Search

Polymorphonuclear neutrophils (PMNs), the main effectors of the innate immune system, have rarely been considered as an anticancer therapeutic tool. However, recent investigations using animal models and preliminary clinical studies have highlighted the potential antitumor efficacy of PMNs. In the current study, we find that PMNs from some healthy donors naturally have potent cancer-killing activity against 4 different human cancer cell lines. The killing activity appears to be cancer cell-specific since PMNs did not kill primary normal epithelial cells or an immortalized breast epithelial cell line. Transfecting the immortalized mammary cells with plasmids expressing activated forms of the rat sarcoma viral oncogene homolog (Ras) and teratocarcinoma oncogene 21 (TC21) oncogenes was sufficient to provoke aggressive attack by PMNs. However, transfection with activated Ras-related C3 botulinum toxin substrate (Rac1) was ineffective, suggesting specificity in PMN-targeting of neoplastic cells. Furthermore, PMNs from lung cancer patients were also found to exhibit relatively poor cancer-killing activity compared to the cytolytic activity of the average healthy donor. Taken together, our results suggest that PMN-based treatment regimens may represent a paradigm shift in cancer immunotherapy that may be easily introduced into the clinic to benefit a subset of patients with PMN-vulnerable tumors.

Ab therapy against surface Ags on tumor cells has demonstrated significant efficacy for some cancers. However, it is costly and patients frequently develop acquired resistance over time. In cases of Ab therapy resistance, T cell responses have been shown to be essential in controlling disease progression. Thus, vaccination that generates a sustained Ab response as well as a T cell response may be more effective and economical. In this article, we have developed a vaccination strategy by targeting protein Ags to B cells via a CD19 single-chain variable fragment miniAb. Using the tumor-associated Ag her-2/neu extracellular domain, we showed that the coengagement of CD19 and BCR induced full B cell activation to produce a high titer of Abs and enhanced CD4 Th2 response and CD8 T cell activation and differentiation. These Abs competitively inhibited humanized her-2/neu Ab binding and were capable of activating the complement and inhibiting human breast cancer growth in vitro. Therapeutic efficacy was demonstrated in vivo using murine mammary carcinoma models. Furthermore, four different extracellular domains of her-2/neu could be targeted to B cells to generate Abs against particular domains with different antitumor properties. This approach may offer a new avenue for vaccine development with significantly lower cost, which may be of use not only for cancer therapy but also for infectious agents.

A variant of the integrin-α-M (CD11b) gene has been linked to the pathogenesis of systemic lupus erythematosus. However, how this genotype results in the lupus phenotype is not fully understood. Here we show that autoreactive B cells lacking CD11b exhibit a hyperproliferative response to B cell receptor (BCR) crosslinking and enhanced survival. In vivo engagement of BCR in CD11b-deficient mice leads to increased autoAb production and kidney Ig deposition. In addition, CD11b-deficient autoreactive B cells have decreased tyrosine phosphorylation including Lyn and CD22 with decreased phosphatase SHP-1 recruitment but increased calcium influx. Results obtained using B cells transfected with the wild type or rs1143679 lupus-associated variant of CD11b suggest that this mutation completely abrogates the regulatory effect of CD11b on BCR signalling. This is through disruption of CD22-CD11b direct binding. These results reveal a previously unrecognized role of CD11b in maintaining autoreactive B cell tolerance.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with suppressive properties that preferentially expand in cancer. Myeloid-derived suppressor cells mainly suppress T-cell proliferation and cytotoxicity, inhibit natural killer cell activation, and induce the differentiation and expansion of regulatory T cells. The wide spectrum of MDSC suppressive activity in cancer and its role in tumor progression have rendered these cells a promising target for effective cancer immunotherapy. In this review we briefly discuss the origin of MDSCs and their main mechanisms of suppression and focus more on the approaches developed up to date targeting of MDSCs in tumor-bearing animals and cancer patients.

A mutation in the Signal Transducer and Activator of Transcription 3 (STAT-3) has been linked to incidence of Autosomal Dominant Hyper Immunoglobulin E Syndrome (AD-HIES), a disease characterized by elevated serum IgE antibody and susceptibility to respiratory and skin infection. However, how this genetic mutation leads to the phenotype has not been fully understood. In this study we investigated the specific role of STAT-3 in the germinal center (GC), the site of B-cell proliferation and isotype switching. Through the use of CD2-Cre or CD19-Cre driven STAT-3 conditional knockout (cKO) mice in a Th2-type immunization model, we aimed to determine the specific cell subset(s) and signaling mechanism by which STAT-3 mediated isotype switching is regulated. We demonstrated in vivo and in vitro that CD2-Cre driven STAT-3 cKO mice showed elevated IgE and decreased IgG1 in the serum, and a reduction in GC formation. Within the GC, IgG1+ GC B cells were decreased while IgE+ GC B cells were more prevalent. Additionally, these mice exhibited reduced IgG1 and elevated IgE populations of antibody-producing plasma cells. Subsequent experiments using a CD19-Cre B-cell specific cKO mouse established this effect to be B-cell dependent. Our data also revealed other transcription factors, including Bcl-6 and Aicda, function as downstream signals of STAT-3 regulation. This model suggests a role for STAT-3 in regulating class switching of the GC B cells from the IgG1 to the IgE producing state, which may serve as a potential therapeutic target for treatment of AD-HIES and other immune disorders.

Emerging evidence has unveiled the contribution of tumor microenvironment to the regulation of chemotherapeutic outcomes. However, the impact of chemotherapy on anti-tumor immunity and immunosuppression still remains elusive. In this study, we investigated the role of chemotherapy-derived inflammatory responses in the differentiation of myeloid-derived suppressive cells (MDSC). The anti-cancer agents Gemcitabine and Carboplatin induced the activation of MAPK pathway in mouse mammary cancer cell line EO771 and also stimulated cytokine and chemokine production including GM-CSF and IL-6. The supernatants of Gemcitabine-treated tumor cells promoted more bone marrow-derived monocytic MDSC differentiation, and enhanced their immunosuppressive activity on effector T cells. The expression of MDSC associated molecules such as Arginase and iNOS significantly increased in M-MDSC induced by the supernatant of Gemcitabine-treated tumor cells. In vivo treatment of EO771 tumors with Gemcitabine increased the suppressive function of MDSC. Our findings also suggest that Dectin-1 activation by β-glucan could significantly reduce MDSC suppressive function. Together, these results describe a role of chemotherapy-derived inflammation in the modification of chemotherapy-treated tumor microenvironment and suggest the targeting of Dectin-1 signaling may benefit cancer patients by reprogramming MDSC in chemotherapy setting.

We show for the first time that signaling through the TLR4/TRIF pathway plays a critical role in allogeneic bone marrow cell (BMC) rejection. This appears to be unique to BMCs as organ allografts are rejected mainly via MyD88 signaling. Using T- or T-/B-cell-deficient mice, we found that BMC allorejection occurred early before T-cell activation and was T- and B-cell independent, suggesting an effector role for innate immune cells in BMC rejection. We further demonstrated the innate immune signaling in BMC allorejection by showing superior engraftment in mice deficient in TRIF or TLR4 but not in MyD88 or TLR3. The restored cytotoxicity in TRIF-deficient recipients transferred with wild-type F4/80+ or NK1.1+ cells suggests TRIF signaling dependence on macrophages or NK cells in early BMC rejection. Production of the proinflammatory cytokine IL-6 and TRIF relevant chemokine MCP-1 was significantly increased early after bone marrow transplantation. In vivo specific depletion of macrophages or NK innate immune cells in combination with anti-CD154/rapamycin resulted in additive-enhanced allogeneic engraftment. The requirement for irradiation was completely eliminated when both macrophages and NK cells were depleted in combination with anti-CD154/rapamycin to target T- and B-cells, supporting the hypothesis that two barriers involving innate and adaptive immunity exist in mediating the rejection of allogeneic BMCs. In summary, our results clearly demonstrate a previously unappreciated role for innate immunity in BMC allorejection via signaling through a unique MyD88-independent TLR4/TRIF mechanism. These findings may have direct clinical impact on strategies for conditioning recipients for stem cell transplantation.

Promoting complement (C) activation may enhance immunological mechanisms of anti-tumor Abs for tumor destruction. However, C activation components, such as C5a, trigger inflammation, which can promote tumor growth. We addressed the role of C5a on tumor growth by transfecting both human carcinoma and murine lymphoma with mouse C5a. In vitro growth kinetics of C5a, control vector, or parental cells revealed no significant differences. Tumor-bearing mice with C5a-transfected xenografted tumor cells had significantly less tumor burden as compared with control vector tumors. NK cells and macrophages infiltrated C5a-expressing tumors with significantly greater frequency, whereas vascular endothelial growth factor, arginase, and TNF-α production were significantly less. Tumor-bearing mice with high C5a-producing syngeneic lymphoma cells had significantly accelerated tumor progression with more Gr-1+CD11b+ myeloid cells in the spleen and overall decreased CD4+ and CD8+ T cells in the tumor, tumor-draining lymph nodes, and the spleen. In contrast, tumor-bearing mice with low C5a-producing lymphoma cells had a significantly reduced tumor burden with increased IFN-γ–producing CD4+ and CD8+ T cells in the spleen and tumor-draining lymph nodes. These studies suggest concentration of local C5a within the tumor microenvironment is critical in determining its role in tumor progression.

Summary Interleukin-23 (IL-23) and CD4 + T helper 17 (Th17) cells are thought to be critical in psoriasis pathogenesis. Here, we report that IL-23 predominantly stimulated dermal γδ T cells to produce IL-17 that led to disease progression. Dermal γδ T cells constitutively expressed the IL-23 receptor (IL-23R) and transcriptional factor RORγt. IL-17 production from dermal γδ T cells was independent of αβ T cells. The epidermal hyperplasia and inflammation induced by IL-23 were significantly decreased in T cell receptor δ-deficient ( Tcrd −/− ) and IL-17 receptor-deficient ( Il17ra −/− ) mice but occurred normally in Tcra −/− mice. Imiquimod-induced skin pathology was also significantly decreased in Tcrd −/− mice. Perhaps further promoting disease progression, IL-23 stimulated dermal γδ T cell expansion. In psoriasis patients, γδ T cells were greatly increased in affected skin and produced large amounts of IL-17. Thus, IL-23-responsive dermal γδ T cells are the major IL-17 producers in the skin and may represent a novel target for the treatment of psoriasis.

β-glucans have been reported to function as a potent adjuvant to stimulate innate and adaptive immune responses. However, β-glucans from different sources are differential in their structure, conformation, and thus biologic activity. Different preparations of β-glucans, soluble versus particulate, further complicate their mechanism of action. Here we show that yeast-derived particulate β-glucan activated dendritic cells (DCs) and macrophages via a C-type lectin receptor dectin-1 pathway. Activated DCs by particulate β-glucan promoted Th1 and cytotoxic T-lymphocyte priming and differentiation in vitro. Treatment of orally administered yeast-derived particulate β-glucan elicited potent antitumor immune responses and drastically down-regulated immunosuppressive cells, leading to the delayed tumor progression. Deficiency of the dectin-1 receptor completely abrogated particulate β-glucan–mediated antitumor effects. In contrast, yeast-derived soluble β-glucan bound to DCs and macrophages independent of the dectin-1 receptor and did not activate DCs. Soluble β-glucan alone had no therapeutic effect but significantly augmented antitumor monoclonal antibody-mediated therapeutic efficacy via a complement activation pathway but independent of dectin-1 receptor. These findings reveal the importance of different preparations of β-glucans in the adjuvant therapy and allow for the rational design of immunotherapeutic protocols usable in clinical trials.

Aims The effects on angiogenesis of a novel CXC chemokine receptor 4 (CXCR4) antagonist, SDF-1βP2G, derived from human stromal cell-derived factor-1β (SDF-1β), were examined in a model of hind limb ischaemia in mice. Methods and results The antagonistic activities of SDF-1βP2G against CXCR4 were evaluated in vitro and in vivo and compared with phosphate-buffered saline and AMD3100 (a small bicyclam antagonist of SDF-1). Angiogenesis, muscle regeneration and the expression of pro-angiogenic factors were evaluated in ischaemic gastrocnemius muscles. Distant toxic effects of SDF-1βP2G were evaluated by inflammatory and apoptotic markers. SDF-1βP2G induced CXCR4 internalization and competitively inhibited the chemotaxis of SDF-1β but did not mediate migration, calcium influx, or the phosphorylation of Akt and extracellular signal-regulated kinase in cultured T-lymphoblastic leukaemia cells or H9C2 cells. SDF-1βP2G enhanced blood flow, angiogenesis, and muscle regeneration in ischaemic hind limbs, and the enhancement was significantly better than that of AMD3100. Markers of angiogenesis and progenitor cell migration, including phosphorylated Akt, vascular endothelial growth factor (VEGF), SDF-1 and CXCR4, were up-regulated by SDF-1βP2G and co-localized with CD31-positive cells. Neutralization of VEGF with its specific antibody abolished SDF-1βP2G-induced blood reperfusion and angiogenesis. No apparent inflammatory and apoptotic effects were found in heart, liver, kidneys, and testes after SDF-1βP2G administration. Conclusion Our findings indicate that the novel CXCR4 antagonist, SDF-1βP2G, can efficiently enhance ischaemic angiogenesis, blood flow restoration, and muscle regeneration without apparent adverse effects, most likely through a VEGF-dependent pathway.

Sensitization is a critical unresolved challenge in transplantation. We show for the first time that blockade of CD154 alone or combined with T-cell depletion prevents sensitization. Allogeneic skin grafts were rejected by recipients treated with anti-αβ T-cell receptor (TCR), anti-CD154, anti-OX40L, or anti–inducible costimulatory pathway (ICOS) mAb alone with a kinetic similar to untreated recipients. However, the production of anti–donor MHC antibody was prevented in mice treated with anti-CD154 mAb only, suggesting a specific role for the CD154-CD40 pathway in B-cell activation. The impairment of T cell–dependent B-cell responses by blocking CD154 occurs through inhibiting activation of T and B cells and secretion of IFN-γ and IL-10. Combined treatment with both anti-CD154 and anti–αβ TCR abrogated antidonor antibody production and resulted in prolonged skin graft survival, suggesting the induction of both T- and B-cell tolerance with prevention of allogeneic sensitization. In addition, we show that the tolerance induced by combined treatment was nondeletional. Moreover, these sensitization-preventive strategies promote bone marrow engraftment in recipients previously exposed to donor alloantigen. These findings may be clinically relevant to prevent allosensitization with minimal toxicity and point to humoral immunity as playing a dominant role in alloreactivity in sensitized recipients.

IL-17 producing γδ T cells (γδ-T17) are major players in promoting several autoimmune inflammatory diseases and cancers. However, γδ-T17 homeostatic regulation has not been fully understood. In naïve, adult IL-17r-/- and IL-17A-/-/F-/- mice we observed higher frequencies of total γδ T cells, predominantly γδ-T17, in the lung, spleen, gut, LN and skin but not in the thymus. We hypothesized that the IL-17-IL-17r axis regulates peripheral γδ-T17 homeostasis. Two different regulatory pathways were identified to be involved in this regulation: extrinsically through microbiota-host interaction and intrinsically through IL-17r-IL-7r signaling interaction. The extrinsic pathway was discovered in vitro using CFSE-labeled IL-17r-/- lymphocytes that had endogenous γδ-T17 proliferation dependent upon DCs, particular CD103+ DCs, and IL-1β. Using mice delivered from IL-17r-/- pregnant females treated either with lymphotoxin beta receptor-Ig to remove LNs or antibiotics (ampicillin, vancomycin, neomycin, and metronidazole) to deplete gut microbiota we found significantly decreased frequency of γδ-T17 in peripheral tissues. Also, intrinsically IL-17r-/- γδ-T17 expressed higher levels of RORγt that suppressed BTLA expression, which is a known inhibitor of IL-7-driven γδ-T17 proliferation. Taken together, our findings suggest that the IL-17-IL-17r axis regulates γδ-T17 via both intrinsic and extrinsic pathways.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that promote tumor progression. Herein, we demonstrate that activation of a C-type lectin receptor, dectin-1, in MDSC differentially modulates the function of different MDSC subsets. Yeast-derived particulate β-glucan (WGP), a ligand to engage dectin-1, oral treatment in vivo significantly decreases tumor weight and splenomegaly in tumor-bearing mice by reducing the accumulation of PMN-MDSC but not M-MDSC in spleens and tumors, and decreases PMN-MDSC suppression in vitro through the induction of respiratory burst and apoptosis. On a different axis, WGP-treated M-MDSC differentiate into F4/80+CD11c+ cells that can uptake and present OVA antigen to OVA-specific CD4+ T cells and cross-present antigen to OVA-specific CD8+ T cells in a dectin-1 dependent manner. In addition, ERK1/2 phosphorylation is required for the acquisition of APC properties in M-MDSC, since M-MDSC treated with ERK inhibitor and WGP do not differentiate into APC. Moreover, WGP-treated M-MDSC do not promote tumor growth in vivo when inoculated subcutaneously with LLC cells. Interestingly, patients with non-small cell lung cancer (NSCLC) that received WGP treatment prior to any treatment for two weeks had a decreased frequency of CD14-HLA-DR-CD11b+CD33+ in the peripheral blood. Overall, these data indicate that WGP may be a potent immune modulator of MDSC suppressive function and differentiation in cancer.

Antibody maturation as well as memory B and plasma cell differentiation occurs primarily in the germinal center (GC). Lupus may develop as a result of enhanced GC activity. Many factors involved in GC reaction, including T follicular helper (Tfh) cells, IL-21, IL-6, play critical roles in lupus pathogenesis. These inflammatory cytokines predominantly activate the STAT3 signaling pathway. Here, we demonstrate that STAT3 signaling in B cells is essential for antibody response as well as GC formation and maintenance. Increased cell apoptosis and down-regulated Bcl-xL expression are found in the STAT3 deficient GC B cells. The Tfh cell response is significantly decreased in B cell STAT3-deficient immunized mice. Further, STAT3 deficiency in autoreactive B cells results in decreased autoantibody production upon immunization with apoptotic cells. Results obtained using B cell STAT3 deficient MRL.Fas(lpr) mice suggest that STAT3 signaling contributes to the pathogenesis of lupus by regulation of autoantibody production. Our findings thus provide new insights into the role of B cell STAT3 signaling in the GC maintenance and pathogenesis of lupus.

To study the effects of dendritic cells (DC) transfected with recombinant adenovirus encoding Epstein-Barr virus(EBV)latent membrane protein 2A(LMP2A)gene, and to provide evidence for further investigation on the therapeutic vaccine against EBV-associated malignancies.DCs were transfected with EBV-LMP2A recombinant adenovirus (Ad5-LMP2A), which was generated by homologous recombination. The expression of LMP2A protein on mature DC transfected with Ad5-LMP2A at different multiplicity of infection(MOI)was analyzed by fluorescence activated cell sorting(FACS), and the dead cells were counted by trypan blue staining. The alteration of surface markers on mature DC including CD1a, CD83, CD40, CD80, HLA?DR was detected by means of FACS before and after transfection. Meanwhile, the functions including stimulating allogenetic T cells reaction and expressing IL12 P40 mRNA on transfected DC were measured by methods of 3H-thymidine uptake and fluorescent semi?quantitative polymerase chain reaction (PCR), respectively.About 80% mature DC expressed LMP2A protein and92% cells were viable after transfection at the MOI of 200 No. significant changes in the surface markers and the cytomorphology of mature DCs were detected during the transfection. Transfected DC still have strong potential to stimulate the proliferation of allogenetic T cells and to express IL-12 P40 mRNA.EBV-LMP2A gene,which was carried by adenovirus vectors, could be transferred into DC with high efficiency. The function of mature DC was not affected significantly by the transfection of Ad5-LMP2A.

Germinal center (GC) B cells and T follicular helper (Tfh) cells are critical for long-lived plasma cell differentiation and high-affinity antibody production in response to T cell-dependent antigens. Previous studies demonstrated that GC formation was intact in the absence of STAT3 in B cells at day 12. However, emerging evidence suggests that IL-21/STAT3 signaling is a potent regulator for B cell differentiation. To better understand the role of STAT3 in the kinetics of GC formation and GC B cell response, we assessed GC B cells at consecutive time points following immunization with antigen OVA. In B cell conditional STAT3-dificient mice, fewer GC and GC B cells were generated upon immunization in the late phase (day 21) but not on day 14 compared with that of control mice. Increased cell death and decreased cell proliferation were found in the STAT3 deficient GC B cells. Consequently, plasma cell differentiation and antibody production were impaired in B cell STAT3-deficient mice. In addition, the Tfh cell response was correlated with the magnitude of the GC B cell response and significantly decreased in B cell STAT3-deficient mice. Furthermore, GC B cells and autoantibody production were also significantly decreased in autoreactive B cell STAT3-deficient mice after apoptotic cell immunization. These data provide new insights into the GC and Tfh differentiation and the function of STAT3 in humoral immune responses.

Whole glucan particles (WGP) are microparticles of 1,3-beta glucan with 1,6-branches purified from the yeast Saccharamyces cerevisiae. Previous studies have reported its therapeutic efficacy in different tumor models through the activation of dendritic cells and macrophages via the c-type lectin receptor dectin-1. In this study, we investigated the effect of WGP treatment on myeloid-derived suppressor cells (MDSC) in mouse tumor models and in patients with non-small cell lung cancer (NSCLC). MDSC are a heterogeneous population of immature myeloid cells with suppressive activity that accumulate in cancer. Here we found that tumor-bearing mice treated orally with WGP have a significantly lower tumor weight with a significant decrease in the percentage of MDSC in the spleen compared to untreated animals in vivo. Patients with NSCLC treated with WGP prior to surgery have a decreased trend in MDSC percentage and increased respiratory burst of neutrophils in the peripheral blood. In addition, in vitro stimulation of MDSC subsets with WGP abrogates STAT3 phosphorylation in polymorphonuclear MDSC and enhances STAT3 phosphorylation in monocytic MDSC, with the activation of Erk1/2 phosphorylation in both subsets, suggesting that WGP differently modulates MDSC subsets. Moreover, MDSC treated with WGP have a decreased suppressive activity when cocultured with OVA-specific OTI and OTII splenocytes. Overall, these data indicate that WGP might be a new modulator of MDSC function in cancer.

The hallmark of systemic lupus erythematosus (SLE) is loss of immunological tolerance and B cell hyperactivity. Recent studies demonstrated an association between the variant of the integrin-α-M (ITGAM, also called CD11b) gene and SLE susceptibility, severity of disease, and pathogenic anti-dsDNA antibody production. However, the cellular and molecular mechanism by which CD11b regulates disease pathogenesis is not understood. We found that autoreactive B cells defective of CD11b exhibited hyperproliferative responsiveness to B cell receptor (BCR) cross-linking. CD11b deficiency promoted cell cycle progression and B cell survival, which was associated with elevated anti-apoptotic gene and protein levels. In vivo engagement of BCR resulted in increased autoAb production and kidney Ig deposition in CD11b-deficient mice. CD11b-deficient autoreactive B cells had the decreased tyrosine phosphorylation including Lyn and CD22 with decreased SHP-1 recruitment but increased calcium influx. Colocalization analysis revealed that the inhibitory effect of CD11b is due to its cis-interaction with CD22. Interestingly, B cells transfected with wildtype CD11b or rs1143679 (R77H) lupus associated variant of CD11b suggest that this mutation completely impairs CD11b regulatory effect on BCR signaling. Taken together, these results reveal the negative regulatory role for CD11b in maintaining autoreactive B cell tolerance.

The immunological hallmark of systemic lupus erythematosus (SLE) is a loss of immune tolerance and B cell hyperactivity. Recent studies demonstrated an association between the variant of the integrin-α-M (ITGAM, also called CD11b) gene and SLE susceptibility. However, how CD11b regulates disease pathogenesis remains elusive. We found surprisingly that B cells from CD11b knock out (KO) mice exhibited hyperproliferative responsiveness to B cell receptor (BCR) cross-linking. CD11b KO autoreactive B cells had the decreased tyrosine phosphorylation including Lyn and PLCγ2 and increased p38 MAKP. Moreover, CD11b deficiency promoted B cell survival which was associated with increased BCL-xl expression. In vivo engagement of BCR by apoptotic cells resulted in significantly increased autoAb production in CD11b KO mice. Furthermore, we found that BCR cross-linking could induce CD11b activation and modulate CD11b-medited phagocytosis, suggesting ‘out-side-in’ activation of CD11b integrin by BCR crosslinking. Taken together, these results reveal the interaction between CD11b integrin and BCR signaling pathways and suggest the critical role for CD11b molecule in maintaining autoreactive B cell tolerance (This work was supported by the Alliance for Lupus Research).

Promoting complement activation may enhance immunological mechanisms of anti-tumor antibodies for tumor destruction. However, complement activation components, such as C5a, trigger inflammation which can promote tumor growth. It has been demonstrated, in one tumor model, that C5a in the tumor can inhibit anti-tumor CD8 T cell responses through effects on myeloid-derived suppressor cells. We address the role of C5a on tumor growth by transfecting tumor cells with mouse C5a: SKOV-3 human ovarian carcinoma and RMA murine lymphoma. The two models allow us to study C5a in immunocompromised and immunocompetent hosts. In vitro growth kinetics of C5a, control vector (CV), or wild-type cells revealed no significant differences. In both models, tumor-bearing mice with C5a-transfected tumor cells have significantly less tumor burden as compared to CV tumors. In the immunocompromised model, DX5 CD11b NK cells and F4/80 macrophages infiltrated C5a expressing tumors with significantly greater frequency while VEGF and arginase production were significantly less. The syngeneic model revealed C5a leads to a greater tumor influx of CD11c and CD4 T cells. A greater percentage of CD4 T cells infiltrating RMA C5a tumors demonstrated an activated status, CD44hi CD62L low. Thus our studies demonstrate tumor C5a chemoattracts innate NK cells and macrophages while downregulating negative mediators, and promotes infiltration of important antigen-presenting cells and activated CD4 T effector cells.

Enhancement of anti-tumor mAb immunological mechanisms for tumor destruction may be obtained by promoting complement activation. However, complement activation components, such as C5a, trigger inflammation and chronic inflammation promotes tumor growth. Recently, it has been shown that C5a in the tumor could recruit and activate myeloid-derived suppressor cells to inhibit the anti-tumor CD8 T cell response. However, this effect was observed in only one tumor model. We chose to address the role of C5a on tumor growth by transfecting tumor cells with C5a. A human ovarian carcinoma, SKOV-3, and a murine lymphoma, RMA, were transfected which allows us to study C5a in an immunocompromised xenograft model and an immunocompetent syngeneic model. In vitro growth kinetics observed revealed no significant difference between C5a, control vector (CV), or wild-type cells. In both models, tumor-bearing mice with C5a-transfected tumor cells have significantly less tumor burden as compared to CV tumors. In the xenograft model, CD11b+DX5+ NK cells were significantly more and VEGF and arginase levels were significantly less in C5a expressing tumors. The syngeneic lymphoma model revealed C5a modulates tumor-infiltrating CD11b+ cells to produce more IL-12 and there is a greater influx of NK and CD4 T cells. Thus our studies demonstrate C5a chemoattracts innate NK cells and downregulates negative mediators in the xenograft model while in the syngeneic model C5a promotes shift to a Th1 response.

Aberrant T and B cell responses to multiple nuclear and cytoplasmic Ags such as small nuclear ribonucleoparticles (snRNPs) are a hallmark of systemic lupus erythematosus. Although Th1 response is considered to be predominant in lupus pathogenesis, the identification of Th17 cells may challenge Th1 paradigm. To investigate the respective roles of Th1, Th17, and Treg cells in lupus development, we analyzed different T cell subsets in spleen and kidney of lupus-prone MRL lpr/lpr mice. With increasing age and disease severity, the IFN-gamma-producing T cells were significantly increased. No significant change was observed in the IL-17-secreting CD4 T cells. Furthermore, the ration of Th1/Treg was significantly increased when mice aged while Th17/Treg ratio remained unchanged. To determine the antigenic epitopes recognized by autoreactive T and B cells, synthesized peptides of autoAg snRNP D protein were used. Two epitopes were recognized by autoreactive T cells while three epitopes were recognized by autoantibodies in MRL lpr/lpr mice. Interestingly, peptide 21-40 of snRNP D protein was recognized by both autoantibodies and T cells. Furthermore, one of T cell hybridomas generated from snRNP D-immunized B10.A Tg mice was also responsive to peptide 21-40. Taken together, our studies demonstrate that Th1 response remains dominant in lupus-prone MRL lpr/lpr mice and p21-40 of snRNP D protein appears to be a pathogenic epitope that can be recognized by both autoreactive B and T cells.

Systemic lupus erythematosus (SLE) is characterized by dysregulated autoreactive B cell activation. Previous studies demonstrate that plasmacytoid dendritic cells (pDCs) play a pivotal role in the pathogenesis of SLE. This study was undertaken to investigate the mechanisms of interactions between pDCs and autoreactive B cells. After co-culture of anti-small nuclear ribonucleoprotein particle (snRNP) B cells with activated pDCs via toll-like receptor (TLR)-7 or -9 agonist, we found that pDCs could significantly enhance autoreactive B cell proliferation and autoantibody production. In addition, autoreactive B cell activation via BCR engagement was also augmented by activated pDCs. This in vitro enhancement effect was further demonstrated by an in vivo B cell adoptive transfer experiment, demonstrating that autoreactive B cell proliferation was significantly decreased in MyD-88-deficient mice compared to that in wildtype mice. Neutralization of IFN-α and IL-6 could abrogate partially enhanced B cell activation via pDCs. Transwell studies demonstrated that pDCs could provide activation signal to B cells in a cell-to-cell contact manner. The involvement of the ICAM-LFA-1 pathway, but not the CD40L-CD40 pathway, was discovered to mainly contribute to this effect. These data suggest that pDCs regulate autoreactive B cell activation by soluble factors as well as cell-to-cell direct interactions. (This work was supported by an Arthritis Foundation Postdoctoral Fellowship)

To test the hypothesis that MUC1 specific T cell responses may be induced by targeting MUC1 to B cells in the presence of CpG ODN adjuvant, we first conjugated a model Ag ovalbumin (OVA) with anti-CD19 mAb as a means to target Ag to B cells. OVA TCR transgenic CD8 T cells (OT-I) and CD4 T cells (OT-II) were used to exam if targeting OVA to B cells through CD19 leads to Ag presentation in the context of MHC class I and II for T cell activation. The results indicated that OVA conjugates could significantly induce CD4 and CD8 T cells proliferation and activation. These expanded T cells were not deleted or anergized, and were characterized by proliferation (OT-II T cells) and IFN-γ release (OT-I T cells) in response to OVA peptide. CpG ODN could significant enhance the survival of CD8+ T cells and IFN-γ production. To further extend these observations to tumor Ag, MUC1 transgenic (Tg) mice that are tolerized to MUC1 Ag, resembling cancer patients, were immunized with anti-CD19 conjugated MUC1 with or without CpG ODN. The data demonstrated that MUC1 conjugates in combination with CpG ODN elicit high-titer anti-MUC1 Abs. In addition, MUC1-specific cytotoxicity and vigorous IFN-γ secretion was generated. In contrast, Tg mice immunized with MUC1 peptide, even in the presence of CpG ODN, did not elicit Ab production and T cell responses. These results indicated that Ag targeted to B cells via CD19 in combination with TLR agonist could reverse immunological tolerance to MUC1 Ag and generate MUC1-specific B cell and T cell responses.