Increased IFN-gamma Secretion Research Articles

MRNA-Lipid Nanoparticle (LNP) Delivery of Humanized EpCAM-CD3 Bispecific Antibody Significantly Blocks Colorectal Cancer Tumor Growth.

The epithelial cell adhesion molecule (EpCAM) is often overexpressed in many types of tumors, including colorectal cancer. We sequenced and humanized an EpCAM mouse antibody and used it to develop bispecific EpCAM-CD3 antibodies. Three different designs were used to generate bispecific antibodies such as EpCAM-CD3 CrossMab knob-in-hole, EpCAM ScFv-CD3 ScFv (BITE), and EpCAM ScFv-CD3 ScFv-human Fc designs. These antibody designs showed strong and specific binding to the EpCAM-positive Lovo cell line and T cells, specifically killed EpCAM-positive Lovo cells and not EpCAM-negative Colo741 cells in the presence of T cells, and increased T cells' IFN-gamma secretion in a dose-dependent manner. In addition, transfection of HEK-293 cells with EpCAM ScFv-CD3 ScFv human Fc mRNA-LNPs resulted in antibody secretion that killed Lovo cells and did not kill EpCAM-negative Colo741 cells. The antibody increased IFN-gamma secretion against Lovo target cells and did not increase it against Colo741 target cells. EpCAM-CD3 hFc mRNA-LNP transfection of several cancer cell lines (A1847, C30, OVCAR-5) also demonstrated functional bispecific antibody secretion. In addition, intratumoral delivery of the EpCAM-CD3 human Fc mRNA-LNPs into OVCAR-5 tumor xenografts combined with intravenous injection of T cells significantly blocked xenograft tumor growth. Thus, EpCAM-CD3 hFc mRNA-LNP delivery to tumor cells shows strong potential for future clinical studies.

Immunopotentiation by linking Hsp70 T-cell epitopes to Gag-Pol-Env-Nef-Rev multiepitope construct and increased IFN-gamma secretion in infected lymphocytes

Therapeutic human immunodeficiency virus (HIV) vaccines can boost the anti-HIV host immunity to control viral replication and eliminate viral reservoirs in the absence of anti-retroviral therapy. In this study, two computationally designed multiepitope Gag-Pol-Env-Nef-Rev and Hsp70-Gag-Pol-Env-Nef-Rev constructs harboring immunogenic and highly conserved HIV T cell epitopes were generated in E. coli as polypeptide vaccine candidates. Furthermore, the multiepitope gag-pol-env-nef-rev and hsp70-gag-pol-env-nef-rev DNA vaccine constructs were prepared and complexed with MPG cell-penetrating peptide. The immunogenicity of the multiepitope constructs were evaluated using the homologous and heterologous prime/boost strategies in mice. Moreover, the secretion of IFN-γ was assessed in infected lymphocytes in vitro. Our data showed that the homologous polypeptide regimens could significantly induce a mixture of IgG1 and IgG2a antibody responses, activate T cells to secret IFN-γ, IL-5, IL-10, and generate Granzyme B. Moreover, IFN-γ secretion was significantly enhanced in single-cycle replicable (SCR) HIV-1 virions-infected splenocytes in these groups compared to uninfected splenocytes. The linkage of heat shock protein 70 (Hsp70) epitopes to Gag-Pol-Env-Nef-Rev polypeptide in the homologous regimen increased significantly cytokines and Granzyme B levels, and IFN-γ secretion in virions-infected splenocytes. Briefly, both designed constructs in the homologous regimens can be used as a promising vaccine candidate against HIV infection.

IMMU-07. CELLULAR IMMUNOTHERAPY TO OVERCOME TEMOZOLOMIDE INDUCED T CELL EXHAUSTION IN GLIOBLASTOMA

Abstract INTRODUCTION We have previously demonstrated that standard dose (SD) temozolomide results in T cell exhaustion in glioblastoma. In this study, we hypothesized that cellular immunotherapies will prevent T cell exhaustion. We tested temozolomide treatment with adoptive T cell transfer alone, dendritic cell (DC) vaccines alone and T cell transfer in combination with DC vaccines. METHOD GL-261-gp100 tumor-bearing mice were treated with SD (50 mg/kg for 5 days) or metronomic dose (MD) (25 mg/kg for 10 days) temozolomide. CD3+ T cells were isolated from Pmel mice and infused intravenously. Antigen-specific DC vaccines (gp-100) were given intradermally at the concentration of 50×104. Peripheral blood T cell populations were evaluated by flow cytometry. IFN-gamma secretion was measured for evaluation of T cells function. Survival was compared between groups. RESULTS Survival analysis demonstrated that naïve T cell transfer alone and DC vaccine alone add no survival benefit to temozolomide treatment for tumor-bearing animals. There were no significant differences in the expression of exhaustion markers of Tim-3 and Lag-3 on T cells of animals that received a combination of activated T cells and DC vaccines in the context of temozolomide therapy compared to the animals that just received temozolomide. Combination of activated T cell transfer and DC vaccines did not increase IFN-gamma secretion from T cells compared to temozolomide treated animals. Combination of naïve T cell transfer and two DC vaccines increased IFN-gamma secretion from T cells of GL-261 gp100 tumor-bearing animals. However, naïve T cell transfer and two DC vaccines in the context of temozolomide was not potent enough to extend the survival of these tumor-bearing animals. CONCLUSION Combinatorial treatment with naïve antigen-specific T cells and three dendritic cell vaccine boosters in the context of temozolomide is a promising approach to preserve T cell function and avoid T cell exhaustion.

Abstract A124: Preclinical characterization of AB154, a fully humanized anti-TIGIT antibody, for use in combination therapies

Abstract TIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an inhibitory receptor that is expressed on natural killer (NK) cells, CD8+ T-cells, and immunosuppressive regulatory T-cells (Treg). DNAM-1 (DNAX Accessory Molecule-1; CD226) is an activating receptor found on NK cells, monocytes and a subset of T-cells. TIGIT and DNAM-1 are paired receptors that compete for shared ligands CD155 (PVR) and CD112 (Nectin-2) expressed by tumor and antigen-presenting cells. TIGIT binding to CD155 or CD112 results in immune suppression, whereas binding of DNAM-1 to the same ligands mediates immune activation. As malignancies progress, high TIGIT expression often occurs alongside the upregulation of other immune checkpoint proteins and markers of T-cell exhaustion such as PD-1 (Programmed Death-1). We have developed AB154 to inhibit TIGIT and shift the balance in the tumor microenvironment towards a more productive anticancer response. Blockade of multiple immune checkpoint proteins can confer effective and durable responses in the treatment of cancer. Data assembled from TCGA (The Cancer Genome Atlas) identified numerous tumor types in which TIGIT is co-expressed with PD-1. In these tumors, TIGIT and PD-1 were significantly upregulated compared to normal adjacent tissue. Immunophenotyping performed on human tumor infiltrating lymphocytes demonstrated a strong correlation between TIGIT and PD-1 co-expression on specific immune cells including CD8+ T-cells and Treg cells. AB154 is a fully humanized antibody that blocks human TIGIT with sub-nanomolar affinity, as determined using a CHO.hTIGIT over-expressing cell line and primary human T-cells. Functional consequences of blocking TIGIT/CD155 interactions in combination with anti-PD-1 or anti-PD-L1 were evaluated using mixed lymphocyte reactions (MLR). Briefly, we show here that co-cultures of GM-CSF/IL-4-differentiated CD155+ PD-L1+ monocytes and TIGIT+ CD4+ T-cells, in the presence of AB154, significantly increased IFN-gamma secretion when combined with anti-PD-1 or anti-PD-L1 blocking antibodies relative to each monotherapy. Understanding pharmacokinetic (PK) and pharmacodynamic (PD) relationships enables the choice of a dosing regimen that provides adequate target coverage. To evaluate the PD effects of AB154 in clinical samples, we developed a multicolor flow cytometry-based assay that utilizes an anti-TIGIT antibody that is competitive with AB154 to determine receptor occupancy. In human whole blood, ex vivo addition of AB154 achieved complete inhibition of TIGIT. Analysis of blood mononuclear cells, including CD8+ and CD4+ T-cells, Treg and NK cells, demonstrated target engagement by AB154 suitable for clinical development. In addition, we examined TIGIT receptor occupancy of AB154 (added to whole blood ex vivo) in a small cohort of non-small cell lung cancer (NSCLC) patients treated with anti-PD-1 antibody pembrolizumab. In these samples, TIGIT receptor occupancy by AB154 was comparable to that obtained with healthy donor blood samples. The data presented here provide: 1) rationale for combining AB154 with our in-house developed anti-PD-1 antibody (AB122) in upcoming clinical trials, and 2) methodology to evaluate TIGIT receptor occupancy in the upcoming AB154 dose escalation studies. AB154 is expected to enter clinical trials in 2018. Citation Format: Amy E. Anderson, Annette Becker, FangFang Yin, Hema Singh, Xiaoning Zhao, Lisa Seitz, Rick Stanton, Nigel P.C. Walker, Joanne B.L. Tan. Preclinical characterization of AB154, a fully humanized anti-TIGIT antibody, for use in combination therapies [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A124.

Abstract 274: Bavituximab modulates tumor microenvironment and activates CD8+ tumor infiltrating lymphocytes in a patient-derived 3D ex vivo system of lung cancer

Abstract Bavituximab is a monoclonal antibody directed against the membrane phospholipid phosphatidylserine exposed on the outer leaflet of tumor and vascular endothelial cells of the tumor microenvironment. Bavituximab modulates the tumor microenvironment by blocking PS-mediated immune suppression and activating cytotoxic T lymphocyte anti-tumor responses. In this study, we tested the immunomodulatory effect of bavituximab using a proprietary 3D ex vivo tumor microsphere technology. Upon obtaining informed consent, fresh tumor tissue from lung cancer patients were collected at the time of surgical resection. Tissue was processed for characterization of the tumor microenvironment and potential immunosuppressive mechanisms such as expression of PD-1, CTLA4, LAG3, TIM3, BTLA, and Adenosine A2AR. 3D tumor microspheres were prepared and cells were treated ex vivo with f(ab)’2 version of bavituximab, bavituximab, docetaxel, and a combination of bavituximab and docetaxel for 36 hours within the 3D tumor microsphere simulating an intact tumor microenvironment made up of tumor infiltrating lymphocytes (TIL) and myeloid cells. At the end of the treatment, TILs were analyzed by flow cytometry for cell activation and changes in CD4, CD8 and Treg (CD25+/CD127-) subpopulations. A multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines, including human IFN-gamma in culture media as a surrogate of TIL activation. Preliminary results indicate the combination of bavituximab and docetaxel can induce TIL activation as demonstrated by a significant increase in IFN-gamma secretion when compared to tumors treated with control or either agent alone. Flow cytometry analysis revealed that this effect was associated with low PD-1 expression on CD8 cells, but did not correlate with other known immune-modulating receptors. This lung patient derived ex-vivo approach indicates that bavituximab in combination with docetaxel can elicit a tumor specific immune response in human adenocarcinoma of the lung. This effect involves, at least in part, activation of CD8+ TIL and increased inflammatory cytokine production by lymphoid and myeloid cells. In addition, we have observed low PD1 expression as a potential prognostic biomarker of positive response to bavituximab treatment. Citation Format: Soner Altiok, Melanie Mediavilla-Valera, Jenny Kreahling, David Noyes, Tiffany N. Razabdouski, Nikoletta L. Kallinteris, Joseph Shan, Scott Antonia. Bavituximab modulates tumor microenvironment and activates CD8+ tumor infiltrating lymphocytes in a patient-derived 3D ex vivo system of lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 274. doi:10.1158/1538-7445.AM2015-274

The role of HIF-1 in up-regulating MICA expression on human renal proximal tubular epithelial cells during hypoxia/reoxygenation

BackgroundHuman major histocompatibility complex class I-related chain A (MICA) plays a dual role in adaptive and innate immune responses. Increasing evidence demonstrates that MICA is closely correlated with acute and chronic kidney allograft rejection. Therefore, understanding the activation mechanisms of MICA is important in kidney transplantation. We previously demonstrated that ischemia/reperfusion injury (IRI) could up-regulate MICA expression on mouse kidney allografts. Since hypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular adaptive responses to hypoxia during IRI, here we investigate whether HIF-1 could up-regulate MICA expression and its influence on NK cell cytotoxicity.ResultsWe find that HIF-1alpha plays an important role in up-regulating MICA expression, inducing IFNgamma secretion and NK cell cytotoxicity during hypoxia/reoxygenation. First, we generated a HIF-1alphaDELTAODD-expressing adenovirus to stably and functionally express HIF-1alpha in human renal proximal tubular epithelial (HK-2) cells under normoxia conditions. HIF-1alpha over-expression in HK-2 cells induces MICA expression and enhances NK cell cytotoxic activity towards cells that express HIF-1alpha. Second, we used a hypoxia/reoxygenation cell model to simulate IRI in vitro and found that the suppression of HIF-1alpha by RNAi induces down-regulation of MICA expression and inhibits NK cytotoxicity. In antibody blocking experiments, an anti-MICA mAb was able to down-regulate NK cell cytotoxic activity towards HK-2 cells that over-expressed HIF-1alpha. Moreover, when NK cells were co-cultured with the HK-2 cells expressing MICA, which was up-regulated by over-expression of HIF-1alpha, there was a significant increase in the secretion of IFNgamma. In the presence of the blocking MICA mAb, IFNgamma secretion was significantly decreased.ConclusionsThese results demonstrate that hypoxia/reoxygenation-promoted MICA expression on HK-2 cells is through a HIF-1 pathway. The increased IFNgamma secretion and enhanced NK cell cytotoxicity was mainly due to the surface expression of MICA induced by over-expression of HIF-1alpha. This study enhances our understanding of MICA activation mechanisms during kidney transplantation and provides insights into how IRI can influence transplant outcome. Moreover, these findings might be also important for developing strategies to reduce the effect of MICA in kidney transplant outcomes in the future.

IL‐10 and TGF‐β Immunoregulatory Cytokines rather than Natural Regulatory T Cells are Associated with the Resolution Phase of Vogt‐Koyanagi‐Harada (VKH) Syndrome

The pro-inflammatory cytokines play a critical role in the initiation and propagation of ocular autoimmune diseases. Regulation of these cytokines is generally mediated by the immunoregulatory cytokine such as IL-10 or TGF-beta. In this study, we investigated the immunoregulatory cytokine profile and frequency of natural regulatory T cells (nTregs) in patients with Vogt-Koyanagi-Harada (VKH). We obtained the peripheral blood mononuclear cells (PBMC) from patients with VKH and healthy controls. The cytokine profile from supernatants of PBMC cultured with or without phytohaemagglutinin (PHA) was measured by ELISA, the percentage of CD4(+) Foxp3(+) and CD25(high)Foxp3(+) T regulatory cells were analysed by flow cytometry, and the transcriptional level of Foxp3 expression was analysed by real-time quantitative PCR. The immunoregulatory cytokines, TGF-beta and IL-10, increased in patients with VKH in the inactive stage of the disease. We observed no significant difference in the CD4(+) Foxp3(+) and CD25(high)Foxp3(+) T cells as well as no reduction in FOXP3 mRNA expression in the patients with VKH when compared to healthy controls. We showed in our work, an increase in IFN-gamma secretion by PBMC of patients with VKH in the active stage of the disease when compared to healthy controls and patients in the inactive stage. Our data suggest that IL-10 and TGF-beta cytokines, rather than nTregs are associated with the resolution phase of the disease and may have a more relevant role in controlling this disease.

Activation drives PD‐1 expression during vaccine‐specific proliferation and following lentiviral infection in macaques

Recent data supports that increased expression of PD-1, a negative regulator of immune function, is associated with T cell exhaustion during chronic viral infection. However, PD-1 expression during acute infection and vaccination has not been studied in great detail in primates. Here, we examine PD-1 expression on CD3(+) T cells following DNA vaccination or lentiviral infection of macaques. Ex vivo peptide stimulation of PBMC from DNA-vaccinated uninfected macaques revealed a temporal increase in PD-1 expression in proliferating antigen-specific CD8(+) T cells. Following the initial increase, PD-1 expression steadily declined as proliferation continued, with a concomitant increase in IFN-gamma secretion. Subsequent examination of PD-1 expression on T cells from uninfected and lentivirus-infected non-vaccinated macaques revealed a significant increase in PD-1 expression with lentiviral infection, consistent with previous reports. PD-1 expression was highest on cells with activated memory and effector phenotypes. Despite their decreased telomere length, PD-1(hi) T cell populations do not appear to have statistically significant uncapped telomeres, typically indicative of proliferative exhaustion, suggesting a different mechanistic regulation of proliferation by PD-1. Our data indicate that PD-1 expression is increased as a result of T cell activation during a primary immune response as well as during persistent immune activation in macaques.

Anti-tumor effects of immunotherapeutic peptide on the treatment of hepatocellular carcinoma with HBc carrier

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. Tumor specific cellular and humoral immunotherapy may be a viable approach for the treatment of HCC. This study investigated specific inhibitory and cytotoxic effects on hepatocellular carcinoma (HCC) induced by the peptide, designated HBc Delta-5L, using HBc carrier with multiple T cell and B cell sequence insertions. We developed the HBc Delta carrier containing insertions of multiple CTL and T helper (Th) epitopes, which were selected from HCC tumor associated antigens (TAAs) including alpha fetoprotein (AFP), melanoma antigen gene (MAGE) and telomerase reverse transcriptase (TERT) antigen, and ligands for EGFR and IGFR, designated HBc Delta-5L. LDH release assay and IFN-gamma ELISPOT assay were carried to determine whether HBc Delta-5L could induce specific cytotoxicity in peripheral blood mononuclear cells (PBMC) of HCC donors. The levels of antibodies and inhibitory effects of sera of immunized mice against HBc Delta-5L were also identified. LDH release assay revealed that PBMC from HCC donor group (n=8) stimulated with HBc Delta-5L could specifically kill target tumor cells and specific lysis was 62.7% (E:T=60:1). ELISPOT assay showed a significant increase in secretion of IFN-gamma from PBMC of HCC donor group in response to HBc Delta-5L. Further, high specific antibody titers were elicited in immunized mice and revealed 42% inhibition of cell growth. These results indicated that inhibitory and cytotoxic effects could be efficiently induced by HBc Delta-5L recombinant particles using HBc Delta as carrier and suggested that it could be important in design of immunotherapeutic approaches.

Natural Killer cell function during foot-and-mouth disease virus infection (46.19)

Abstract Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. Immunological knowledge to assess more rapid acting vaccines against FMDV is presently limited. We examined the reactivity of swine and cattle NK cells following infection for their capability to express intracellular perforin, to kill a human tumor cell line target in vitro, and to secret IFN gamma. The cytotoxicity of NK cells from non-infected animals against the K562 cells is low with baseline levels at 5–15% in swine and 15–20% in cattle. Stimulation with rhIL-2 or rhIL-12 plus rhIL-15, increased the lytic activity against K562 cells. Infection with FMDV inhibited swine NK cell lytic activity but did not significantly increase IFN gamma secretion during the acute infection. Perforin expression increased but this did not correlate with the killing capability of the swine NK cells. Infection of cattle with FMDV initially activated the NK cells to increase target cell lysis. NK cell IFN gamma secretion and perforin expression were slightly elevated upon infection and coincided with the lytic activity in cattle. These results are a further indication of immune evasion by FMDV by inhibiting or limiting NK cell function. The potential to manipulate the innate immune response to block this evasion is discussed in the context of designing rapid acting vaccines for foot-and-mouth disease.

Reduced interleukin-18 secretion in Brucella abortus 2308-infected murine peritoneal macrophages and in spleen cells obtained from B. abortus 2308-infected mice

Th1 immune responses in which gamma interferon (IFN-gamma) production predominates are associated with protective immunity against intracellular bacteria. Following infection, interleukin-18 (IL-18) may contribute, in association with IL-12, to optimal IFN-gamma production. In this study, the secretion of IL-18 following intracellular infection with virulent Brucella abortus 2308 in CD-1 cultured peritoneal macrophages and splenocyte cultures was investigated. The production of IL-18 was reduced in both CD-1 mouse peritoneal macrophages infected with B. abortus 2308 and splenocyte cultures obtained from B. abortus 2308-infected mice at 3, 6 and 10 days post-infection (p.i.). In contrast, splenocyte cultures obtained from B. abortus 2308-infected mice at 3 days p.i. secreted significant amounts of IFN-gamma. Stimulation of these cells with recombinant IL-18 (rIL-18) and/or rIL-12 did not significantly increase IFN-gamma secretion at the splenocyte level. These data suggest that once the infection has been established, B. abortus 2308 selectively limits IL-18 secretion without affecting endogenous IFN-gamma production.

Neutralization of interleukin-10 significantly enhances gamma interferon expression in peripheral blood by stimulation with Johnin purified protein derivative and by infection with Mycobacterium avium subsp. paratuberculosis in experimentally infected cattle with paratuberculosis.

Monoclonal antibody neutralization of interleukin-10 (IL-10) increased Johnin purified protein derivative-induced whole-blood gamma interferon (IFN-gamma) secretion 23-fold and also increased IFN-gamma secretion ninefold following in vitro Mycobacterium avium subsp. paratuberculosis infection of peripheral blood mononuclear cells. These results demonstrate the suppressive effect of IL-10 on immune responses to M. avium subsp. paratuberculosis infection in cattle.

CD4 and CD8 T-lymphocyte recognition of prostate specific antigen in granulomatous prostatitis.

In order to develop immunotherapies for prostate cancer, many groups are exploring vaccination strategies to induce an immune response against prostate specific antigen (PSA). To determine if T-cell recognition of PSA might be a feature of a naturally occurring human disease, we have studied patients with prostatitis, a poorly understood clinical syndrome of men in which there is evidence that an immune response directed against the prostate may be occurring. We wished to determine if a T-cell response to PSA might be occurring in these patients. We generated long-term T-cell lines from peripheral blood mononuclear cells (PBMC) of one patient with granulomatous prostatitis using purified PSA as an antigen. Several CD4+ and CD8+ TcR alpha/beta+ T-cell lines were selected for PSA reactivity as measured by at least a threefold increase in IFN-gamma secretion in response to PSA presented by irradiated autologous PBMC. CD4 and CD8 T-cell lines recognized PSA in the context of HLA-DRbeta1*1501 and HLA-B*0702, respectively. The specificity and HLA restriction of the lines was confirmed using EBV-B cell lines infected with a recombinant PSA-expressing vaccinia virus and also engineered to express PSA by retroviral transfection. HLA-matched targets infected by control vector as well as HLA-mismatched PSA-expressing targets did not induce the response. The data demonstrate that PSA-specific T cells are present in the PBMC of this patient with granulomatous prostatitis, who may be manifesting naturally the type of immune response directed at the prostate that is the goal of prostate cancer immunotherapy. However, the Class I-restricted epitope has not yet been demonstrated to be expressed on the surface of prostate cancer cells. To our knowledge, this is the first demonstration of HLA-DRB1*1501- or HLA-B*0702-restricted responses to PSA and extends the number of HLA molecules accommodating the use of PSA antigen as a candidate vaccine for prostate cancer immunotherapy.

Ipsilateral submandibular lymphadenectomy does not prolong orthotopic corneal graft survival in mice.

To explore the impact of selective lymphadenectomy on the outflow from the outer eye and corneal graft survival in mice. BALB/c mice underwent submandibular lymphadenectomy either on the left side 7 or 21 days before the experiments or bilaterally 7 days in advance. First, (99m)Tc colloidal albumin (Nanocoll) was given as a subcutaneous lower-lid, upper-lid or subconjunctival injection, and count rates were determined 24 h later in the eyes, submandibular lymph nodes, spleen, liver and blood. Second, corneal graft survival was assessed in lymphadenectomised and control mice, and IFN-gamma secretion was determined in draining lymph nodes at the time of transplant rejection. Following subconjunctival Nanocoll injection, count rates/min/mg tissue were significantly higher in the ipsilateral submandibular lymph node than in the other tissues or blood (<0.01). Ipsilateral lymphadenectomy prior to injection resulted in considerably increased count rates in contralateral nodes ( P<0.01), which were higher after injections into upper than into lower lids ( P=0.004). Bilateral submandibular lymphadenectomy led to enhanced count rates in the eyes, blood, spleen and liver (all P<0.01). Removal of the ipsilateral lymph node prior to corneal transplantation did not prolong allograft survival ( P>0.05) and considerably increased IFN-gamma secretion in contralateral nodes after prior removal of the ipsilateral ones paralleled transplant rejection. In mice, removed submandibular lymph nodes are functionally completely replaced by the contralateral nodes. These studies demonstrate for the first time lymphatic drainage crossing the midline of the body. Consequently, unilateral lymphadenectomy does not improve corneal graft survival.

Immunogenicity of recombinant BCG vaccine strains overexpressing components of the antigen 85 complex of Mycobacterium tuberculosis.

The components of antigen 85 complex of Mycobacterium tuberculosis (Ag 85A, Ag 85B and Ag 85C), due to their immunodominant and secretory nature, represent promising protective antigen candidates and have been used in numerous vaccine preparations. We have used recombinant Bacille Calmette Guerin (BCG) strains overexpressing Ag 85A and Ag 85C to immunize BALB/c mice to investigate the immunogenicity of these strains. Mice immunized with recombinant BCG strains exhibited an increased humoral immune response when compared to mice immunized with wild-type BCG. The recombinant BCG strain overexpressing Ag 85A also induced an increased Th1-like response, characterized by elevated levels of IFN-gamma in antigen stimulated splenocyte cultures and a strong IgG2a antibody response, when compared to wild-type BCG. Immunization with recombinant BCG strain overexpressing Ag 85C, on the other hand did not elicit increased IFN-gamma secretion on restimulation of splenocytes in vitro.

Normal Breast Milk Limits the Development of Colitis in IL-10-Deficient Mice.

This study examined the role of breast milk in neonatal bacterial colonization of the colon and disease progression in IL-10-deficient mice. IL-10-deficient mice were cross-fostered at birth and raised until weaning with a normal mother. Results were compared with normal pups cross-fostered to an IL-10-deficient mother. Mice were examined at various ages for histologic disease, levels of colonic bacteria, and proinflammatory cytokine secretion. IL-10-deficient mice that had been cross-fostered to a normal mother demonstrated normal levels of colonic adherent bacteria and reduced TNFalpha and IFN gamma secretion at 2 to 12 weeks of age. Histologic disease was significantly reduced up to 12 weeks of age. Normal mice cross-fostered to an IL-10-deficient mother had increased levels of adherent bacteria at 2 and 4 weeks and increased IFN gamma secretion. This group also demonstrated slight inflammation up until 12 weeks of age. Breast milk has a role in neonatal bacterial colonization. Changing the luminal environment of IL-10-deficient mice during the neonatal period alters the natural disease course.

IL-12 and neutralization of endogenous IL-10 revert the in vitro antigen-specific cellular immunosuppression of paracoccidioidomycosis patients.

Treatment of patients with paracoccidioidomycosis is still a challenge. Patients present defective lymphoproliferation and IFN-gamma responses to the main Paracoccidioides brasiliensis antigen (gp43), which correlates with disease severity. Here, we demonstrated that the patients show also a defective synthesis of interleukin (IL)-12. Therefore, we attempted to revert this immune disfunction by adding IL-12 and neutralizing anti-IL-10 antibody to gp-43-stimulated peripheral blood mononuclear cell cultures. Both treatments increased IFN-gamma secretion to levels observed with healthy sensitized individuals, but affected proliferation only modestly. When combined, the treatments further increased IFN-gamma synthesis and cell proliferation. The addition of suboptimal concentrations of IL-2 also further increased the IL-12-mediated secretion of IFN-gamma. Interestingly, the immune modulation was mostly antigen-specific, since the responses to Candida albicans' antigen were not affected. These results suggest that appropriate immune intervention with cytokines and/or anti-cytokines may help in the treatment of PCM.

Tributyrin enhances the cytotoxic activity of interleukin-2/interleukin-12 stimulated human natural killer cells against LS 174T colon cancer cells in vitro.

Tributyrin has been shown to be cytostatic to tumor cells by inducing differentiation and apoptosis. On the other hand, immunological NK cells can kill tumor cells, particularly when stimulated with interleukin-2 (IL-2) and/or interleukin-12(IL-12). However, little is known about whether and how both antitumor mechanisms act together, although in vivo such an interaction must exist. Here we demonstrate in vitro, that pretreatment of human LS 174T colon cancer cells with nontoxic concentrations of tributyrin augments the sensitivity to spontaneous NK cell activity two-fold. However, when NK cells have been activated with an optimized combination of IL-2 and IL-12, the immunocytotoxicity increases up to five-fold (from 14% to 70%), versus a 3.8-fold increase against untreated cancer cells. These effects are accompanied by increased IFN-gamma secretion and decreased TGF-beta1 secretion. Tributyrin is found to be a potent inducer of ICAM-1, LFA-3 and Fas on target cells corresponding to an increase of the FasL expression by IL-2/IL-12 on the effector cells. Our data suggest a synergistic link between induction of tumor cell differentiation and immunological defense mechanisms that may provide a rational basis for the improvement of clinical protocols, especially for colon cancer.

Oral administration of recombinant cholera toxin subunit B inhibits IL-12-mediated murine experimental (trinitrobenzene sulfonic acid) colitis.

Trinitrobenzene sulfonic acid (TNBS)-induced colitis is an IL-12-driven, Th1 T cell-mediated colitis that resembles human Crohn's disease. In the present study, we showed initially that the oral administration of recombinant subunit B of cholera toxin (rCT-B) at the time of TNBS-induced colitis by intrarectal TNBS instillation inhibits the development of colitis or, at later time when TNBS-induced colitis is well established, brings about resolution of the colitis. Dose-response studies showed that a majority of mice (68%) treated with rCT-B at a dose of 100 microg (times four daily doses) exhibited complete inhibition of the development of colitis, whereas a minority (30%) treated with rCT-B at a dose of 10 microg (times four daily doses) exhibited complete inhibition; in both cases, however, the remaining mice exhibited some reduction in the severity of inflammation. In further studies, we showed that rCT-B administration is accompanied by prevention/reversal of increased IFN-gamma secretion (the hallmark of a Th1 response) without at the same time causing an increase in IL-4 secretion. This decreased IFN-gamma secretion was not associated with the up-regulation of the secretion of counterregulatory cytokines (IL-10 or TGF-beta), but was associated with a marked inhibition of IL-12 secretion, i.e., the secretion of the cytokine driving the Th1 response. Finally, we showed that rCT-B administration results in increased apoptosis of lamina propria cells, an effect previously shown to be indicative of IL-12 deprivation. From these studies, rCT-B emerges as a powerful inhibitor of Th1 T cell-driven inflammation that can conceivably be applied to the treatment of Crohn's disease.

Characterization of the T cell receptor repertoire in patients with common variable immunodeficiency: oligoclonal expansion of CD8(+) T cells.

Common variable immunodeficiency is characterized by an impairment in antibody production of at least two immunoglobulin isotypes, and abnormalities of both B and T cells have also been described. In this study, a profound alteration of the T cell receptor repertoire was noted, especially in CD8(+) T cells, in CVID patients when compared to a control group. However, the higher number of oligoclonally expanded populations seen in the patient group was not dependent on age, whereas in the control group an age-related accumulation of these populations was noted. Expansion of CD8(+) CD28(-) T cells in these patients is strongly correlated with T cell receptor repertoire restriction. Furthermore, analysis of cytokine production showed a statistically significant increase in IFN-gamma secretion in the patient group. Lastly, CD94 and perforin were expressed at increased frequencies on CD8(+) T cells in the patient group when compared to the control group. We speculate that these findings support the concept that CD8(+) T cells may play an important regulatory role in CVID.