Supplementary Data from The Folate Pathway Inhibitor Pemetrexed Pleiotropically Enhances Effects of Cancer Immunotherapy
David SchaerSandaruwan GeeganageNelusha AmaladasZhao Hai LuErik RasmussenAndreas SonyiDarin ChinAndrew CapenYanxia LiCatalina M. MeyerBonita D. JonesXiaodong HuangShuang LuoCarmine CarpenitoKenneth D. RothAlexander NikolayevBo TanManisha BrahmacharyKrishna ChodavarapuFrank C. DorseyJason R. ManroThompson N. DomanGregory P. DonohoDavid SurguladzeGerald E. HallMichael KalosRuslan D. Novosiadly
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<p>Figure S1. Efficacy of pemetrexed in Lewis Lung carcinoma model and metabolomic analysis of MC38 tumor bearing mice treated with 50 mg/kg of pemetrexed Figure S2. T cell inflamed phenotype induced by pemetrexed is modestly reduced in tumors upon combination with cisplatin Figure S3. Combination Effects of pemetrexed -/+ anti-PD-L1 in MC38, LLC and Colon26 tumor models Figure S4 (A) Mice bearing Colon26 tumors were treated starting 10 days after tumor implantation with pemetrexed (50 mg/kg, 5 days on, 2 days off, IP) and/or anti-PD-L1 (αPD-L1) (500 ug/mouse, weekly IP), and tumors were isolated after 14 days of treatment and single cell suspensions were subjected to flow cytometry analysis. FACS plots show representative data of intra-tumor CD45+ tumor-infiltrating lymphocyte (TIL) T cell (CD3+) and Myeloid cell (CD11b+) subsets frequencies; and percentage of Ki67+ CD4+ T effector cells based on the gating scheme indicated. (B) Spleens, TDLNs and tumors were isolated and antigen specific T cell responses were measured by IFNγ ELISpot, gp70 Tetramer staining (Tet) and Intracellular cytokine staining. (Left) CD8 T cells isolated from pooled spleens and TDLNs from individual mice were cultured alone (control) with irrelevant BALB/c tumor EMT6 or cognate Colon26 tumor cells overnight and number of IFNγ producing T cells was measured by ELISpot. (Middle) Freshly isolated CD8+ T cells from spleens and TDLNs were assessed for % of gp70 Tet+ cells. (Right) Freshly isolated tumors single cell suspensions were stimulated with PMA/ phorbol myristate acetate (PMA) and ionomycin with monensin for 4 hrs and the % of TNF-alpha positive and gp70 Tet+ CD8 T cells were measured from the Live CD45+, CD3+ T cells by FACS. Figure S5. Concordance between QuantiGene and nCounter gene expression data for in vitro activated T cells treated in the presence or absence of pemetrexed (related to Figure 6F). Supplementary Table 1. Ingenuity pathway analysis (IPA) of MC38 tumors treated with pemetrexed or paclitaxel with or without carboplatin Supplementary Table 2: IPA results for Colon26 experiment in Figure 4 sorted by functional class. Pemetrexed monotherapy in this experiment at this timepoint did not have enough differentially expressed genes to generate IPA results.</p>Keywords:
ELISPOT
Pemetrexed
In this report we describe a system for the generation of functional, class I MHC-restricted, T-T hybridomas. The BW5147 cell line was transfected with the CD8 gene. BW5147 transfectants were obtained that stably expressed CD8 and this expression was maintained after somatic cell hybridization with activated T lymphocytes. To determine whether the stable expression of CD8 would facilitate the generation of class I MHC-specific T-T hybridomas, the transfected cells were fused with alloreactive T cells and the resultant hybrids were screened for their ability to produce lymphokines in response to antigenic stimulation. Somatic cell hybridizations with BW5147-CD8 transfectants give rise to a much higher frequency of class I MHC-specific T-T hybridomas relative to parallel fusions with BW5147. To determine whether the BW5147-CD8 transfectants would also support the generation of Ag-specific, class I MHC-restricted T-T hybridomas, they were fused with OVA-specific CTL. Several T-T hybrid clones were identified that produced lymphokines after stimulation with a transfected APC that was synthesizing OVA, or with a tryptic digest of OVA in the presence of syngeneic APC. The stimulation by Ag was MHC-restricted and mapped to the Kb molecule. An anti-CD8 mAb inhibited the stimulation of these hybridomas by Ag plus APC, whereas their stimulation by mitogen was unaffected. Cytolytic activity was not detected when several of the OVA-specific or alloreactive hybridomas were tested for their ability to kill target cells bearing the appropriate Ag. These results demonstrate that the BW5147-CD8 transfectants allow the generation of class I MHC-restricted T-T hybridomas. The potential utility of this system is discussed.
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Pemetrexed
Folinic acid
Antifolate
Carboplatin
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Background: To evaluate the clinical efficacy and toxicity of single pemetrexed treatment compared with platinum-based pemetrexed doublet pemetrexed-based as first-line treatment for advanced nonsquamous nonsmall cell lung cancer (NS-NSCLC) in elderly Chinese patients. Methods: The study retrospectively reviewed 175 elderly Chinese patients with NS-NSCLC from June 2010 to September 2013: 90 patients received single pemetrexed treatment, 45 received pemetrexed plus oxaliplatin, and 40 received pemetrexed plus carboplatin. Clinical efficacy was assessed using disease control rate (DCR), overall survival (OS), and progression-free survival (PFS). Results: DCR, OS, and PFS did not significantly differ between single pemetrexed treatment (OS: 14.9 months; DCR: 62.2%; PFS: 3.3 months), pemetrexed plus oxaliplatin (OS: 16.5 months; DCR: 71.1%; PFS: 4.5 months), and pemetrexed plus carboplatin (OS: 15.5 months; DCR: 70.0%; PFS: 4.6 months) groups. Pemetrexed treatment caused significantly lower incidences of adverse events, such as hepatotoxicity and peripheral nerve injury. Performance status (PS), TNM stage, and Thymidylate synthase (TS) expression were predictive factors of DCR. Pemetrexed chemotherapy cycles, PS, and TNM stage were independent prognostic factors. Conclusions: Single pemetrexed was noninferior to platinum-based pemetrexed doublet for clinical efficacy and safety in elderly Chinese patients with advanced NS-NSCLC. Chemotherapy cycles, performance status, and TNM stage were independent prognostic factors.
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Carboplatin
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To explore the role of IL-22 on the recovery and function of thymus from graft-versus host disease (GVHD) mice after allogeneic bone marrow transplantation (allo-BMT).GVHD model was established by using of recipient male BALB/c and donor male C57BL/6 mice(6-8 W) respectively. The mice were divided into normal group, GVHD with IL-22 group (BS+IL-22) and without IL-22 group (BS+PBS). Numbers of thymus cells were detected at different time points. The ratio of T cell subsets from thymus was observed by flow cytometry. Percentages of IFN-γ-producing and IL-17-producing CD4+ T or CD8+ T cells were detected.The total number of thymus cells in BS+IL-22 mice [(14.6±5.1)×10⁴] was significantly higher than that in BS+PBS mice [(6.2±2.9)×10⁴] at 14 days after allo-BMT. Thymus cells in BS+IL-22 mice expanded continuously and reached at the level of normal mice, which were still higher than that in BS+PBS group. Although there was no impact on the ratio of mature CD4+ and CD8+ T cell from thymus, the percentage of immature CD4+CD8+ T cell increased obviously in mice treated with IL-22. Percentages of IFN-γ+CD4+ T cell [Th1:(2.42±0.75)%] and IFN-γ+CD8+ T cell [Tc1:(5.44±0.47)%] were up-regulated by IL-22 treatment, whereas no changes were detected in IL-17+CD4+ T cell (Th17) and IL-17+CD8+ T cell (Tc17).IL-22 accelerates the progress of thymus recovery, and increases the IFN-γ-producing ability of thymus CD4+ and CD8+ T cells from GVHD mice.
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Carboplatin
Pleural disease
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Background. The in vivo effects of immunosuppressants on T cells are classically determined using animal models of organ transplantation. These methods are technically difficult and time consuming. A simple in vivo method is needed for screening new immunosuppressants. Methods. Donor mouse spleen cells were labeled with a fluorescent dye, carboxy-fluorescein diacetate succinimidyl ester (CFSE), and then injected into the blood of recipient severe combined immunodeficiency mice. Three days after the injection, spleen cells of the recipient mice were isolated and the proliferating alloreactive T cells were analyzed by flow cytometry. Results. In control recipient mice, 50% of the T cells were proliferating, consisting of both CD4+ and CD8+ T cells. In cyclosporine- or FK506-treated mice, T-cell proliferation was suppressed in the CD4 subset but not in the CD8 subset. On the contrary, T-cell proliferation was significantly reduced in the CD8 subset but not in the CD4 subset in recipient mice treated with rapamycin. Conclusion. The present mouse model using carboxy-fluorescein diacetate succinimidyl ester labeling is simple and fast. It is useful for screening new immunosuppressants and for examining the effect on T-cell subsets.
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Antifolate
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Objecfive To study T cell subsets distribution in peripheral blood from patients with ankylosing spondylitis(AS)and the role of cell immunity in AS.Methods 30 patients with untreated active phase AS and 30 healthy volunteers were enrolled.The expression of CD4+ and CD8+ T cell subsets were evaluated by flow eytome try.The correlation among T cell subsets and Bath AS function index(BASFI).Bath AS measurement index(BASMI),course of disease,age,ESR,hyper-sensitive C-reactive protein (hs-CRP) were analyzed.Results The level of CD4+ T cell and CD4/CD8+ratio were significantly lower than that of healthy volunteers[(29.24±9.22)% vs.(40.09±6.86) %,(0.96±0.49 ) vs.(1.70±0.67 ),P ＜ 0.01 ],and CD8+ T cell were significantly higher than that of healthy volunteers [(32.91±6.86) % vs.(25.60± 5.97 ) %,P ＜ 0.01 ].The level of CD4+ T cell subsets in peripheral blood of AS patients was negatively correlated with BASMI (r =-0.479,P ＜ 0.01 ),and CD8+ T cell subsets were positively correlated with ESR,hs-CRP,BASFI and BASMI ( r = 0.373,0.430,0.462,0.530,P ＜0.05 ).The ratio of CD4+/CD8+ was negatively correlated with hs-CRP,BASFI and BASMI (r = -0.465,- 0.473,- 0.426,P ＜ 0.05 ).CD4+,CD8+,ratio of CD4/CD8 were not significantly correlated with age and course of disease in patients with AS.Conclusion T cell subsets are significantly abnormal in peripheral blood of patients with AS,and the imbalance degree of T cell is correlated with severity and activity of AS,suggesting that T cell subsets imbalance plays an important role in the pathologensis of AS.
Key words:
Ankylosing spondylitis; T cell subsets; CD4+; CD8+
BASFI
Spondylitis
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Abstract The immunology of vertical HIV transmission differs from that of adult infection in that the immune system of the infant is not fully matured, and the factors that influence the functionality of CD8+ T cell responses against HIV in children remain largely undefined. We have investigated CD8+ T cell responses in 65 pediatric subjects with vertically acquired HIV-1 infection. Vigorous, broad, and Ag dose-driven CD8+ T cell responses against HIV Ags were frequently observed in children who were older than 3 years of age and maintained CD4+ T cell counts >400 cells/μl. In contrast, younger age or a CD4+ T cell count <400 cells/μl was associated with poor CD8+ T cell responses and high HIV loads. Furthermore, subjects with a severely depleted and phenotypically altered CD4+ T cell compartment had circulating Gag-specific CD8+ T cells with impaired IFN-γ production. When viral load was not suppressed by antiviral treatment, subjects that fell below the putative age and CD4+ T cell count thresholds had significantly reduced CD8+ T cell responses and significantly higher viral loads. Thus, the data suggest that fully effective HIV-specific CD8+ T cell responses take years to develop despite an abundance of Ag in early life, and responses are further severely impaired, independent of age, in children who have a depleted or skewed CD4+ T cell compartment. The results are discussed in relation to differences between the neonatal and adult immune systems in the ability to respond to HIV infection.
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