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Cancer immunotherapy represents a potential treatment approach through non-specific and specific enhancement of the immune responses. Adoptive cell therapy (ACT) is a potential modality of immunotherapy that depends on harvesting T cells from the tumor-bearing host, activating them in vitro and infusing them back to the same host. Several cytokines, in particular IL-2, IL-7 and IL-15, have been used to enhance survival T cells in vitro. Although effective, conditioning of T cells in vitro with these cytokines requires long-term culture which results in the loss of expression of their trafficking receptors mainly CD62L. It also results in exhaustion of the activated T cells and reduction in their functions upon adoptive transfer in vivo. Our recent studies and those of other groups showed that brief (3 days) conditioning of CD8+ T cells by IL-12 in vitro can result in enhancing function of tumor-reactive CD8+ T cells. Adoptive transfer of these IL-12-conditioned CD8+ T cells into tumor-bearing mice, preconditioned with cyclophosphamide, 1 day before ACT, induced tumor eradication that was associated with generation of tumor-specific memory response. In this review, we summarize studies that indicated to the superiority of IL-12 as a potential cytokine for conditioning T cells for ACT. In addition, we discuss some of the cellular and molecular mechanisms that govern how IL-12 programs CD8+ T cells to enhance their functionality especially in vitro and its implication in combination with other ACT modalities, opening a avenue for the clinical application of this cytokine.

Introduction:Leukopenia is one of the major side effects of myelosuppressive chemotherapy such as cyclophosphamide (CTX). We and others have used CTX either alone or in combination with G-CSF for the mobilization of hematopoietic stem cells (HSCs). This mobilization can induce expansion of myeloid cells with immunosuppressive phenotype. In this pilot study, we aimed to test whether bone marrow lysate (BML)/CTX, a rich source of growth factors, can lower the expansion of myeloid cells with immunosuppressive phenotypes in tumor-bearing mice without interfering with the anti-tumor effects of CTX or with the mobilization of HSCs.Methods:Female CD1 mice were treated on day 0 with an i.p. injection of Ehrlich ascites carcinoma (EAC). On day 7, the mice were i.p. injected with CTX followed by s.c. injection of G-CSF for 5 consecutive days, single s.c. injection of BML/PBS or BML/CTX or single i.v. injection of BMC/PBS or BMC/CTX.Results:Treatment of EAC-bearing mice with BML/PBS or BML/CTX did not interfere with the anti-tumor effect of CTX. EAC increased the numbers of immature polymorphonuclear cells (iPMN; neutrophils) in both blood and spleen. Treatment of EAC-bearing mice with CTX further increased the numbers of these cells, which were decreased upon treatment with BML/CTX. Treatment with BML/PBS or BML/CTX increased the numbers of stem cells (C.Kit+Sca-1+) in BM; the effect of BML/CTX was higher, but with no significant effect on the numbers of HSCs. Future studies are needed to analyze the molecular components in BM lysate and to determine the underlying mechanisms.

In a series of our preclinical studies, we have reported that conditioning of α/β CD8We aimed in this study to use IL-12 to enhance the expansion and cytotoxic functions of ZOL-expanded Vγ9Peripheral blood mononuclear cells (PBMCs) were separated from healthy donors and stage II breast cancer patients. PBMCs (1 × 10In healthy subjects, the addition of IL-12 to ZOL/IL2-stimulated PBMCs increased the expansion and the cytotoxic activity of Vγ9The addition of IL12 at the start of the expansion protocol can enhance the activity of γδ T cells which might be mediated in part by the activation of αβ T cells.

Background Relapse remains a critical challenge in children with acute lymphoblastic leukemia (ALL). The emergence of immunoregulatory cells, including myeloid-derived suppressor cells (MDSCs), and T regulatory (Treg) cells, has been considered one potential mechanism of relapse in children with ALL. Aim This study aimed to address the microRNAs (miRNAs) related to MDSCs and Treg cells and to explore their targeted immunoregulatory pathways. Methods Affymetrix microarray was used for global miRNA profiling in B-ALL pediatric patients before, during, and after induction of chemotherapy. Bioinformatics analysis was performed on MDSCs and Treg cells-related dysregulated miRNAs, and miR-Pathway analysis was performed to explore their targeted immunoregulatory pathways. Results 516 miRNAs were dysregulated in ALL patients as compared to the healthy donor. Among them, 13 miRNAs and 8 miRNAs related to MDSCs and Treg cells, respectively, were common in all patients. Besides, 12 miRNAs were shared between MDSCs and Treg cells; 4 of them were common in all patients. Four immune-related pathways; TNF, TGF-β, FoxO, and Hippo were found implicated. Conclusion Our pilot study concluded certain miRNAs related to MDSCs and Treg cells, these miRNAs were linked to immunoregulatory pathways. Our results open avenues for testing those miRNA as molecular biomarkers for the immunosuppressive tumor microenvironment.

Background: Psoriasis is a complex immune-mediated inflammatory disease that occurs in a genetically susceptible individual. Aim: In this study, we aimed to test whether the use of autovaccine containing different attenuated inflammatory cells could ameliorate the inflammatory process in a psoriasis-like skin inflammation model. Materials and methods: After six days of IMQ application, the psoriasis-like skin inflammation was developed. After six days of IMQ application, the psoriasis-like skin inflammation was developed. Results: After seven days of treatment (14 days of IMQ), the mice treated with the autovaccine showed the most significant improvement as reflected by PASI score. Treatment with autovaccine also induced the best significant decrease of the epidermal thickness as compared to the other treatments (P

Objective: The main objective of this study is to analyze and evaluate the association of IL6-174 G/C gene polymorphisms with response to interleukin-6 blockade in Egyptian patients with systemic juvenile idiopathic arthritis, and to assess the effect of other factors on treatment response as well as long-term efficacy of treatment with Tocilizumab. Methods: Sixty systemic JIA patients (37 males and 23 females with median age at onset 5.2 years) who received IL-6 blockade (Tocilizumab) were recruited. The overall response to treatment with IL-6 blockade was assessed according to: 1) Clinical response, 2) switch (or no switch) to another biological DMARD therapy following IL-6 blockade, 3) achievement of clinically inactive disease within 6 months of IL-6 blockade, 4) improvement in disease activity measured using the modified JADAS-10, and 5) achievement of a glucocorticoid-free state. In addition, basic demographic, laboratory data, characteristics of the disease course and IL6 polymorphisms were assessed.Results: Three IL6 -174 genotypes, including, GG, GC, and CC were found with higher frequencies of GC genotype. These genotypes had non-significant association with the response of sJIA patients to IL-6 blockade in this cohort study. Moreover, a longer time frame from disease onset to diagnosis was associated with poorer long-term treatment response. Conclusion. No significant impact of IL6 -174 G/C gene polymorphisms on treatment response to IL6 blockade therapy in sJIA Egyptian patients, providing evidence of a “window of opportunity” for improved long-term treatment response with shorter time from the disease onset to diagnosis.

MSc Thesis Abstract Background: The use of vaccination in the control of schistosomiasis at a population level induced low cure rates and also resistance to drugs has been recently recorded in Egyptian patients. For these reasons, a search for new drugs with adjuvant and anti-schistosomal activity is urgently needed. Aim of the study: The study focused on newly synthesized ligands that play a central role as inducers of innate and adaptive immune responses and are therefore considered as potential targets for vaccine adjuvants. These ligands include Polyinosinic: polycytidylic acid (Poly (I:C) (TLR3L) and Zymosan (TLR2L) which have displayed an impressive array of biological activities including antitumor and antiviral effects. The present study investigated their adjuvant and anti-schistosomal effects in S. mansoni-infected mice. Materials and Methods: The study was divided into two experimental settings including the vaccination and treatment settings. In the vaccination settings, the mice were assigned into 8 groups. Mice were vaccinated with the ligands 2 times at 2 weeks intervals, then infected with S. mansoni. Five weeks post-infection, the mice were euthanized for analysis. In the non-vaccination settings, the mice were assigned into 8 groups. The determination of worm burden, tissue eggs count, assessment of liver function tests, and parameters of oxidative stress were done. Results: Vaccination and treatment by Poly (I:C) and Zymosan increased the counts of platelets, segmented neutrophils, eosinophils, and monocytes while decreases the lymphocytes count, indicating to the immunological effect of these ligands. The current study showed that Poly (I:C) and Zymosan displayed an adjuvant with soluble worm antigen protein (SWAP) antigen that associated anti-schistosomal activity and with improvement in the liver enzymes and oxidative stress as compared to the vaccination with SWAP antigen alone. These ligands also showed an apparent effect on the granuloma volume. These TLR ligands had no side effects on normal healthy mice and reduced the adverse effects caused by S. mansoni through the improvement of liver enzymes, oxidative stress, reduction of worm burden, and ova count. Conclusion: This study is the first study that assesses TLR ligands as adjuvants and treatment for S. mansoni infection. Our recommendation is to use vaccination and increase the treatment time of both ligands for significant reduction of worm and ova to preserve normal liver function parameters. (This MSc thesis was approved by the Faculty of Science, Tanta University, Egypt by November 26, 2012).

Thesis Abstract Background: Hepatitis C virus (HCV) is a major leading cause of pathogenesis of liver disease which was treated with IFN-based therapy and recently with Sovaldi®. There is a challenge to explore new drugs. Probiotics are the most common natural therapeutic that can ameliorate liver dysfunction. Aim: study the antiviral and antibacterial responses of the probiotics in patients with chronic (HCV). Materials and Methods: patients were treated with IFN/ribavirin, or both IFN and probiotics capsule that was containing L. acidophilus and Bifidobacterium spp. and administered as one capsule per day for a month. Blood and urine samples were collected before and after treatment strategies and were processed for quantitative determination of HCV PCR assay, identification of bacteria by VITEK2 system and 16S r RNA gene sequencing, estimation of probiotics antibacterial activity, as well as counts of leukocytes and CD3+ T cells and CD56+ natural killer cells. Results: Treatment of patients with L. acidophilus and Bifidobacteria spp. enhanced the responses of the patients to IFN-α and ribavirin treatment, as shown by the viral titter of HCV PCR assay that clarified the antiviral activity and also the antibacterial activity against the most common bacterial infections in patients with chronic HCV. Additionally, it increased the numbers of CD3+ and CD56+, Conclusions: Treatment with probiotics before IFN-α and ribavirin therapy can act as a supportive supplement with antiviral, positive immune response and antibacterial activities

Thesis Abstract Background: Using of experimental animal as models to mimic human pathological conditions has facilitated ]understanding the mechanisms of different processes involved in these diseases. Among the experimental animals, mice are the mostly widely used animals in different experimental biology since it has a small size, easy breading, low cost, high fertility rate, and the extensive knowledge of its genome. The differences in the immune cell compmenents between young and adult mice, however, have not been well investigated. Aim: The present study aimed to compare aging on the immune cell components in outbreed and inbreed mice as well as to evaluate the their responses to the inflammatory stimuli Polyinosinic:polycytidylic acid (poly(I:C)). Results: The study investigated whether the aging or the poly(I:C) has the more damaging effect on the cell histology of the thymus gland and spleen. This study confirms that the alteration in the histology of the spleen of aged mice as compared to young mice. Furthermore, poly(I:C) had more tendency to induce alteration in teh archetecture and cellular components in the thymus and spleen of old mice than those in young mice. Conclusion: Immune compartments in old mice are more susciptable to inflammation than those in young mice.

Since December 2019, COVID-19 is a pandemic disease which associates with severe acute respiratory syndrome due to infection with the new coronavirus SARS-Cov-2. The SARS-Cov-2 infection has been recorded in more than 6.4 million patients and associated with over 380,000 deaths. Several scenarios have been suggested to explain the pathogenesis behind the rapid death of patients who SARS and treatment options have been presented to minimize the complication and safe the life of patients. The latest scenario behind the pathogenicity caused by SARS-Cov-2 infection is the capability of the virus to bind with an iron molecule in hemoglobin, resulting in anemia and thrombosis. This scenario might explain the efficacy of the antimalarial drug hydroxychloroquine in the protection of the patients since it competes with the virus to bind with the iron molecule. Due to the side effects of hydroxychloroquine, it is of paramount significance to explore new drugs that can safely block the binding of SARS-Cov-2 to iron molecules. Lactoferrin is an iron-binding glycoprotein related to the transferrin family, which has several besides its anti-viral and immunomodulatory effects. Our hypothesis is that lactoferrin can be used as an alternative to hydroxychlroquine in the treatment of SARS-Cov-2.

Background: About 80% to 85% of lung cancers are non-small cell lung cancer (NSCLC), which are believed to secrete mediators that may lead to disorders in the immune system and tumor immune surveillance. This disorder may include dysfunction in CD8+ T cells and NK cells, which play critical role in fighting against cancer. Aim: Evaluate the impact of lung cancer on the numbers and function of CD8+ T cells, NK and NK T cells as well as the inflammatory milieu. Materials and Methods: Peripheral blood samples were collected from healthy volunteers (n=5) and early diagnosed lung cancer patients (NSCLCs; n=5). The numbers of CD8+ T cells, NK cells and NK T cells were analyzed by flow cytometry after staining with the relevant antibodies. Plasma was prepared from the blood to measure the proinflammatory cytokines and chemokines by Lumiex® protein array. Results: Compared to healthy controls, lung cancer patients showed significant decrease in numbers of CD8+ T cells and NK cells. NK cells showed considerable reduction in their expression levels of the cytolytic molecule granzyme-B. Marked higher levels of IL-1 and CCL2 was detected in the plasma of lung cancer patients as compared to healthy controls. Conclusion: Early diagnosed lung cancer patients showed dysfunction not only in the numbers of cytolytic immune cells but also their function that correlated with inflammatory microenvironment.

Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children. The precise mechanism behind the relapse in this disease is not clearly known. One possible mechanism could be the accumulation of immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs) and T regulatory cells (TIn this study, we aimed to analyze the numbers of these cells in a population of B-ALL pediatric patients.Peripheral blood samples withdrawn from B-ALL pediatric patients (n = 45 before, during and after the induction phase of chemotherapy. Using multi parametric flow cytometric analysis. MDSCs were identified as LinEarly diagnosed B-ALL patients showed significant increases in the numbers of MDSCs and TOur results indicate that B-ALL patients harbor high numbers of both MDSCs and T

Hepatocellular carcinoma (HCC) is the 3rd most common cause of cancer-related death worldwide. It has evolved different immune escape mechanisms, which might include emergence of lymphoid and myeloid regulatory cells. Aim of this work: To determine the numbers of Myeloid-derived suppressor cells (MDSCs) in peripheral blood and ascitic fluid in cirrhosis and HCC and their relation to IFN-γ and α-fetoprotein (α-FP).Sixty individuals were enrolled in this study; forty cirrhotic patients with ascites; twenty without HCC (Group I), and twenty with HCC (group II) as well as twenty healthy individuals as a control group (group III). The phenotype and numbers of MDSCs were analyzed in peripheral blood of all the individuals and ascitic fluid of the patients using flow cytometry. Intracellular IFN-γ and serum alfa-fetoprotein were measured.Significant increases in the relative and the mean number of peripheral blood MDSCs were found in the cirrhosis and HCC groups than in the control group, with the HCC group showing the highest number. MDSC count was negatively correlated with IFN-γ levels, while α-FP was positively correlated with MDSC% in the HCC group. MDSC count was low in ascitic fluid of both HCC and cirrhosis groups with no significant difference between the 2 groups.A high frequency of MDSCs was detected in the peripheral blood of cirrhotic and HCC patients, indicating presence of immunosuppressive arms. These cells could be targeted to develop a new effective immunotherapy or an adjuvant to current therapies.

One of the activating factors of the cells of the innate immune system is the agonists of toll-like receptors (TLRs). Our earlier publications detailed how poly(I:C), a TLR3 agonist, elevates the NK cell population and the associated antigen-specific CD8+ T cell responses. This study involved a single treatment of the B6 mice with poly(I:C) intraperitoneally. To perform a detailed phenotypic analysis, mononuclear cells were prepared from each of the liver, peripheral blood, and spleen. These cells were then examined for their NK cell population by flow cytometric analysis following cell staining with indicated antibodies. The findings of the study showed that the NK cell population of the liver with an NK1.1highCD11bhighCD11chigh B220+Ly6G- phenotype was elevated following the treatment with poly(I:C). In the absence of CD11b molecule (CR3-/- mice), poly(I:C) can still increase the remained numbers of NK cells with NK1.1+CD11b- and NK1.1+Ly6G- phenotypes in the liver while their numbers in the blood decrease. After the treatment with anti-AGM1 Ab, which induced depletion of NK1.1+CD11b+ cells and partial depletion of CD3+NK1.1+ and NK1.1+CD11b- cell populations, poly(I:C) normalized the partial decreases in the numbers of NK cells concomitant with increased numbers of NK1.1-CD11b+ cell population in both liver and blood. Regarding mice with a TLR3-/- phenotype, their injection with poly(I:C) resulted in the partial elevation in the NK cell population as compared to wild-type B6 mice. To summarise, the TLR3 agonist poly(I:C) results in the elevation of a subset of liver NK cells expressing the two myeloid markers CD11c and CD11b. The effect of poly(I:C) on NK cells is partially dependent on TLR3 and independent of the presence of CD11b.

Granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor, is a standard supportive therapy given during cancer treatment. It induces acceleration in neutrophil recovery through stimulation of mobilization of hematopoietic progenitors. Given that the latter is also induced by chemotherapy itself, the timing of administration of G-CSF postchemotherapy might impact the resultant overall effects. The present study aimed to determine the optimal timing of G-CSF postchemotherapy to exert its optimal effects on the immune cell recovery and its impact on antigen-specific CD8+ T-cell response. B6 mice were treated once with cyclophosphamide (4 mg/mouse; CTX) and then daily with G-CSF (5 g/mouse) from Days 1-5, 2-5 or 5-9 post-CTX treatment. The total numbers of various immune cell types were analyzed on Days 7, 9 and 12 post-CTX treatment. To evaluate effects on CD8+ T-cell response, a pmel-1 transgenic mouse model was used in combination with prime boost peptide vaccination therapy. The total number of white blood cells (WBC), neutrophils, monocytes, lymphocytes, granulocytes and dendritic cells (DC) were significantly increased after G-CSF treatment in particular when G-CSF was administered from Days 2-5 post-CTX treatment. Application of this timing of G-CSF and CTX treatment after adoptive transfer of T-cells followed by prime-boost vaccination with antigenic peptide did not block the expansion of the donor pmel-1 CD8+ T-cells. In conclusion, adjusting the timing of treatment with G-CSF postchemotherapy can optimize its promoting effects on recovery of myeloid cells without altering the associated antigen-specific immunity.

Given the self nature of cancer, anti-tumor immune response is weak. As such, acute inflammation induced by microbial products can induce signals that result in initiation of an inflammatory cascade that helps activation of immune cells. We aimed to compare the nature and magnitude of acute inflammation induced by toll-like receptor ligands (TLRLs) on the tumor growth and the associated inflammatory immune responses. To induce acute inflammation in tumor-bearing host, CD1 mice were inoculated with intraperitoneal (i.p.) injection of Ehrlich ascites carcinoma (EAC) (5 × 10(5) cells/mouse), and then treated with i.p. injection on day 1, day 7 or days 1 + 7 with: (1) polyinosinic:polycytidylic (poly(I:C)) (TLR3L); (2) Poly-ICLC (clinical grade of TLR3L); (3) Bacillus Calmette Guerin (BCG) (coding for TLR9L); (4) Complete Freund's adjuvant (CFA) (coding for TLR9L); and (5) Incomplete Freund's Adjuvant (IFA). Treatment with poly(I:C), Poly-ICLC, BCG, CFA, or IFA induced anti-tumor activities as measured by 79.1%, 75.94%, 73.94%, 71.88% and 47.75% decreases, respectively in the total number of tumor cells collected 7 days after tumor challenge. Among the tested TLRLs, both poly(I:C) (TLR3L) and BCG (contain TLR9L) showed the highest anti-tumor effects as reflected by the decrease in the number of EAc cells. These effects were associated with a 2-fold increase in the numbers of inflammatory cells expressing the myeloid markers CD11b(+)Ly6G(+), CD11b(+)Ly6G(-), and CD11b(+)Ly6G(-). We concluded that Provision of the proper inflammatory signal with optimally defined magnitude and duration during tumor growth can induce inflammatory immune cells with potent anti-tumor responses without vaccination.

Background and aim Cancer stem cells (CSCs) are rare cell population present in the tumor bulk that are thought to be the reason for treatment failure following chemotherapy in terms of their intrinsic chemo-resistance. Our study aimed to develop an effective therapeutic strategy to target chemo-resistant cancer stem – like cells population in solid Ehrlich carcinoma (SEC) mice model using dendritic cells (DCs) loaded with enriched tumor cells lysate bearing CSC-like phenotype as a vaccine. Materials and methods Ehrlich carcinoma cell line was exposed to different concentrations of cisplatin, doxorubicin, or paclitaxel. Drug treatment that resulted in drug surviving cells with the highest expression of CSCs markers (CD44+/CD24−) was selected to obtain enriched cell cultures with resistant CSCs population. Dendritic cells were isolated from mice bone marrow, pulsed with enriched CSC lysate, analyzed and identified (CD11c, CD83 and CD86). SEC-bearing mice were treated with loaded or unloaded DCs either as single treatment or in combination with repeated low doses of cisplatin. IFN- γ serum level and p53gene expression in tumor tissues were determined by ELISA and real-time PCR, respectively. Results and conclusion The results revealed that vaccination with CSC loaded DCs significantly reduced tumor size, prolonged survival rate, increased IFN-γ serum levels, and upregulated p53gene expression in SEC bearing mice. These findings were more evident and significant in the group co-treated with CSC-DC and cisplatin rather than other treated groups. This study opens the field for combining CSC-targeted immunotherapy with repeated low doses chemotherapy as an effective strategy to improve anticancer immune responses.

Although the majority of cancers respond to chemotherapy, most cancer types relapse, at least in part, due to the poor immunogenicity of most tumor. We have reported before that treatment of tumor bearing mice with a combination of the anti-cancer chemotherapy cyclophosphamide (CTX) and immunotherapy can result in complete tumor regression using T-cell receptor (TCR) transgenic CD8+ T cells specific to antigens. This study aimed to determine whether chemotherapy can cure immunogenic tumor which expresses non-self-tumor antigen and result in antitumor immunity. Either EL4 cell line, a poorly immunogenic thymoma, or EG7, a clone of EL4 cells transfected with ovalbumin (OVA), as a non-self-antigen were inoculated subcutaneously into wild type or splenectomized C57BL/6 mice and then treated once with intraperitoneal (i.p.) injection of 4 mg CTX/mouse. In certain experiments, the mice were rechallenged with the same tumor type 1–2 months after the primary challenge. Treatment of EL4 bearing mice with CTX induced transient antitumor effect followed by tumor progression. Interestingly, however, treatment of EG7-bearing mice with CTX resulted in regression of early and advanced tumors. EG7 tumor-free mice rejected the second and the third challenges with EG7 cells, but not with challenge EL4 cells. These antitumor effects did not require spleen, since splenectomized mice showed similar antitumor effects of CTX on EG7 cells. Taken together, these data indicate that expression of non-self-antigen by poorly immunogenic tumor might be a reliable means to increase its immunogenicity and its response to chemotherapy.

The current study investigated the combinatorial effect of pravastatin (PRAV) and bone marrow mononuclear cells (BM-MNC) on acute myocardial infarction (AMI) induced experimentally in rats. After induction of MI, rats were given oral PRAV (20 mg/kg/day) for 28 days or a bolus intravenous injection (via lateral vein) of a total of 14 × 10(6) autologous BM-MNC or a combination of both. Serum brain natriuretic peptide (BNP) and histologic changes in cardiac tissues were assessed. Cardiac contents of lipid peroxides, superoxide dismutase (SOD) and inflammatory biomarkers including tumor necrosis factor (TNF)-α and interleukin (IL)-1β as well as vascular endothelial growth factor (VEGF) and nitric oxide (NO) were also measured. Combined PRAV and BM-MNC treatment significantly suppressed serum BNP. Cardiac cell apoptosis and inflammatory cell infiltration in heart tissue decreased significantly in both the PRAV and the PRAV + BM-MNC groups. Cardiac lipid peroxides along with TNFα and IL-1β levels were significantly reduced in both the PRAV and PRAV + BM-MNC hosts with an increase in SOD levels. However, the combined treatment increased cardiac NO levels and did not modify cardiac VEGF levels. The current results indicated that administration of BM-MNC improved the therapeutic efficacy of PRAV treatment by improving the morphology of infarcted hearts as well as decreasing inflammation in a host, but did not do so by inducing therapeutic angiogenesis.

Mouse CD8(+) T cells conditioned with interleukin (IL)-12 ex vivo mediate the potent regression of established melanoma when transferred into lymphodepleted mice. However, the quantitative and qualitative changes induced by IL-12 in the responding mouse CD8(+) T cells have not been well defined. Moreover, the mechanisms by which IL-12-conditioning impacts human CD8(+) T cells, and how such cells might be expanded prior to infusion into patients is not known. We found that ex vivo IL-12-conditioning of mouse CD8(+) T cells led to a tenfold-100-fold increase in persistence and anti-tumor efficacy upon adoptive transfer into lymphodepleted mice. The enhancing effect of IL-12 was associated with maintenance of functional avidity. Importantly, in the context of ongoing ACT clinical trials, human CD8(+) T cells genetically modified with a tyrosinase-specific T cell receptor (TCR) exhibited significantly enhanced functional activity when conditioned with IL-12 as indicated by heightened granzyme B expression and elevated peptide-specific CD107a degranulation. This effect was sustainable despite the 20 days of in vitro cellular expansion required to expand cells over 1,000-fold allowing adequate cell numbers for administration to cancer patients. Overall, these findings support the efficacy and feasibility of ex vivo IL-12-conditioning of TCR-modified human CD8(+) T cells for adoptive transfer and cancer therapy.

Introduction: Hepatitis C virus (HCV) infection causes chronic hepatitis, which is often associated with suppressed anti-HCV immune responses. We have recently reported accumulation of myeloid-derived suppressor cells (MDSCs) and suppressed immunity in cancer patients.Aim: The main aim of this study was to determine whether chronic HCV patients harbor high of MDSCs in general and in nonresponders to IFN-based therapy in particular as well as to analyze the immune suppressive molecules.Methods: Peripheral blood samples withdrawn from 154 patients with chronic HCV infection and were categorized into responders and nonresponders based on viral titer upon IFN-α treatment.Results: The relative and absolute numbers of MDSCs defined as Lin−/HLA-DR−/CD33+/CD11b+ increased in all HCV patients, where they were higher in nonresponders than in responders. Additionally, the levels of MDSCs after 4–6 months of treatment in responders were lower than during the course of treatment. The responders also showed hig...

In order to get mature dendritic cells (DC) that is a crucial prerequisite for success in tumor immunotherapy protocols. Herein, we assumed that administration of murine bone marrow (BM)-derived DC (BM-DC), loaded ex vivo with whole Ehrlich ascites carcinoma (EAC) lysate, in the context of systemic chemotherapy cyclophosphamide (CTX) to induce antitumor immune responses, may be a good strategy to improve the presentation of tumor-specific antigens to the immune system. In the first series of experiments, BM cells generated either from BM of naive mice or from BM of EAC-bearing mice were cultured in the presence of GM-CSF and IL-4 for 6 days. At day 7, cells were loaded for 48 h with one of the following maturation agents: EAC lysate (1 mg/ml), poly-inosinic: polycytidylic acid [poly(I:C)] (25 μg/ml) or mRNA encoding human telomerase reverse transcriptase (hTERT-mRNA) (2 μg/ml). In the second series of experiments, EAC-bearing mice were intraperitoneally (i.p.) injected with CTX followed by i.p. vaccination with DC, loaded ex vivo with EAC lysate. DC yield and the phenotypic expression of maturity-related surface markers of DC ( i.e. CD11b and CD11c) in both series of experiments were investigated. As a result, a significant decrease in the number of DC generated from poly(I:C)-supplemented BM culture from EAC-bearing mice has been detected. Loading of BM cells with poly(I:C), EAC lysate or hTERT-mRNA could induce the expression of CD11b and CD11c. Additionally, vaccination of EAC-bearing mice with DC loaded ex vivo with EAC lysate following CTX treatment, resulted in increases in the percentage of multiple populations of CD11b + CD11c + in BM, spleen and peripheral blood (PB). To conclude, further researches to clarify the mechanism involved in DC maturation are crucial not only to comprehend DC biology but also to optimize DC immunotherapy protocols.

Background: Acute lymphocytic leukemia (ALL) is biologically and clinically considered as a heterogeneous neoplasm of lymphoid progenitor cells in the bone marrow (BM). 15- 20 % of children with ALL who achieve an initial remission, will show relapse. One potential mechanism behind this relapse could be the emergence of cancer stem cells (CSCs), which are considered the driving force of tumorigenesis due to their ability of self-renewal as well as the emergence of immune regulatory cells including myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Treg). Aim: The main aim of this study was to analyze the numbers of CSCs and correlate these numbers with the numbers of blasT-cells, MDSCs and Treg cells in children with B-ALL before and after induction of chemotherapy. Materials and Methods: CSCs were defined as CD45dimCD19+CD10+CD34+CD38-, MDSCs were defined as Lin-HLA-DR-CD33+CD11b+ and Treg cells were defined as CD4+CD25+CD127-. The frequencies of these cells were analyzed in the peripheral blood of B-ALL patients before (n= 10) and after (n= 10) induction of chemotherapy using flow cytometry. Results: Significant increases in the numbers of CSCs were shown in B-ALL patients after induction of chemotherapy as compared to newly diagnosed patients (7.6± 8.3 vs. 2.7± 2.4, P Citation Format: Mohamed Labib Salem, Mohamed Attia, Said Abdou, Abdel-Aziz A. Zidan, Mona F. Zidan. Higher numbers of cancer stem cells in the peripheral blood of children with B-ALL after chemotherapy [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 A106.

Liver is considered the target of hepatitis C virus (HCV), which has marked tropism for hepatocytes. In this study, we investigated changes in bone marrow (BM) and blood and their correlation with viremia level in 30 pale patients with chronic HCV who were selected before antiviral therapy. Patients with BM positive for HCV RNA (53.33 %) showed moderate to high viremia, while patients with BM negative for RNA (46.67 %) had low viremia. There was no significant difference in the liver histopathology between patients with HCV-RNA-negative and positive BM. Patients with BM positive for HCV RNA showed significant changes in BM cells, including the degree of immune complex deposition and alterations in peripheral blood counts compared to patients with BM negative for RNA and healthy controls, suggesting that BM changes could be a sequel or a reservoir for HCV viremia.

Background Clinical application of adoptive T cell therapy has been hindered by an inability to generate adequate numbers of nontolerized, functionally active, tumor-specific T cells, which can persist in vivo. In order to address this, we evaluated the impact of interleukin (IL)-12 signaling during tumor-specific CD8+ T cell priming in terms of persistence and antitumor efficacy using an established B16 melanoma tumor adoptive therapy model. Study Design B6 mice were injected subcutaneously with B16 melanoma tumor cells. On day 12 of tumor growth, mice were preconditioned with cyclophosphamide (4mg dose, intraperitoneally), and 1 day later were treated by adoptive transfer of tumor-specific pmel-1 CD8+ T cells primed ex vivo 3 days earlier with both IL-12 and antigen (hGP10025-33 peptide) or antigen only. Tumors were measured biweekly, and infused donor T cells were analyzed for persistence, localization to the tumor, phenotype, and effector function. Results Adoptive transfer of tumor-specific CD8+ T cells primed with IL-12 was significantly more effective in reducing tumor burden in mice preconditioned with cyclophosphamide compared with transfer of T cells primed without IL-12. This enhanced antitumor response was associated with increased frequencies of infused T cells in the periphery and tumor as well as elevated expression of effector molecules including granzyme B and interferon-γ (IFNγ). Conclusions Our findings demonstrate that ex vivo priming of tumor-specific CD8+ T cells with IL-12 dramatically improves their in vivo persistence and therapeutic ability on transfer to tumor-bearing mice. These findings can be directly applied as novel clinical trial strategies.

Recently, we showed that post cyclophosphamide (CTX) microenvironment benefits the function of transferred T cells. Analysis of the kinetics of cellular recovery after CTX treatment showed that a single 4 mg/mouse CTX treatment decreased the absolute number of leukocytes in the peripheral blood (PBL) at days 3-15, and in the spleen and bone marrow (BM) at days 3-6. The absolute numbers of CD11c(+)CD11b(-) and CD11c(+)CD11b(+) dendritic cells (DCs), CD11b(+) and Ly6G(+) myeloid cells, T and B cells, CD4(+)CD25(+) T regulatory (T(reg)) cells, and NK1.1(+) cells also decreased. The cell numbers returned to control levels during the recovery phase. The absolute numbers of B cells remained low for 3 weeks. The numbers of DCs increased in PBL and spleen at day 9 but returned to control levels at day 15. These data indicate that CTX alters the cellular microenvironment in kinetics that might be precisely targeted to benefit the host.

Traditionally, expression of toll-like receptors (TLRs) has been associated with innate immune cells in particular professional antigen presenting cells and natural killer cells. This led to the concept that the adjuvant effects of ligation of TLR in a host occur mainly in innate immune cells. However, this concept has been challenged by recent studies including ours demonstrating that T cells express appreciated levels of different TLRs, which can serve as costimulatory co-receptors during polyclonal and antigen-specific stimulation of T cells. Because T cells express low levels of TLRs as compared to innate immune cells, increasing the expression levels of TLRs in T cells can significantly maximize their responses to the costimulatory effects of TLR ligation. This review article focuses on the potential role of TLR expression in T cells in their responses to vaccination regimen containing TLR agonists and how it can be modulated to optimize anti-tumor immunity.

Preconditioning a recipient host with lymphodepletion can markedly augment adoptive T cell therapy. However, the precise mechanisms involved are poorly understood. In a recent study, we observed a significant increase in the circulating levels of dendritic cells (DCs; CD11c+CD11b+) during the recovery from cyclophosphamide (CTX)-induced lymphodepletion. Herein, we demonstrate that the CTX-induced DC expansion was not altered by adjuvant chemotherapy or tumor burden but was augmented by coadministration of granulocyte-colony stimulating factor. Although the increase in the number of DCs was preceded by a systemic expansion of a population expressing the phenotype of myeloid-derived suppressor cells (Gr-1+CD11b+), depletion of these Gr-1+ cells had no effect on the noted expansion. Moreover, when Gr-1highCD11bhigh cells were sorted from CTX-treated mice and adoptively transferred into control or CTX-treated recipients, they did not differentiate into DCs. Post-CTX expansion of DCs was associated with proliferation of DCs in bone marrow (BM) during the lymphopenic phase and in the blood and spleen during the recovery phase. Furthermore, adoptive transfer of BM cells from CTX-treated mice produced equal numbers of DCs in the blood of either CTX-treated or untreated recipients. CTX induced a dynamic surge in the expression of growth factors and chemokines in BM, where CCR2 and Flt3 signaling pathways were critical for DC expansion. In sum, our data suggest that CTX induces proliferation of DCs in BM prior to their expansion in the periphery. Targeting DCs at these phases would significantly improve their contribution to the clinical application of lymphodepletion to adoptive immunotherapy.

Pancreatic cancers are classified as either exocrine or endocrine tumors depending on which type of tissue they arise from within the gland. Endocrine tumors of the pancreas are very rare, accounting for only 5% of all pancreatic cancers. The majority of endocrine pancreatic tumors are functional adenocarcinomas that overproduce a specific hormone. Pancreatic cancer is a devastating disease and has an extremely poor prognosis. Most patients die within a year of diagnosis and the overall 5-year survival rate is

Adoptive transfer of autologous tumor-reactive T cells holds promise as a cancer immunotherapy. In this approach, T cells are harvested from a tumor-bearing host, expanded in vitro and infused back to the same host. Conditioning of the recipient host with a lymphodepletion regimen of chemotherapy or radiotherapy before adoptive T cell transfer has been shown to substantially improve survival and anti-tumor responses of the transferred cells. These effects are further enhanced when the adoptive T cell transfer is followed by vaccination with tumor antigens in combination with a potent immune adjuvant. Although significant progress has been made toward an understanding of the reasons underlying the beneficial effects of lymphodepletion to T cell adoptive therapy, the precise mechanisms remain poorly understood. Recent studies, including ours, would indicate a more central role for antigen presenting cells, in particular dendritic cells. Unraveling the exact role of these important cells in mediation of the beneficial effects of lymphodepletion could provide novel pathways toward the rational design of more effective anti-cancer immunotherapy. This article focuses on how the frequency, phenotype, and functions of dendritic cells are altered during the lymphopenic and recovery phases post-induction of lymphodepletion, and how they affect the anti-tumor responses of adoptively transferred T cells.

Cytokines play crucial roles in all aspects of health and diseases. They influence immune expression, the development of immunologic memory, and regulation of antigen-specific and nonspecific immune activation. Based on their production by T helper (TH) cells, cytokines are categorized into TH1 and TH2 types. TH1 type includes inflammatory cytokines and growth factors. The former induces signal three that bridge innate and adaptive immunity, whilst the latter sustains proliferation and survival of T cells. Therefore, TH1 cytokines profoundly mediate generation of preventive immune responses toward infectious diseases and cancer. By contrast, TH2 cytokines are anti-inflammatory through suppression of THl-mediated immune responses, but they also accelerate TH2-mediated diseases. This article describes the impact of the THl/TH2 cytokines on shaping the nature of the immune cell responses to inflammation, infection, and cancer. We predict that THl/TH2 could be considered as a fingerprint for probing the identity of diseases. Fundamental knowledge of the design of cytokine-based therapeutic strategies is now reaching the phase of application and a number of cytokines are already on the market and many others are under clinical investigation.

Recent preclinical studies suggest that vaccination following adoptive transfer of CD8+ T cells into a lymphopenic host can augment the therapeutic antitumor responses of the transferred cells. However, the mechanism by which the lymphopenic microenvironment benefits Ag-specific CD8+ T cell responses remains elusive. We show herein that induction of lymphodepletion by a single 4 mg cyclophosphamide (CTX) treatment induces a marked expansion of immature dendritic cells (DCs) in the peripheral blood on days 8–16 post-CTX (termed restoration phase). In vitro, these DCs were functional, because they showed normal phagocytosis and effective Ag presentation capability upon activation. In vivo, administration of the TLR3 agonist poly(I:C) at the peak of DC expansion (day 12 postlymphopenia) induced inflammatory cytokine production and increases in the number of activated DCs in lymph nodes. Importantly, boosting with gp10025–33 melanoma peptide combined with poly(I:C) 12 days after an initial priming with the same regimen significantly increased the expansion and the antitumor efficacy of adoptively transferred pmel-1 CD8+ T cells. These responses were abrogated after depletion of activated DCs during Ag boosting. In conclusion, our data show that CTX treatment induces, during the restoration phase, expansion of immature DCs, which are functional and can be exploited in vivo to foster more effective antitumor adoptive immunotherapy strategies.

Interleukin-12 can act as a potent adjuvant for T cell vaccines, but its clinical use is limited by toxicity. Paracrine administration of IL-12 could significantly enhance the response to such vaccines without the toxicity associated with systemic administration. We have developed a novel vaccine delivery system (designated F2 gel matrix) composed of poly-N-acetyl glucosamine that has the dual properties of a sustained-release delivery system and a potent adjuvant. To test the efficacy of paracrine IL-12, we incorporated this cytokine into F2 gel matrix and monitored the response of OT-1 T cells in an adoptive transfer model. Recipient mice were vaccinated with F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12 (paracrine IL-12), or F2 gel/SIINFEKL plus systemic IL-12 (systemic IL-12). Systemic levels of IL-12 were lower in paracrine IL-12-treated mice, suggesting that paracrine administration of IL-12 may be associated with less toxicity. However, paracrine administration of IL-12 was associated with an enhanced Ag-specific T cell proliferative and functional response. Furthermore, paracrine IL-12 promoted the generation of a stable, functional memory T cell population and was associated with protection from tumor challenge. To study the mechanisms underlying this enhanced response, wild-type and gene-deficient mice were used. The enhanced immune response was significantly reduced in IFN-γ−/− and IL-12Rβ2−/− recipient mice suggesting that the role of IL-12 is mediated, at least in part, by host cells. Collectively, the results support the potential of F2 gel matrix as a vaccine delivery system and suggest that sustained paracrine release of IL-12 has potential clinical application.

Systemic interleukin-2 (IL-2) therapy has been shown to enhance the clinical efficacy of peptide-based cancer vaccines. However, the mechanisms involved in this complex response remain poorly defined. IL-2 is known to be a potent T cell growth factor, but recent studies suggest that IL-2 is also involved in the regulation of T cell immune responses by increasing the susceptibility of proliferating T cells to apoptosis. Using an adoptive transfer model, we demonstrate that the administration of systemic IL-2 significantly enhances the primary and memory immune responses following peptide-based vaccination. In order to define the mechanisms of IL-2 therapy on the antigen-specific T cell response, the kinetics of T cell proliferation, apoptosis, and trafficking were explored. Systemic IL-2 therapy did not appear to alter the kinetics of T cell proliferation immediately following vaccination, but did prolong the proliferative response. Furthermore, IL-2 therapy did not significantly influence apoptosis of proliferating T cells. Such therapy did, however, potentiate l -selectin (CD62L) downregulation on activated antigen-specific T cells, and altered their trafficking confirming their potential therapeutic value. Our findings support the use of systemic IL-2 following peptide-based vaccination, and suggest that IL-2 therapy enhances the primary and memory immune responses by prolonging the proliferative response and altering the trafficking of antigen-specific T cells.

The adoptive transfer of tumor-specific T cells expanded in vitro can be of significant therapeutic value in select cancer patients. This strategy is limited though, as it is often difficult, if not impossible, to obtain T cells of clinical value. The transfer of TCR genes to mature T cells to generate tumor-reactive T cells provides a potential mechanism to overcome these limitations. To evaluate the feasibility of such an approach and the quality of the resulting T cells, we generated replication-deficient retroviral vectors using the well-characterized OT-1 TCR genes. After transducing murine T cells, we were able to expand large numbers of Ag-specific T cells that were functionally active against tumor cells expressing the relevant Ag. Furthermore, we found that T cells expressing retrovirally encoded TCR had avidity that was similar to that of the parental clone. This maintenance of avidity was despite variable expression of the retrovirally encoded TCR and the presence of potentially competing endogenous TCRs. These results suggest that the inherent qualities of the TCR, as dictated by the coding sequence, are the most critical parameters in the generation of high-avidity T cells.

Background : Poly(I:C) is a Toll-like receptors 3 agonist, which induces potent innate immune responses, and as a consequence adaptive immunity. However, the rapid degradation of poly(I:C) influences its half-life and its adjuvant effects in preclinical and clinical uses. Aims : We aimed in this study to conjugate poly(I:C) with polyethylene glycol/poly D, L-actide-co-glycolide (PLGA/PEG) polymers as an approach for better delivery and immunomodulatory effects. Materials and Methods : Female CD1 mice were treated once with PEG/PLGA, poly(I:C)/PLGA/PEG (50 μg), poly(I:C)/ PLGA/PEG (10 μg) or PEG via intraperitoneal injection and mice were sacrificed 1 day later for complete blood count analysis and analysis of the immune cells by flow cytometry. Results : Treatment with PEG/PLGA, poly(I:C)/PLGA/PEG (50 μg), poly(I:C)/PLGA/PEG (10 μg) or PEG administration resulted in significant (P = 0.0197) increases (1.89, 1.76, 1.69, and 1.42-fold, respectively) in the absolute number of neutrophils as compared to naive mice. Conclusion : Conjugation of poly(I:C) with polymers does not hamper its immunomodulatory effects, instead it enhances its effects on increasing the number of immune cells opening an avenue for further studies on the beneficial effects of this conjugate.

In this study, antiviral effect of black seed oil (BSO) from Nigella sativa was investigated using murine cytomegalovirus (MCMV) as a model. The viral load and innate immunity mediated by NK cells and Mphi; during early stage of the infection were analyzed. Intraperitoneal (i.p.) administration of BSO to BALB/c mice, a susceptible strain of MCMV infection, strikingly inhibited the virus titers in spleen and liver on day 3 of infection with 1x10(5) PFU MCMV. This effect coincided with an increase in serum level of IFN-gamma. Although BSO treatment decreased both number and cytolytic function of NK cells on day 3 of infection, it increased numbers of Mphi; and CD4(+) T cells. On day 10 of infection, the virus titer was undetectable in spleen and liver of BSO-treated mice, while it was detectable in control mice. Although spleen of both control and BSO-treated mice showed similar CTL activities on day 10 after infection, serum level of IFN-gamma in BSO-treated mice was higher. Furthermore, BSO treatment upregulated suppressor function of Mphi; in spleen. These results show that BSO exhibited a striking antiviral effect against MCMV infection which may be mediated by increasing of Mphi; number and function, and IFN-gamma production.

Mice treated with a contraceptive dose of beta-estradiol (E2) demonstrated changes in their macrophage (Mphi) number and functions. While E2 increased and decreased the Mphi number in PBMC and PEC respectively, it enhanced the in vitro phagocytosis of FITC-labeled beads by both cells. E2 treatment also enhanced the phagocytic function of Mphi as assessed by the in vivo carbon clearance assay. In contrast, the in vitro intracellular killing function of adherent cells in peritoneal exudate cells (PEC) against Listeria monocytogenes decreased after E2 treatment. In line with the decrease in the intracellular killing function, the E2-treated mice showed an impaired protection against L. monocytogenes infection. To clarify the mechanism of the E2-mediated suppression of the protective response against L. monocytogenes infection, we next analyzed the cytokine expression by PEC in E2-treated L. monocytogenes-infected mice. On day 5 of the infection, the expression of IL-12, TNF-alpha and IL-10 by adherent PEC from the E2-treated mice was lower than that from the control-infected mice. The decrease in the cytokine expression by adherent PEC of E2-treated mice coincided with the decrease of IFN-gamma expression, and the increase in the IL-4, IL-10 and TGF-beta expressions by non-adherent PEC. These results revealed two aspects of the effects of E2 on Mphi. Even though E2 was found to enhance Mphi phagocytosis, the anti-bacterial function was suppressed. This suppression may be mediated by the inhibition of both IL-12 and TNF-alpha which play important roles in the protective response against intracellular bacteria.

Effective antitumor immunity requires the generation and persistence of functional tumor-specific T-cell responses. Among the critical factors that often control these responses is how the antigen is delivered and presented to T cells. The use of peptide-based vaccination has been found to be a promising means to induce antitumor T-cell responses but with limited effects even if the peptide is co-delivered with a potent adjuvant. This limited response could be due to cancer-induced dysfunction in dendritic cells (DC), which play a central role in shaping the quantity and quality of antitumor immunity. Therefore, DC-based peptide delivery of tumor antigen is becoming a potential approach in cancer immunotherapy. In this approach, autologous DC are generated from their precursors in bone marrow or peripheral blood mononuclear cells, loaded with tumor antigen(s) and then infused back to the tumor-bearing host in about 7 days. This DC-based vaccination can act as an antigen delivery vehicle as well as a potent adjuvant, resulting in measurable antitumor immunity in several cancer settings in preclinical and clinical studies. This chapter focuses on DC-based vaccination and how this approach can be more efficacious in cancer immunotherapy.Effective antitumor immunity requires the generation and persistence of functional tumor-specific T-cell responses. Among the critical factors that often control these responses is how the antigen is delivered and presented to T cells. The use of peptide-based vaccination has been found to be a promising means to induce antitumor T-cell responses but with limited effects even if the peptide is co-delivered with a potent adjuvant. This limited response could be due to cancer-induced dysfunction in dendritic cells (DC), which play a central role in shaping the quantity and quality of antitumor immunity. Therefore, DC-based peptide delivery of tumor antigen is becoming a potential approach in cancer immunotherapy. In this approach, autologous DC are generated from their precursors in bone marrow or peripheral blood mononuclear cells, loaded with tumor antigen(s) and then infused back to the tumor-bearing host in about 7 days. This DC-based vaccination can act as an antigen delivery vehicle as well as a potent adjuvant, resulting in measurable antitumor immunity in several cancer settings in preclinical and clinical studies. This chapter focuses on DC-based vaccination and how this approach can be more efficacious in cancer immunotherapy.

CD62L governs the circulation of CD8(+) T cells between lymph nodes and peripheral tissues, whereby the expression of CD62L by CD8(+) T cells promotes their recirculation through lymph nodes. As such, CD62L participates in the fate of adoptively transferred CD8(+) T cells and may control their effectiveness for cancer immunotherapy, including settings in which host preconditioning results in the acute lymphopenia-induced proliferation of the transferred cells. Indeed, previous studies correlated CD62L expression by donor CD8(+) cells with the success rate of adoptive cell therapy (ACT). Here, we analyzed the functions and fate of ex vivo-activated, tumor-specific CD62L(-/-) CD8(+) T cells in a mouse melanoma model for ACT. Unexpectedly, we observed that CD62L(-/-) CD8(+) T cells were functionally indistinguishable from CD62L(+/+) CD8(+) T cells, i.e., both greatly expanded in cyclophosphamide preconditioned animals, controlled subcutaneously and hematogenously spreading tumors, and generated anti-tumor-specific CD8(+) T cell memory. Moreover, even in hosts with rudimentary secondary lymphoid organs (LT(-/-) animals), CD8(+) T cells with and without CD62L expanded equivalently to those adoptively transferred into wild-type animals. These results put into question the utility of CD62L as a predictive biomarker for the efficacy of ex vivo-expanded T cells after ACT in lymphopenic conditions and also offer new insights into the homing, engraftment, and memory generation of adoptively transferred ex vivo-activated CD8(+) T cells.

Meeting abstracts Inflammation is a vital process induced by microbial infection or tissue injury. The main function of inflammation is to resolve the infection or repair the damage and return the body to a state of homeostasis. Acute inflammation can induce signals that result in two main

We have reported recently that Interleukin-12 (IL-12) released from poly-N-acetyl glucosamine gel matrix (F2 gel/IL-12) is more effective than free IL-12 to enhance vaccination of mice with Schistosoma soluble worm antigen preparation. The aim of this study is to evaluate the effect of F2 gel/IL-12 on the inflammatory responses in mice undergoing schistosomiasis infection in absence of vaccination. To achieve this, mice undergoing Schistosoma mansoni infection or cured from this infection, after treatment with praziquantil (PZQ), were treated with subcutaneous injection of IL-12 for 3 consecutive days or once with F2 gel loaded with IL-12 (F2 gel/IL-12). The treatment was started on day 35 days after infection. For infection, mice were infected with 100 cercariae of S. mansoni using tail immersion method. We found that treatment with F2 gel/IL-12 induced significant decreases in the egg burden with a moderate reduction in the size of granuloma and decrease in the cellular granulomatous reaction in the lung as compared to infected mice treated with IL-12. These effects of F2 gel/IL-12 were more pronounced in infected mice previously treated with the anti-schistosomal drug PZQ. The total numbers of white blood cells in all treated mice showed similar profile. Treatment with IL-12 or F2 gel/IL-12, however, showed significant reduction in the number of mononuclear cells when compared with non-treated infected mice. In conclusion, this study showed the ability of IL-12 released from F2 gel to lower the inflammatory response to Schistosoma infection even in absence of vaccination.

Background: Integrating immunotherapy and chemotherapy is most likely to be the basis for new optimism in targeting cancer therapies to form local tumor microenvironment and attack tumors early in their development. This regimen has some potential risks such as myelo- and immunosuppression and chemo-resistant tumor cells. Aim: The present study aimed to investigate the combination of chemotherapy, immunotherapy, and the prospective of mobilized stem cells for optimization and modulation of the immune system to overcome immunosuppression and kill distant cancer cells. Materials and Methods: Ehrlich ascetic carcinoma (EAC) cell line-bearing mice treated with cyclophosphamide (CTX) followed by adoptively transferred with in vitro -activated T-cells either harvested from naive or EAC-bearing host with or without unfractionated bone marrow (BM) cells or granulocyte-colony stimulating factor-mobilized hematopoietic stem cells (HSCs) 1-day post-CTX treatment. All mice were vaccinated with EAC lysate and Hiltonol. Results: Cotransfer of activated T-cells obtained from EAC-bearing mice with HSC-progenitors induced the highest antitumor effect through increasing the percentage of apoptosis and decreasing DNA replication in S phase of EAC cells. Besides, marked an increase in the percentage of myeloid cells in spleen and stem cell populations in BM cells. Interestingly, Adoptive T-cell transfer (ACT) derived from EAC-bearing host with or without BM cells induced mobilization of stem cells from BM to circulation increasing their expansion. Conclusion: Combination of chemotherapy with ACT plus vaccination may constitute a potent antitumor therapy that provides more efficacious antitumor responses when it is combined with BM cells fostering more effective antitumor immunotherapy strategies.

Recent preclinical and clinical studies provide evidence that adoptive transfer of in vitro activated T cells can results in significant antitumour responses in vivo upon acquisition of certain survival and homing properties during in vitro activation. Based on recent studies showing in vivo antioxidant effects of thymoquinone (TQ), the active ingredient of Nigella sativa seeds, this study aims to determine whether or not TQ can increase survival and sustain the expression of the homing receptor CD62L in antigen-specific T cells in vitro. The results showed that stimulation of OT-1 (transgenic CD+) T cells with OVA antigen resulted in activation, as shown by a decrease in the surface expression of CD62L which coincided with significant apoptosis measured three and five days after antigen stimulation. Addition of low concentrations of TQ during CD85+ T-cell activation resulted in enhanced survival of the activated T cells and sustained expression of CD62L. These effects coincided with enhancement in the capability of CD8+ T cells to produce the effector cytokine interferon-gamma (IFNgamma). These results suggest that TQ has a beneficial effect in conditioning T cells in vitro for adoptive T-cell therapy against cancer and infectious disease.

Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer. © 2011 Fox et al; licensee BioMed Central Ltd.

Constituents of the Nigella sativa seed are reported to possess potent antioxidant effects. Treatment with anticancer drugs such as cyclophosphamide (CTX) is associated with significant toxicity due to over-production of reactive oxygen species, resulting in increased levels of oxidative stress. The aim of this study is to test whether or not N. sativa L oil (NSO) or its active ingredient, thymoquinone (TQ), can reduce CTX-induced toxicity. Male albino rats were treated with intraperitoneal administration of phosphate buffered saline (PBS) or 200 mg/Kg CTX followed by intragastric administration of NSO or TQ on alternate days for 12 days. Administration of NSO and TQ was initiated 6 h before or after CTX injection. Twenty-four hours after the last NSO and TQ treatment, blood and liver were harvested to analyse toxicity-related parameters. Treatment with CTX induced significant toxicity as shown by decrease in haemoglobin concentration and increases in blood sugar levels, activities of liver enzymes, bilirubin, urea, creatinine, lipids (triglyceride, cholesterol and low-density lipoprotein (LDL)-cholesterol) and lipid peroxidation in the liver. Treatment with NSO or TQ induced significant reduction in overall toxicity. The antitoxic effects of NSO and TQ were associated with induction of antioxidant mechanisms. These results suggest that administration of NSO or TQ can lower CTX-induced toxicity as shown by an up-regulation of antioxidant mechanisms, indicating a potential clinical application for these agents to minimise the toxic effects of treatment with anticancer drugs.

Hepatitis C virus (HCV) infection is a major public health concern with approximately 3% of the world's population is infected, posing social, economical and health burden. Less than 20% of the infected individuals clear the virus during the acute infection, while the rest develop chronic infection. The treatment of choice for HCV infection is pegylated interferon-alpha (IFN-alpha) in combination with ribavarin. Despite the cost and side effects of this treatment regimen, many patients fail this therapy and develop persistent HCV infection, leading to cirrhosis and hepatocellular carcinoma. Although the mechanisms underlying the failure to resolve HCV infection are poorly understood, the incapability of patients to develop effective anti-HCV immunity is a potential cause. We hypothesize that the dysfunctional anti-HCV immunity is due to the emergence of immunosuppressive cells coinciding with a decrease in the stimulatory dendritic cells (DCs) and natural killer (NK) cells. We further hypothesize that applying agents that can correct the imbalance between the immunosuppressive cells and stimulatory cells can results in resolution of chronic HCV. In this review article, we will discuss potential approaches, focusing on the use of Toll-like receptor agonists, to block the suppressive effects of the regulatory cells and restore the stimulatory effects of DCs and NK cells.

We have shown recently that cyclophosphamide (CTX) treatment induced a marked increase in the numbers of immature dendritic cells (DCs) in blood, coinciding with enhanced antigen-specific responses of the adoptively transferred CD8(+) T cells. Because this DC expansion was preceded by DC proliferation in bone marrow (BM), we tested whether BM post CTX treatment can generate higher numbers of functional DCs. BM was harvested three days after treatment of C57BL/6 mice with PBS or CTX and cultured with GM-CSF/IL-4 in vitro. Compared with control, BM from CTX-treated mice showed faster generation and yielded higher numbers of DCs with superior activation in response to toll-like receptor (TLR) agonists. Vaccination with peptide-pulsed DCs generated from BM from CTX-treated mice induced comparable adjuvant effects to those induced by control DCs. Taken together, post CTX BM harbors higher numbers of DC precursors capable of differentiating into functional DCs, which be targeted to create host microenvironment riches in activated DCs upon treatment with TLR agonists.