Sabry A. El-Naggar

Defining the ability of cyclophosphamide preconditioning to enhance the antigen-specific CD8+ T-cell response to peptide vaccination: creation of a beneficial host microenvironment involving type I IFNs and myeloid cells.

Although cyclophosphamide (CTX) has been clearly shown to enhance active specific and adoptive immunotherapies, the mechanism(s) underlying these beneficial effects have not been clearly defined. To define the impact of CTX preconditioning on the antigen-specific CD8 T-cell response to peptide vaccination, we used an adoptive transfer model based on the OT-1 T-cell receptor transgenic mouse. CTX preconditioning dramatically enhanced the antigen-specific CD8 T-cell response to peptide vaccination. Specifically, CTX significantly enhanced the expansion and function of responding CD8 T cells as demonstrated by flow cytometry and cytokine production. In parallel experiments, we attempted to define the mechanism(s) underlying these beneficial effects of CTX therapy. CTX therapy increased the relative number and activation status of myeloid dendritic cells, and was associated with the induction of significant levels of the inflammatory cytokines interferon-α, monocyte chemoattractant protein-1, and IL-6. Adoptive transfer experiments into type I IFNR−/− and CR3−/− mice confirmed that the beneficial effects of CTX were at least partially dependent on type I interferons and myeloid cells. Adoptive transfer of up to 150×106 naive spleen cells at the time of antigen-specific CD8 T-cell transfer did not abrogate the effects of CTX therapy, suggesting that the creation of a niche in the immune system may not be required. CTX decreased the absolute, but not relative number of CD4+CD25+ Treg cells, consistent with the possibility that regulatory T cells may be targeted by CTX therapy. Of note, combination therapy with CTX and a synthetic TLR3 agonist further enhanced the antigen-specific CD8+ T-cell response. Taken together, our data suggest that CTX modulates specific components of the innate immune system resulting in a beneficial host microenvironment. Specific targeting of these components may enhance the effectiveness of CTX preconditioning for adoptive immunotherapy.

Recovery from Cyclophosphamide-Induced Lymphopenia Results in Expansion of Immature Dendritic Cells Which Can Mediate Enhanced Prime-Boost Vaccination Antitumor Responses In Vivo When Stimulated with the TLR3 Agonist Poly(I:C)

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.

Cyclophosphamide Induces Dynamic Alterations in the Host Microenvironments Resulting in a Flt3 Ligand-Dependent Expansion of Dendritic Cells

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.

The TLR3 agonist poly(I:C) targets CD8+ T cells and augments their antigen-specific responses upon their adoptive transfer into naive recipient mice

We have recently reported that the toll-like receptor 3 (TLR3) agonist poly(I:C) induces adjuvant effects to post vaccination CD8+ T cells responses through rapid induction of innate mediators, including NK cells, macrophages, dendritic cells (DCs), and inflammatory cytokines. However, whether this TLR3 agonist directly targets CD8+ T cells needs to be carefully investigated. In this study, we found that optimal post vaccination CD8+ T cell responses to ex vivo DC-based vaccination requires triggering of TLR3 signaling pathway in DCs in vitro as well as in the recipient host, indicating a role for other cell types. Real-time PCR analysis revealed that TLRs (TLR1-TLR13) are expressed in purified (>99% pure) CD4+ and CD8+ T cells from C57BL/6 and BALB/c mice, where the magnitude of the expression was strain and cell type dependent. In vitro, treatment of these purified T cells with poly(I:C) modulated the expression of TLRs including TLR3. Furthermore, non-specific and antigen-specific stimulation of CD8+ T cells by phorbol myristate acetate and MHC class I peptide-pulsed splenocytes, respectively, modulated TLR expression in purified CD4+ and CD8+ T cells. Importantly, brief conditioning of purified naïve TCR transgenic OT-1 (CD8+) T cells in vitro with poly(I:C) induced activation of these cells in absence of antigen stimulation. Interestingly, when these in vitro poly(I:C)-conditioned OT-1 cells were adoptively transferred into naïve recipient followed by peptide vaccination, they showed superior expansion and activation to their naïve counterparts. These results suggest that CD8+ T cells can be activated by triggering their TLR3. Furthermore, the data support the notion of direct involvement of TLRs in adaptive immune responses.

Cyclophosphamide induces bone marrow to yield higher numbers of precursor dendritic cells in vitro capable of functional antigen presentation to T cells in vivo

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.

Ameliorative effect of propolis against cyclophosphamide-induced toxicity in mice.

Abstract Context: Cyclophosphamide (CTX) is a common anticancer agent used for the treatment of several malignancies. However, upon treatment, it induces severe toxicity due to its oxidative stress capability. Propolis, a natural product collected by honey bees, has shown several biological activities, such as free radical scavenging and antioxidant agent. Objective: This study elucidates the protective effects of propolis against CTX-induced changes in mice. Materials and methods: Forty-eight male Swiss albino mice were divided into four groups; group 1 was intraperitoneally (i.p.) injected with 200 µL of phosphate buffer saline (PBS), group 2 was injected with 100 mg/kg/d propolis, group 3 was injected with a single dose of CTX (200 mg/kg), and group 4 was injected with a single dose of CTX (200 mg/kg) followed by propolis (100 mg/kg) for 7 consecutive days. After 12 d, mice were bled and then sacrificed to analyze the hematological, biochemical, and histological parameters. Results: The results indicated that CTX-injected mice showed an increase in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine and a decrease in the total number of white blood cells (WBCs) and platelets. Moreover, dramatically changes in the histological architectures of the liver and kidney were observed. The mice that were injected with CTX/propolis showed an improvement in the levels of ALT, AST, urea, creatinine, WBCs, and platelets. Moreover, the histological picture of the liver and kidney was significantly improved. Conclusions: In conclusion, propolis might be considered an effective agent in ameliorating the toxicity resulted from CTX treatment.

Growth, hydrolases and ultrastructure of Fusarium oxysporum as affected by phenolic rich extracts from several xerophytic plants

Fusarium oxysporum, the causal agent of rot and wilt diseases, is one of the most detrimental phytopathogens for the productivity of many economic crops. The present study was conducted to evaluate the potentiality of some xerophytic plants as eco-friendly approach for management of F. oxysporum. Phenolic rich extracts from five plants namely: Horwoodia dicksoniae, Citrullus colocynthis, Gypsophila capillaris, Pulicaria incisa and Rhanterium epapposum were examined in vitro. The different extracts showed high variability in their phenolic and flavonoid contents as well as total antioxidant capacity. A strong positive correlation existed between the antifungal activity of the tested extracts and their contents of both total phenolics and flavonoids (r values are 0.91 and 0.82, respectively). Extract of P. incisa was the most effective in reducing the mycelial growth (IC50=0.92mg/ml) and inhibiting the activities of CMCase, pectinase, amylase and protease by 36, 42, 58 and 55%, respectively. The high performance liquid chromatography analysis of P. incisa extract revealed the presence of eight phenolic acids along with five polyphenolic compounds. The flavonol, quercetin and its glycosides rutin and quercetrin were the most abundant followed by the phenolic acids, t-cinnamic, caffeic, ferulic and vanillic. P. incisa extract not only affects the growth and hydrolases of F. oxysporum but also induces ultrastructure changes in the mycelium, as revealed by transmission electron microscopy. To our knowledge, this is the first study to investigate the mechanisms underlying the antifungal activity of P. incisa.

Efficacy of Rosmarinus officinalis leaves extract against cyclophosphamide-induced hepatotoxicity.

Abstract Context Cyclophosphamide (CTX) is used to treat different cancer types, although it causes severe hepatotoxicity due to its oxidative stress effect. Rosmarinus officinalis, L. (Lamiaceae) has a therapeutic potential against hepatotoxicity due to its antioxidant activity. Objective The objective of this study is to investigate the phytochemical analysis of the methanol extract of Rosmarinus officianalis leaves (MEROL) and its efficacy against CTX-induced hepatotoxicity. Materials and methods The phytochemical analyses were assessed spectrophotometericaly. To assess the MEROL efficacy, 72 Swiss albino mice were divided into six groups. Group 1 was control, groups 2 and 3 included mice which were injected intraperitoneally (i.p.) with 100 or 200 mg/kg of MEROL at days 1, 4, 7, 10, 13 and 16; group 4 was injected (i.p.) with CTX (200 mg/kg) at day 17, groups 5 and 6 were injected (i.p.) with MEROL as groups 3 and 4 followed by 200 mg/kg CTX at day 17, respectively. At day 22, six mice from each group were sacrificed and the others were sacrificed at day 37. Results MEROL has a high content of total phenolics, saponins, total antioxidant capacity and DPPH radical scavenging activity. The median lethal dose (LD50) value of MEROL was 4.125 g/kg b.w. The inhibitory concentration 50 (IC50) value for DPPH radical scavenging was 55 μg/mL. Pretreatment with 100 mg/kg MEROL for 16 d ameliorated CTX-induced hepatotoxicity represented in lowering the levels of the aspartate aminotransferase (AST) and lipid profile and minimizing the histological damage. Conclusions Pretreatment with 100 mg/kg b.w. MEROL mitigated CTX-induced hepatotoxicity due to its antioxidant activity.

Cyclophosphamide eradicates murine immunogenic tumor coding for a non-self-antigen and induces antitumor immunity

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.

Treatment with Nigella sativa oil ameliorated the hepato-renal toxicities induced by cyclophosphamide in splenectomized mice

Cyclophosphamide (CTX) treatment in splenectomized (SPLNZ) mice led to a decrease in leukocytes (W.B.Cs) count and did not restore to the normal value until day 21 post-injection. This study addressed the potential role of Nigella sativa oil (NSO) as a protective agent against CTX-induced toxicity in SPLNZ mice. Some hematological, biochemical parameters and histological examination of liver and kidney tissues were assessed. Thirty (30) Swiss male mice were divided into five groups (6 mice/group). Control mice (G1) were sham-operated treated with normal saline. The other four SPLNZ groups were treated with normal saline (G2), a single dose of 200 mg/kg CTX (G3), NSO (1 mL/kg) on alternate days for 14 days (G4) and the same NSO protocol after 200 mg/kg CTX (G5). Normal saline and CTX were injected intraperitoneally (i.p.), NSO was administered orally. G2 (SPLNZ) mice, compared to G1, showed significantly increased W.B.C. counts, platelet counts and cholesterol levels, while aspartate aminotransferase (AST) levels decreased. Group (G3) (SPLNZ/CTX) mice had significantly decrease W.B.C counts and increase AST, ALT, cholesterol and triglycerides. G5 (SPLNZ/CTX/NSO) mice partially restored W.B.Cs count, increased platelet counts and enhanced AST and ALT enzymes. Histopathology of livers and kidneys from G3 (SPLNZ/CTX) showed necrosis, cytoplasmic degeneration of hepatocytes, cellular infiltration with pyknotic nuclei. SPLNZ/CTX/NSO (G5) improved the histological architecture of the livers and kidneys. No changes showed in G4 mice (SPLNZ/ NSO). In summary, NSO treatment ameliorated the hepato-renal toxicities after CTX injection in SPLNZ mice.