Interferon-γ Release Research Articles

The contribution of cyclophilin A to immune-mediated central nervous system inflammation

Cyclophilin A has multiple known functions in inflammation. Intracellular cyclophilin A modulates T helper 2 response (Th2) and extracellular cyclophilin A functions as a leukocyte chemotactic factor. The contribution of cyclophilin A to central nervous system (CNS) inflammation has not been reported. To test the hypothesis that inhibition of cyclophilin A would ameliorate immune-mediated CNS inflammation, we compared the course and neuroimmunology of experimental allergic encephalomyelitis (EAE) in cyclophilin A knockout mice and wild type littermates. There was a trend towards lower incidence of EAE in cyclophilin A knockout mice, but the clinical course of EAE among animals that manifested clinical signs of EAE was similar in cyclophilin A knockout and wild type littermates. Antigen recall response to myelin oligodendrocyte glycoprotein (MOG) peptide showed that interferon-γ release was lower in cyclophilin A knockout mice. Analysis of CNS inflammatory cells showed that CD3+ T cell infiltration into the CNS was lower in cyclophilin A knockout mice. These results showed that the loss of cyclophilin A results in altered peripheral immune activation and CNS leukocyte infiltration, but these changes did not result in a substantial change in the clinical course of EAE.

PO 8518 LATENT TUBERCULOSIS INFECTION DETECTION BY IGRA USINGMYCOBACTERIUM TUBERCULOSISALTERNATIVE DESATURASE A1 IMMUNE-DOMINANT ANTIGEN

BackgroundDespite an efficient treatment and a widely-used vaccine, one third of the world’s population is estimated to be latently infected (LTBI) withMycobacterium tuberculosis(Mtb) and are at risk of progressing to contagious active tuberculosis. New TB vaccines and improved detection of LTBI are therefore urgently required for global TB control. As a first step towards these goals, a better understanding of host recognition and response to immunogenic Mtb antigens is needed.MethodsIn this study, interferon-γ release in response to the immunodominant antigens ESAT-6, TB10.4, Ag85A, Rv2031 and DesA1 was assessed by ELISPOT on PBMC from 55 newly diagnosed pulmonary TB patients, 121 of their household contacts and 123 matched community controls, enrolled in a high-burden area.ResultsESAT-6 and PPD ELISPOT responses were higher in the TB patient group compared to both contacts and controls (p<0.05 repectively). These responses increased with time after recruitment in the contacts and fell after successful treatment in the patients consistent with the hypothesis that responses to these antigens reflect antigenic or bacterial load. However, the response to DesA1 was significantly lower in the controls compared to the contacts (p<0.05 respectively). Receiver Operating Characteristic curve analysis showed that PPD/ESAT-6 best segregated TB patients from the other groups, while DesA1 best segregated contacts from controls.ConclusionThe present study therefore identifies DesA1 as an immunodominant antigen with the potential to contribute to improved immunodiagnosis.

Comparative analysis of CEACAM1 expression in thin melanomas with and without regression.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a key molecule in several intracellular and intercellular signaling pathways, with multiple functional and structural roles. CEACAM1 expression in melanoma is often described in the invading part of the tumor and has been associated with increased melanoma cells invasion and migration. We studied CEACAM1 expression in regressing versus non-regressing thin melanomas, knowing that phenomenon of regression represents a valuable model for understanding tumor immunity. In melanoma, through homophilic interactions, CEACAM1 inhibits natural killer cell activity, inhibits effector functions of tumor infiltrating lymphocytes, such as cytotoxicity and interferon-γ release. We present a retrospective study including 53 consecutive cases of thin melanoma, 21 with regression and 32 without regression. Comparative analysis of CEACAM1 expression in regressed and non-regressed areas from melanomas with regression and in non-regressed melanomas was performed. We used three different clones of CEACAM1: AA 1-428, extracellular domain, rabbit; AA 1-428, mouse, clone 8B6E2F4; and AA 1-468, full length, mouse, clone 2F6. All three clones had similar reactivity. We identified membrane positivity of tumor cells in non-regressed melanomas and in non-regressed areas in melanomas with regression. Remaining tumor cells in regressed areas were mostly negative for CEACAM1. In non-regressed lesions, there was a stronger positivity of CEACAM1 in the deep invasive front. In thin melanomas, CEACAM1 overexpression is related with invasiveness, suggesting that CEACAM1-positive melanomas are more aggressive. Also, in areas of regression tumor cells lose CEACAM1 expression, probably correlated with the presence of natural killer cells.

Administration of Dendritic Cells and Anti-PD-1 Antibody Converts X-ray Irradiated Tumors Into Effective In situ Vaccines

Danger signals and release of tumor-specific antigens after exposure to ionizing radiation can convert an irradiated tumor into an in situ vaccine. However, radiation alone is not sufficient to induce an effective systemic immune response. In this study, we investigated whether a combination of x-ray irradiation with bone marrow-derived dendritic cells (BM-DCs) and anti-PD-1 antibody (αPD1-ab) administration can enhance both local tumor control and the systemic abscopal effect in murine subcutaneous tumor models. B16/BL6 melanoma and Lewis lung carcinoma cells were examined for radiosensitivity and expression of H-2kd and PD-L1 before and after irradiation. The tumor cells were implanted subcutaneously in the left thigh of C57BL/6 mice as primary tumors. BM-DCs were induced from mouse bone marrow cells using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The primary tumors were treated with 8Gy of x-ray, followed by simultaneous intratumoral injection of BM-DCs and intraperitoneal injection of αPD1-ab. To examine the abscopal effect, the same tumor cells were also inoculated in the right thigh as metastatic tumors 4days after the primary tumor inoculation, and only the primary tumors were treated with the same protocols. Invivo analyses of tumor growth and survival rates and in vitro analyses of splenic T-cell proliferation and interferon-γ release were performed. The triple-combination treatment of x-ray irradiation with BM-DC and αPD1-ab administration inhibited primary tumor growth and significantly extended survival time in association with significant increase of T-cell proliferation and interferon-γ release. In addition, this triple-combination treatment significantly inhibited the growth of metastatic tumors. The results indicated that BM-DC and αPD1-ab administration led to the conversion of irradiated tumors into effective in situ vaccines. This combination therapy can be a promising approach to develop a novel individualized therapy for patients with solid cancers.

Antibody to Poly-N-acetyl glucosamine provides protection against intracellular pathogens: Mechanism of action and validation in horse foals challenged with Rhodococcus equi.

Immune correlates of protection against intracellular bacterial pathogens are largely thought to be cell-mediated, although a reasonable amount of data supports a role for antibody-mediated protection. To define a role for antibody-mediated immunity against an intracellular pathogen, Rhodococcus equi, that causes granulomatous pneumonia in horse foals, we devised and tested an experimental system relying solely on antibody-mediated protection against this host-specific etiologic agent. Immunity was induced by vaccinating pregnant mares 6 and 3 weeks prior to predicted parturition with a conjugate vaccine targeting the highly conserved microbial surface polysaccharide, poly-N-acetyl glucosamine (PNAG). We ascertained antibody was transferred to foals via colostrum, the only means for foals to acquire maternal antibody. Horses lack transplacental antibody transfer. Next, a randomized, controlled, blinded challenge was conducted by inoculating at ~4 weeks of age ~106 cfu of R. equi via intrabronchial challenge. Eleven of 12 (91%) foals born to immune mares did not develop clinical R. equi pneumonia, whereas 6 of 7 (86%) foals born to unvaccinated controls developed pneumonia (P = 0.0017). In a confirmatory passive immunization study, infusion of PNAG-hyperimmune plasma protected 100% of 5 foals against R. equi pneumonia whereas all 4 recipients of normal horse plasma developed clinical disease (P = 0.0079). Antibodies to PNAG mediated killing of extracellular and intracellular R. equi and other intracellular pathogens. Killing of intracellular organisms depended on antibody recognition of surface expression of PNAG on infected cells, along with complement deposition and PMN-assisted lysis of infected macrophages. Peripheral blood mononuclear cells from immune and protected foals released higher levels of interferon-γ in response to PNAG compared to controls, indicating vaccination also induced an antibody-dependent cellular release of this critical immune cytokine. Overall, antibody-mediated opsonic killing and interferon-γ release in response to PNAG may protect against diseases caused by intracellular bacterial pathogens.

Identification of an HLA-A2-restricted CD147 epitope that can induce specific CTL cytotoxicity against drug resistant MCF-7/Adr cells.

Cluster of differentiation (CD)147 is highly expressed in drug-resistant tumor cell lines and is involved in the formation of tumor drug resistance. Therefore, immunotherapy utilizing CD147 epitope peptides is a promising approach for the elimination of drug-resistant tumor cells. However, like most tumor-associated antigens (TAAs), CD147 belongs to the autoantigen category, and T cells that recognize high affinity, immunodominant epitopes from autoantigens are deleted though thymic negative selection. Furthermore, wild-type autoantigen peptides cannot effectively activate and expand T lymphocytes with lower affinity T cell receptors in vivo. However, mutations of TAA peptides have been demonstrated to increase the affinity of major histocompatibility complex molecules and their binding to T cell receptor molecules, leading to activation of T lymphocytes in vitro. In the present study, a high-affinity point mutation peptide, CD147126-134L2, was predicted by the human leukocyte antigen (HLA) binding prediction algorithm and its affinity was testified using a T2 binding assay. In addition, when peptide-specific cytotoxic T lymphocytes (CTLs) were stimulated with dendritic cells loaded with the CD147126-134L2 peptide under HLA-A*02:01 restriction, interferon-γ release and cytotoxicity assays showed that peptide-specific CTLs effectively cross-recognized and lysed T2 target cells loaded either with the wild-type (CD147126-134) or mutated peptide (CD147126-134L2). Moreover, the CD147126-134L2 peptide-specific CTLs exerted strong cytotoxic activity against drug-resistant MCF-7/Adr cells, which express a high level of CD147 and are HLA-A*02:01-positive, but not against normal MCF-7 cells. Thus, this suggests that the wild-type peptide (CD147126-134) is naturally presented on HLA-A*02:01 of CD147-expressing MCF-7/Adr cells and is cross-recognized by CTLs. In conclusion, an HLA-A*02:01-restricted CD147-point mutant epitope peptide was identified that induces CTLs to efficiently lyse drug-resistant MCF-7 cells that highly express CD147. Therefore, this immunotherapeutic approach should be explored as a potential treatment for drug-resistant tumors.

CD45RO+ T Cells and T Cell Activation in the Long-Lasting Immunity after Leishmania infantum Infection.

Manifestations of Leishmania infantum infection range from asymptomatic to symptomatic visceral leishmaniasis (VL). People with symptomatic VL (sVL) have suppressed immune responses against Leishmania antigens that are reversed after clinical cure. The intradermal leishmanin skin test (LST) is negative during sVL, but it becomes positive after treatment. The aim of this study was to compare T cell responses in individuals with sVL, recovered VL (RecVL), and endemic controls. Endemic controls were household contacts of a VL case and they were grouped by their LST results, either positive (LST+) or negative (LST-). Mononuclear cells were studied ex vivo or after stimulation with soluble Leishmania antigens (SLA); cell surface markers and cytokines were determined. T cells, ex vivo, from individuals with sVL and from LST+ individuals presented a higher activation for CD4+ and CD8+ cells expressing CD69. However, lymphocytes from sVL stimulated with SLA had lower percentages of CD4+ and CD8+ cells expressing CD69 and CD8+ cells expressing CD25, with no release of interferon-γ or tumor necrosis factor. sVL subjects had lower percentage of memory cells (CD4+ CD45RO+), ex vivo, without SLA stimulation than RecVL, LST+, or LST- (P = 0.0022). However, individuals with sVL had fewer regulatory cells after SLA stimulation (CD4+ CD25HIGH, P = 0.04 and CD4+ FOXP3+, P = 0.02) than RecVL. The decrease in specific memory and activated CD4+ and CD8+ cells, as in response to Leishmania antigens, could explain, in part, the immune impairment during sVL. Finally, protective T cell responses are long lasting because both RecVL or LST+ individuals maintain a specific protective response to Leishmania years after the primary infection.

A multicentre verification study of the QuantiFERON®-TB Gold Plus assay

ObjectivesThe aim of this verification study was to compare the QuantiFERON®-TB Gold Plus (QFT-Plus) to the QuantiFERON®-TB Gold In Tube (QFT-GIT). The new QFT-Plus test contains an extra antigen tube which, according to the manufacturer additionally elicits a CD8+ T-cell response above the CD4+ T-cell response. We assessed the value of this tube in detecting recent latent tuberculosis infections. MethodsBetween May 2015 and December 2016, 1031 subjects underwent QFT-Plus and QFT-GIT test. Overall agreement between both tests and performance for different test indications and/or immune states was assessed. A difference of >0.6 IU/mL interferon-γ release between the two antigen tubes of the QFT-Plus assay was considered a true difference and used as estimation for CD8+ T-cell response. ResultsAnalysis of the QuantiFERON tests resulted in an overall agreement between assays of 95%. Subjects considered to be recently exposed to tuberculosis had significantly more often a true difference in interferon-γ release compared to all other subjects (p = 0.029). ConclusionResults of QFT-Plus are highly comparable to QFT-GIT. Although there is an indication that a true difference in interferon-γ release between the antigen tubes is associated with recent latent tuberculosis infection, the QFT-Plus could not be used to exclude recent exposure.

Combination therapy of hTERTR and FAM96A for hepatocellular carcinoma through enhancing apoptosis sensitivity.

Avoidance of apoptosis induced by anticancer drugs is an essential factor of carcinogenesis and a hallmark of resistance to cancer therapy. Human telomerase reverse transcriptase receptor (hTERTR) is a potential anti-cancer agent for inhibiting tumor growth. Family with sequence similarity 96 member A (FAM96A) is a ubiquitous, conserved protein and possesses apoptosome-activating and pro-apoptotic tumor suppressor potential in hepatocellular carcinoma (HCC). In the present study, hTERTR and FAM96A were identified as efficient anti-cancer agents for activating apoptosomes and reducing tumor growth. The potential tumor suppressor function of combination treatment with hTERTR and FAM96A in HCC was also investigated. hTERTR and FAM96A proteins were expressed by genetic engineering and their anti-cancer function was explored in vitro and in vivo. Effects of hTERTR and FAM96A on improvement of apoptotic sensitivity and inhibition of migration and invasion were examined in cancer cells and in a mouse model. The present results demonstrated that the therapeutic effects of hTERTR and FAM96A were effective for inhibiting tumor growth and inducing apoptosis of HCC cells in H22-bearing nude mice compared with single agent treatment. hTERTR and FAM96A were found to bind with apoptotic protease activating factor 1 and human telomerase reverse transcriptase, which enhanced the apoptosis of tumor cells and apoptosis sensitivity. In addition, hTERTR and FAM96A therapy enhanced cytotoxic effects by cytotoxic T lymphocyte responses, interferon-γ release, T lymphocytes infiltration and apoptosis on tumor cells. Furthermore, hTERTR and FAM96A protein inhibited tumor growth in HCC mice. In conclusion, the present findings suggested that combination therapy with hTERTR and FAM96A may serve as novel tumor suppressor agents.

Abstract 3997: Interleukin-4 receptor-targeted cytotoxic T cells enhances the therapeutic efficacy of adoptive T cell therapy against melanoma

Abstract Melanoma, the deadliest form of skin cancer, has long been a major focus of immunotherapy. Adoptive T cell therapy-based cancer immunotherapy has been used to enhance the specificity and potential of host immune system to treat melanoma. However, the infiltration and antitumor function of cytotoxic T cells (CTLs) are blocked or down-regulated by various signals derived from tumor cells and cells in tumor microenvironment. A novel approach to specify and enhance the homing ability of CTLs to tumor is of high demand. Interleukin-4 receptor (IL-4R) is over-expressed on many types of cancer cells, including melanoma, and has been widely employed for targeted drug delivery. In this work, we examined whether the tumor homing efficiency and anti-tumor therapeutic efficacy of CTLs could be heighten by labeling CTLs with IL4RPep-1, a targeting peptide that binds to IL-4R. The labeling was empowered by conjugating IL4RPep-1 with Dioleylphosphatidylethanolamine-biological anchor for membrane (DOPE-BAM), an oleyl acid-derived membrane anchor that incorporates into lipid membrane of cells. Tumor-specific CTLs isolated from the mice immunized by irradiated B16F10 melanoma cells were highly populated with CD62L+CD44+ activated T cells. After labeling with IL4RPep-1 via DOPE-BAM, CTLs were still functionally active, as determined by intracellular interferon-γ and granzyme release assays. Moreover, such labeling did not affect the proliferation of T cells. Adoptive transfer of IL4RPep-1-labeled, IL-4R-targeted CD45.1+CTLs into CD45.2 mice bearing B16F10 melanoma enhanced their accumulation at tumor compared to that of unlabeled T cells. Tumor homing of IL4RPep-1-labeled T cells was further validated by in vivo fluorescence imaging of mice bearing 4T1 tumor at mammary gland. Adoptive transfer of IL-4R-targeted CTLs showed remarkable anti-tumor growth activity in mice bearing B16F10 tumor compared to untargeted CTLs. After the transfer, flow cytometric study of immune cells showed significant increase of CD8+ T cells and F4/80+CD86+M1-polarized macrophages with noticeable decrease in tumor-promoting immune cells, such as F4/80+CD206+M2-polarized macrophages, Gr1+CD11b+ myeloid derived suppressor cells, and CD3+CD4+ T cells. The CTLs accumulated at tumor were mostly exogenous CD45.1+ T cells. These results implicate that IL-4R-targeted CTLs, which is enabled by labeling of with IL4RPep-1, can be a promising strategy in the field of adoptive T cell therapy against IL-4R-overexpressing tumor. Citation Format: Gunassekaran Gowri Rangaswamy, Poongkavithai Vadevoo Sri Murugan, Guruprasath Padmanaban, Lianhua Chi, Ha-Jeong Kim, Byung Heon Lee. Interleukin-4 receptor-targeted cytotoxic T cells enhances the therapeutic efficacy of adoptive T cell therapy against melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3997. doi:10.1158/1538-7445.AM2017-3997

A multi-target protein of hTERTR-FAM96A presents significant anticancer potent in the treatment of hepatocellular carcinoma.

The abilities to escape apoptosis induced by anticancer drugs are an essential factor of carcinogenesis and a hallmark of resistance to cancer therapy. In this study, we identified hTERTR-FAM96A (human telomerase reverse transcriptase-family with sequence similarity 96 member A) as a new efficient agent for apoptosome-activating and anti-tumor protein and investigated the potential tumor suppressor function in hepatocellular carcinoma. The hTERTR-FAM96A fusion protein was constructed by genetic engineering and its anticancer function of hTERTR-FAM96A was explored in vitro and in vivo by investigating the possible preclinical outcomes. Effects of hTERTR-FAM96A on improvement of apoptotic sensitivity and inhibition of migration and invasion were examined in cancer cells and tumors. Our results showed that the therapeutic effects of hTERTR-FAM96A were highly effective for inhibiting tumor growth and inducing apoptosis of hepatocellular carcinoma cells in H22-bearing nude mice. The hTERTR-FAM96A fusion protein could specifically bind with Apaf-1 and hTERT, which further induced apoptosis of hepatocellular carcinoma cells and improved apoptosis sensitivity. Our results indicated that hTERTR-FAM96A treatment enhanced cytotoxic effects by upregulation of cytotoxic T lymphocyte responses, interferon-γ release, and T lymphocyte infiltration. In addition, hTERTR-FAM96A led to tumor-specific immunologic cytotoxicity through increasing apoptotic body on hepatocellular tumors. Furthermore, hTERTR-FAM96A dramatically inhibited tumor growth, reduced death rate, and prolonged mice survival in hepatocellular carcinoma mice derived from three independent hepatocellular carcinoma mice cohorts compared to control groups. In summary, our data suggest that hTERTR-FAM96A may serve as an efficient anti-tumor agent for the treatment of hepatocellular carcinoma.

Abstract B67: Antibody-mediated delivery of viral peptides selectively activates CD8+ T cells to eliminate tumor cells in vitro and in vivo

Abstract Recent success of T cell based cancer immunotherapy approaches like chimeric antigen receptor T cells (CAR-Ts) and especially bispecific T cell engagers (BiTEs) has suggested great potential for cancer therapy. However, these technologies have several drawbacks like high risks for side-effects, such as autoimmunity, as well as inappropriate T cell activation due to a non-natural synapse. Hence, we sought to use antibody-mediated delivery of viral peptides to tumor cells in order to mimic viral infection and induce tumor cell lysis by virus-specific cytotoxic CD8+ T cells. Here, we generated immunoconjugates called antibody-targeted pathogen-derived peptides (ATPPs), where mature virus-derived MHC class I peptides from Influenza A or Epstein-Barr virus were conjugated to a tumor-associated antigen-specific antibody via a disulfide bond. Using optical imaging in combination with fluorescence resonance energy transfer, we revealed that after ATPP binding to the target antigen and subsequent internalization, the peptides are released in endosomes and loaded onto recycling MHC class I complexes. These peptide-MHC complexes are subsequently presented on the cell surface and mediate activation of human peptide-specific CD8+ T cells as shown by Interferon-γ release using enzyme-linked immunosorbent assay. Moreover, ATPP-loaded cancer cells from different tumor types were efficiently killed in vitro by human CD8+ T cells in a dose-dependent manner, reaching up to 100% killing after 24h at 0,13nM with an effector-to-target ratio of 3:1. Tumor cell death was assessed by the xCELLigence system as well as lactate dehydrogenase quantification in the supernatant. Interestingly, ATPPs were about 1000-times more potent than free peptide, while antibody alone, a non-cleavable ATPP construct, as well as peptides conjugated to a non-binding antibody exhibited no effects. Importantly, all used antibodies carried the P329G/LALA mutation for abolished Fc-receptor binding. In addition, we performed an experimental subcutaneous xenograft in vivo study with PDL1-expressing MDA-MB231 breast cancer cells in NOG mice in combination with anti-PD1 treatment. We observed that ATPPs successfully recruited adoptively transferred human peptide-specific CD8+ T cells into the tumor and induced 60% tumor growth inhibition of an established tumor after 3 weeks in the anti-PD1 combination setting. Altogether, we generated first in class immunoconjugates for antibody-targeted loading of cancer cells with foreign MHC-I restricted peptide antigens. Our results demonstrate potent ATPP-mediated antitumor efficacy in vitro and in vivo, by loading tumor cells with viral peptide antigens and redirecting virus-specific cytotoxic T cells against cancer. Citation Format: Julian Peter Sefrin, Lars Hillringhaus, Valeria Lifke, Alexander Lifke. Antibody-mediated delivery of viral peptides selectively activates CD8+ T cells to eliminate tumor cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B67.

HIV Interferes with Mycobacterium tuberculosis Antigen Presentation in Human Dendritic Cells

HIV coinfection is the most prominent risk factor for progression of Mycobacterium tuberculosis (Mtb) infection into active tuberculosis (TB) disease. The mechanisms behind the increased transition from latent to active TB in coinfected individuals have not been well elucidated at the cellular level. We hypothesized that HIV infection contributes to Mtb pathogenesis by interfering with the dendritic cell (DC)-mediated immune control. Mtb-antigen processing and presentation are key events in the immune response against TB. Human immature DCs coinfected with HIV/Mtb had decreased expression of human leukocyte antigen antigen D related and the costimulatory molecules CD40, CD80, and CD86. In addition, Mtb-infected DCs triggered a significant release of the proinflammatory cytokines IL-6, IL-1β, and tumor necrosis factor-α, whereas coinfected DCs did not. To assess the DC antigen presentation capacity, we measured interferon-γ from co-cultures of DCs and autologous Mtb antigen-specific CD4+ T cells. Interferon-γ release was significantly reduced when purified protein derivative- and Ag85B-specific CD4+ T cells had been activated with coinfected DCs compared to Mtb-infected DCs, and this effect was attributed to Mtb antigen processing rather than peptide-major histocompatibility complex class II loading. Evaluating autophagy as a measure of vesicular processing and maturation further revealed that HIV efficiently blocks initiation of this pathway during coinfection. Overall, our results demonstrate that HIV impairs Mtb antigen presentation in DCs, thereby suppressing an important cell linking innate and adaptive immune response in TB.

The molecular mechanism of thalidomide analogs in hematologic malignancies.

Thalidomide was sold in the 1950s as a sedative and was also used by pregnant women to treat morning sickness. It became notorious for causing severe birth defects and was removed from the market. More than four decades later, thalidomide had a renaissance in the treatment of cancer. Thalidomide and its more potent analogs, lenalidomide and pomalidomide, are nowadays approved treatments for multiple myeloma and myelodysplastic syndrome with deletion of chromosome 5q. In addition, thalidomide and its analogs inhibit release of tumor necrosis factor-α and increase interleukin-2 (IL-2) and interferon-γ release from T cells. The underlying molecular mechanisms for these pleiotropic effects remained obscure until the identification of the cereblon (CRBN) E3 ubiquitin ligase as the primary target of thalidomide and its analogs in 2010. Binding of thalidomide or lenalidomide increases the affinity of CRBN to the transcription factors IKZF1 and IKZF3 and casein kinase 1α (CK1α). Ubiquitination and degradation of these neo-substrates results in IL-2 release and growth arrest of multiple myeloma and MDS cells. The discovery of this previously undescribed mechanism for an approved drug provides a proof-of-concept for the development of new therapeutics that exploit ubiquitin ligases for specific degradation of disease-associated proteins.

Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity

ABSTRACTPediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8+ T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1130 peptide-driven stimulations with HLA-A*02:01+ dendritic cells (DC), allo-restricted HLA-A*02:01− CD8+ T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and β-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01− primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2−/−γc−/− mouse model. Initially generated transgenic T cells specifically recognized STEAP1130-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01+ ES lines more effectively than HLA-A*02:01− ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1130-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01+ ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors.

Type I-polarized BRAF-pulsed dendritic cells induce antigen-specific CD8+ T cells that impact BRAF-mutant murine melanoma.

Existing therapies targeting the mutated BRAF oncodriver (BRAF(V600E)) successfully treat melanoma but are susceptible to resistance. This study assessed the potential of a dendritic cell-based BRAF(V600E) vaccine for the treatment of BRAF(V600E)-mutant melanoma. Type 1-polarized dendritic cells (DC1) pulsed with affinity-modified BRAF(V600E) peptide were administered to C57Bl/6 mice both before (prevention) and twice weekly after (treatment) the development of established tumor with B16 melanoma transfected to express BRAF(V600E) (B16(V600E)). The efficacy of the BRAF(V600E)-pulsed DC1 vaccine was corroborated in a novel transplantable BRAF(V600E)-mutant murine melanoma model (BRAF(V600E-/+); PTEN(-/-); CDK2NA(-/-)). Three-dimensional tumor measurements and survival were determined. Induction of BRAF(V600E)-specific CD8(+) T-cell responses after brief in-vitro sensitization was assessed by interferon-γ enzyme-linked immunosorbent assay and/or enzyme-linked immunospot. Mice receiving BRAF(V600E)-pulsed DC1 vaccines before B16(V600E) tumor challenge demonstrated increased tumor-doubling times (P<0.001) and improved survival (P=0.0186) compared with those that received ovalbumin (control)-pulsed DC1 vaccines. In mice bearing established B16(V600E) tumors (mean 32 mm(3)), BRAF-pulsed DC1 vaccines delayed tumor growth (P<0.001) and improved survival (P=0.0008), compared with untreated mice. Likewise, in mice bearing BRAF(V600E-/+); PTEN(-/-); CDK2NA(-/-) tumors, compared with controls, BRAF-DC1 vaccination recapitulated these effects by delaying tumor growth (P<0.001) and improving survival (P=0.002). Vaccination elicited specific CD8(+) T-cell recognition of BRAF(V600E)-pulsed antigen-presenting cells (P<0.05), as well as BRAF(V600E)-expressing cancer cells (P<0.001), measured by interferon-γ release in vitro. BRAF-(V600E)pulsed DC1 vaccines induce oncogene-specific CD8(+) T-cell immune responses that impact tumor growth and survival in preclinical models of BRAF(V600E)-mutant melanoma. Exploration of BRAF(V600E)-targeted vaccines, in combination with BRAF-targeted therapies and checkpoint inhibitors, is warranted.

3,5,6,7,8,3',4'-Heptamethoxyflavone reduces interleukin-4 production in the spleen cells of mice.

In our previous studies, we reported anti-inflammatory functions of 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF), which is a polymethoxyflavone rich in various citrus fruits. Here, we investigated the immunomodulatory function of HMF in mice. HMF administration (50 mg/kg, i.p., 2 times/week) tended to reduce the production of antigen-specific IgE induced by ovalbumin in combination with aluminum hydroxide gel. Fluorescence-activated cell sorting analysis revealed the reduction of interleukin-4(+)CD4(+) spleen cells and sustained presence of interferon-γ(+)CD4(+) spleen cells in mice administered HMF, whereas the ratio of CD4(+)CD8(-) versus CD4(-)CD8(+) spleen cells was not affected. Interleukin-4 release from CD3/CD28-stimulated spleen cells of mice administered HMF was reduced, whereas interferon-γ release was not affected. These results suggest that HMF has an immunomodulatory function via reduced interleukin-4 expression.

Impact of Kindlin-3 in NK Cell-Mediated Anti-Tumor Cytotoxicity and Inflammatory Cytokine Production

Natural Killer (NK) cells are innate lymphocytes that play a central role in anti-viral and anti-tumor responses through direct cytotoxicity and production of inflammatory cytokines. Tumors can evade T-cell mediated immune-surveillance by down-regulation of the Class I Major Histocompatibility Complex (MHC-I) (Haworth et al, Ped Blood and Cancer, 2015). However, this lack of 'self' MHC-I serves as an activation stimulus for NK cells to recognize tumor cells. The molecular mechanism for 'self' recognition and destruction of 'missing self' are poorly understood. Integrins facilitate cell-to-cell interactions and are hypothesized to play a role in the 'self' versus 'missing self' recognition (Crozat et al, Blood, 2011). One of the critical regulators of integrin activation is Kindlin-3, which helps in their 'inside-out' signaling. Kindlin-3 binds to the cytoplasmic tail of β2-integrin and induces a conformational change increasing ligand affinity (Ye et al, Curr Biol, 2013). Consequently, Kindlin-3 is localized at the immunologic synapse and has been shown to interact with Adhesion and Degranulation Adaptor Protein (ADAP) (Kasirer-Friede et al, Blood 2014). As our group has shown, ADAP plays a central role in the signaling transduction for inflammatory cytokine production in NK cells (Rajasekaran et al, Nat Immunol, 2013). Clinically, defects in Kindlin-3 functions in humans are manifested by severe immune deficiency and bleeding disorder defined as Leukocyte Adhesion Deficiency-III (LAD-III). Based on these observations, we hypothesized Kindlin-3-dependent integrin function is critical for NK cell-mediated anti-tumor cytotoxicity and production of inflammatory cytokines.To define the role of Kindlin-3 in NK cell effector functions, we utilized a murine model. Kindlin-3 knock-in (K3KI) mice carry a double substitution mutation disrupting the binding of Kindlin-3 to β2-integrin (Xu et al, Arterioscler Thromb Vac Biol, 2014). NK cells from K3KI mice were evaluated for development and effector functions. Flow cytometry was utilized to identify maturation and developmental populations. Inflammatory cytokine production was assessed by Interferon-γ release following NK cell and tumor co-culture as well as plate-bound antibody activation. Cytotoxicity was assessed by 51Cr-release assay and the following tumor cells were used: cells representing, 1) 'self' (RMA and EL4 thymomas with autologous MHC-I); 2) 'missing-self' (RMA/S with decreased MHC expression relative to RMA); 3) 'induced self' (EL4 thymomas stably expressing H60, an activating ligand for NKG2D; 4) 'non-self' (YAC1 lymphoma with allogeneic MHC).Our results show an overall increase in the peripheral NK populations collected from spleens of K3KI mice, as seen in patients with LAD-III. However, no significant maturational defects were noted in the bone marrow of the K3KI mice. In vitro analyses reveal that K3KI NK cells have significantly impaired anti-tumor cytotoxicity relative to wild type controls (Figure 1). There was a significant reduction in the cytotoxic ability of K3KI NK cells towards 'induced self' or 'missing self' recognition (p<0.05). In contrast, K3KI NK cells have augmented inflammatory interferon-γ cytokine production compared to wild type controls when co cultured with the same tumor models in which cytotoxicity was significantly impaired (Figure 2).These data reveal the essential role of Kindlin-3 interaction with integrin for the effector functions of the NK cell. Currently, we are delineating the signaling mechanisms which mediate this divergence in NK cell functions dependent on Kindlin-3. Our studies reveal an undefined role for Kindlin-3 in NK cells and may help to identify novel therapeutic targets to modulate NK cell effector functions. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The MICA-129Met/Val dimorphism affects plasma membrane expression and shedding of the NKG2D ligand MICA.

The MHC class I chain-related molecule A (MICA) is a ligand for the activating natural killer (NK) cell receptor NKG2D. A polymorphism causing a valine to methionine exchange at position 129 affects binding to NKG2D, cytotoxicity, interferon-γ release by NK cells and activation of CD8+ T cells. It is known that tumors can escape NKG2D-mediated immune surveillance by proteolytic shedding of MICA. Therefore, we investigated whether this polymorphism affects plasma membrane expression (pmMICA) and shedding of MICA. Expression of pmMICA was higher in a panel of tumor (n = 16, P = 0.0699) and melanoma cell lines (n = 13, P = 0.0429) carrying the MICA-129Val/Val genotype. MICA-129Val homozygous melanoma cell lines released more soluble MICA (sMICA) by shedding (P = 0.0015). MICA-129Met or MICA-129Val isoforms differing only in this amino acid were expressed in the MICA-negative melanoma cell line Malme, and clones with similar pmMICA expression intensity were selected. The MICA-129Met clones released more sMICA (P = 0.0006), and a higher proportion of the MICA-129Met than the MICA-129Val variant was retained in intracellular compartments (P = 0.0199). The MICA-129Met clones also expressed more MICA messenger RNA (P = 0.0047). The latter phenotype was also observed in mouse L cells transfected with the MICA expression constructs (P = 0.0212). In conclusion, the MICA-129Met/Val dimorphism affects the expression density of MICA on the plasma membrane. More of the MICA-129Met variants were retained intracellularly. If expressed at the cell surface, the MICA-129Met isoform was more susceptible to shedding. Both processes appear to limit the cell surface expression of MICA-129Met variants that have a high binding avidity to NKG2D.

The MICA‐129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation

The MHC class I chain-related molecule A (MICA) is a highly polymorphic ligand for the activating natural killer (NK)-cell receptor NKG2D. A single nucleotide polymorphism causes a valine to methionine exchange at position 129. Presence of a MICA-129Met allele in patients (n = 452) undergoing hematopoietic stem cell transplantation (HSCT) increased the chance of overall survival (hazard ratio [HR] = 0.77, P = 0.0445) and reduced the risk to die due to acute graft-versus-host disease (aGVHD) (odds ratio [OR] = 0.57, P = 0.0400) although homozygous carriers had an increased risk to experience this complication (OR = 1.92, P = 0.0371). Overall survival of MICA-129Val/Val genotype carriers was improved when treated with anti-thymocyte globulin (HR = 0.54, P = 0.0166). Functionally, the MICA-129Met isoform was characterized by stronger NKG2D signaling, triggering more NK-cell cytotoxicity and interferon-γ release, and faster co-stimulation of CD8+ T cells. The MICA-129Met variant also induced a faster and stronger down-regulation of NKG2D on NK and CD8+ T cells than the MICA-129Val isoform. The reduced cell surface expression of NKG2D in response to engagement by MICA-129Met variants appeared to reduce the severity of aGVHD.