased response to antigen, confirming the major role of prostaglandins in generating the colonic I$\sb{\rm sc}$. To determine the effects of early stages of colon carcinogenesis on this mucosal immune response, mice were immunized with T. spiralis 1 day after or 8 wk prior to the first of 6 weekly injections of the procarcinogen 1,2-dimethylhydrazine (DMH). Responsiveness to antigenic challenge was suppressed in the distal colon 4-6 wk after the final injection of DMH. One injection of DMH was not sufficient to inhibit antigen responsiveness. The colonic epithelium remained sensitive to direct stimulation by exogenous Cl$\sp-$ secretagogues. Decreased antigen-induced $\rm\Delta I\sb{sc}$ in the distal colon was not due to systemic immune suppression by DMH, as the proximal colon and jejunum maintained responsiveness to antigen. Also, rejection of a secondary T. spiralis infection from the small intestine was not altered. Tumors eventually developed 25-30 wk after the final injection of DMH only in the distal portions of the colon. These results suggest that early stages of DMH-induced colon carcinogenesis manipulate the microenvironment such that mucosal immune function, as measured by immune-regulated Cl$\sp-$ secretion, is suppressed in the distal colon, but not in other regions of the gut. Future elucidation of the mechanisms by which this localized inhibition of immune-mediated ion transport occurs may provide possible clues to the microenvironmental changes necessary for tumor progression in the distal colon. ^

Immune responses against major histocompatibility complex and interleukin-2 gene transfected K1735 murine melanoma: Potential vaccines for the immunotherapy of cancer

Tumor specific immunity is mediated by cytotoxic T lymphocytes (CTL) that recognize peptide antigen (Ag) in the context of major histocompatibility complex (MHC) class I molecules and by helper T (Th) lymphocytes that recognize peptide Ag in the context of MHC class II molecules. The purpose of this study is (1) to induce or augment the immunogenicity of nonimmunogenic or weakly immunogenic tumors by genetic modification of tumor cells, and (2) to use these genetically altered cells in cancer immunotherapy. To study this, I transfected a highly tumorigenic murine melanoma cell line (K1735) that did not express constitutively either MHC class I or II molecules with syngeneic cloned MHC class I and/or class II genes, and then determined the tumorigenicity of transfected cells in normal C3H mice. K1735 transfectants expressing either $\rm K\sp{k}$ or $\rm A\sp{k}$ molecules alone produced tumors in normal C3H mice, whereas most transfectants that expressed both molecules were rejected in normal C3H mice but produced tumors in nude mice. The rejection of K1735 transfectants expressing $\rm K\sp{k}$ and $\rm A\sp{k}$ Ag in normal C3H mice required both $\rm CD4\sp+$ and $\rm CD8\sp+$ T cells. Interestingly, the $\rm A\sp{k}$ requirement can be substituted by IL-2 because transfection of $\rm K\sp{k}$-positive/A$\sp{\rm k}$-negative K1735 cells with the IL-2 gene also resulted in abrogation of tumorigenicity in normal C3H mice but not in nude mice. In addition, 1735 $(\rm I\sp+II\sp+)$ transfected cells can function as antigen presenting cells (APC) since they could process and present native hen egg lysozyme (HEL) to HEL specific T cell hybridomas. Furthermore, the transplantation immunity induced by K1735 transfectants expressing both $\rm K\sp{k}$ and $\rm A\sp{k}$ molecules completely cross-protected mice against challenge with $\rm K\sp{k}$-positive transfectants but weakly protected them against challenge with parental K1735 cells or $\rm A\sp{k}$-positive transfectants. Finally, I demonstrated that MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells can function as anti-cancer vaccines since they can abrogate the growth of established tumors and metastasis.^ In summary, my results indicate that expression of either MHC class I or II molecule alone is insufficient to cause the rejection of K1735 melanoma in syngeneic hosts and that both molecules are necessary. In addition, my data suggest that the failure of $\rm K\sp{k}$-positive K1735 cells to induce a primary tumor-rejection response in normal C3H mice may be due to their inability to induce the helper arm of the anti-tumor immune response. Finally, the ability of MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells to prevent growth of established tumors or metastasis suggests that these cell lines can serve as potential vaccines for the immunotherapy of cancer. (Abstract shortened by UMI.) ^

The effects of ultraviolet B radiation on immune responses to {\it Borrelia burgdorferi}, the causative agent of Lyme disease

Ultraviolet B (UVB) radiation, in addition to being carcinogenic, is also immunosuppressive. Immunologically, UVB induces suppression locally, at the site of irradiation, or systemically, by inducing the production of a variety of immunosuppressive cytokines. Systemic effects include suppression of delayed-type hypersensitivity (DTH) responses to a variety of antigens (e.g. haptens, proteins, bacterial antigens, or alloantigens). One of the principal mediators of UV-induced immune suppression is the T helper-2 (Th2) cytokine interleukin-10 (IL-10); this suggests that UV irradiation induces suppression by shifting the immune response from a Th1 (cellular) to a Th2 (humoral) response. These "opposing" T helper responses are usually mutually exclusive, and polarized Th1 or Th2 responses may lead to either protection from infection or increased susceptibility to disease, depending on the infectious agent and the route of infection.^ This study examines the effects of UVB irradiation on cellular and humoral responses to Borrelia burgdorferi (Bb), the causative agent of Lyme disease (LD) in both immunization and infectious disease models; in addition, it examines the role of T cells in protection from and pathology of Bb infection. Particular emphasis is placed on the Bb-specific antibody responses following irradiation since UVB effects on humoral immunity are not fully understood. Mice were irradiated with a single dose of UV and then immunized (in complete Freund's adjuvant) or infected with Bb (intradermally at the base of the tail) in order to examine both DTH and antibody responses in both systems. UVB suppressed the Th1-associated antibodies IgG2a and IgG2b in both systems, as well as the DTH response to Bb in a dose dependent manner. Injection of anti-IL-10 antibody into UV-irradiated mice within 24 h after UV exposure restored the DTH response, as well as the Th1 antibody (IgG2a and IgG2b) response. In addition, injecting recombinant IL-10 mimicked some of the effects of UV radiation.^ Bb-specific Th1 T cell lines (BAT2.1-2.3) were generated to examine the role of T cells in Lyme borreliosis. All lines were CD4$\sp+,$ $\alpha\beta\sp+$ and proliferated specifically in response to Bb. The BAT2 cell lines not only conferred a DTH response to naive C3H recipients, but reduced the number of organisms recovered from the blood and tissues of mice infected with Bb. Furthermore, BAT2 cell lines protected mice from Bb-induced periarthritis. ^

Antigenic variation in Lyme disease spirochetes by segmental recombination of VMP-like sequence cassettes

Lyme disease is a multisystemic disorder caused by tick-borne infection of humans or other mammalian hosts with Borrelia burgdorferi. If untreated, the spirochetes can persist in the mammalian host for months or years. The mechanisms by which Lyme disease spirochetes evade the immune response have not been determined. In this study, we have identified and characterized an elaborate genetic system in the Lyme disease spirochete B. burgdorferi that promotes extensive antigenic variation of a 34-kDa surface-exposed lipoprotein, VlsE. A 28-kilobase linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. The presence of lp28-1 correlates with the high-infectivity phenotype in B. burgdorferi strains tested. Segments of the 15 non-expressed (silent) vls cassette sequences located upstream of vlsE are able to recombine into the centra vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. When compared to parental VlsE, VlsE variants progressively accumulate sequence changes during the period of 4, 7, 14, 21, and 28 days post infection in C3H/HeN mice. However, no recombination was detected during the period of 28-day in vitro culture, suggesting in vivo induction of VlsE antigenic variation. Adaptive immune responses do not appear to play a significant role in this induction, since similar recombination events were also observed in immunodeficient SCID mice. The $5\sp\prime$ and $3\sp\prime$ noncassette regions of vlsE are apparently not subject to recombination and sequence variation. The structure and sequence of the silent vls cassette locus is preserved during the process of the VlsE antigenic variation, consistent with a nonreciprocal recombination mechanism. This combinatorial form of antigenic variation could potentially yield millions of VlsE variants in the mammalian host, and thereby contribute to immune evasion, long-term survival, and pathogenesis of B. burgdorferi. ^

Activation of dendritic cells from patients with epithelial ovarian cancer

Dendritic cells (DCs) are the most potent antigen-presenting cells for inducing immune responses to tumor cells. Lin−HLA-DR + DC populations in peripheral blood mononuclear cells (PBMCs) and in ascites mononuclear leukocytes (MNLs) of patients with epithelial ovarian cancer (EOC) are phenotypically immature. Lin−HLA-DR + DCs from PBMCs of normal subjects and EOC patients and MNLs from ascites cells of patients were examined for specific cell surface markers or indicators of differentiation or activation. Separating Lin− HLA-DR+ DCs into subsets based on their HLA-DR intensity provided an additional method for identifying the two major lineages of DCs, myeloid and plasmacytoid. The activation potential of these DCs following exposure to the maturation agents CD40 ligand (CD40L) and lipopolysaccharide (LPS) was examined by measurement of IL-12 and IL-10 concentrations in DC culture supernatants in addition to their ability to stimulate allogeneic T cells. DCs from PBMCs of normal subjects and EOC patients and DCs isolated from ascites MNLs of EOC patients were separated into subsets based on CD11c and CD123 cell surface marker expression identifying the major DC types. These subsets were then compared with cells sorted on the basis of HLA-DR intensity. The in vivo behavior of DCs and DC subsets in peripheral blood and ascites following treatment of peritoneal carcinoma patients with the growth factor fins-like tyrosine kinase 3 ligand (Flt3L) was also examined. Increases in proportions and total numbers of DCs from peripheral blood and ascites were associated with increased secretion of IL-12 and IL-10 following in vitro activation of cultured DCs. There were differences between DCs from PBMCs and ascites and between DC subsets in expression of cell surface markers, cytokine profile, and the ability of Lin−HLA-DR + cells to stimulate proliferation of allogeneic T cells from EOC patients. These Lin−HLA-DR+ cells have certain functional properties that suggest that they could have the potential to facilitate an adaptive anti-tumor immune response. ^

Mechanisms involved in suppression of the elicitation of immune responses by ultraviolet light

The ultraviolet radiation (UVR) present in sunlight is the primary cause of nonmelanoma skin cancer and has been implicated in the development of cutaneous malignant melanoma. Ultraviolet radiation also suppresses the immune response. In the majority of studies investigating the mechanisms regulating UV-induced immune suppression, UV is used to suppress the induction of immune responses. Equally important, is the ability of UVR to suppress established immune responses, such as the recall reaction in humans, which protects against microbial infections. We established a murine model to help elucidate the immunological mechanisms governing UV-induced suppression of the elicitation of immune responses. 80 kJ/m2 of UVR nine days after sensitization consistently suppressed the elicitation of delayed type hypersensitivity reaction to C. albicans . We found ultraviolet A (320±400 nm) radiation was as effective as solar-simulated ultraviolet A + B (290±400 nm) in suppressing the elicitation of an established immune response. The mechanisms involved in UV-induced suppression of the induction & elicitation of the immune response are similar. For example, mice irradiated with UV after immunization generated antigen-specific T suppressor cells. Injection of monoclonal antibodies to IL-10 or recombinant IL-12 immediately after exposure to UVR blocked immune suppression. Liposomes containing bacteriophage T4N5 to the skin of mice also prevented immune suppression, demonstrating an essential role for ultraviolet-induced DNA damage in the suppression of established immune reactions. ^ In addition to damaging DNA, UV initiates immune suppression through the isomerization of urocanic acid in the epidermis. Here we provide evidence that cis-UCA induces systemic immunosuppression via the serotonin (5-hydroxyyryptamine; 5-HT) receptor. Biochemical and immunological analysis indicate that cis-UCA binds to, and activates, the serotonin receptor. Moreover, serotonin specific antibodies block UV- and/or cis-UCA-induced immune suppression. Our findings identify cis-UCA as novel serotonin receptor ligand and indicate that serotonin receptor engagement can activate immune suppression. Cumulatively, our data suggest that similar immune regulatory mechanisms are activated regardless of whether we expose mice to solar-simulated UV (UVA + UVB) radiation or UVA only, and that ultraviolet radiation activates similar immunologic pathways to suppress the induction or the elicitation of the immune response. ^