文章題目：An HIF-1a/VEGF-A Axis in Cytotoxic T Cells Regulates Tumor Progression細胞毒性T 細胞HIF-1a/VEGF-A 軸調節腫瘤進展
文章出處：Cancer Cell 2017 11 13;325(5) ；10.1016/j.ccell.2017.10.003；劍橋大學生理髮育與神經科學
工作站使用情況：Ruskinn Sci-Tive
Abstract
Cytotoxic T cells infiltrating tumors are thought to utilize HIF transcription factors during adaptation to the hypoxic tumor microenvironment. Deletion analyses of the two key HIF isoforms found that HIF-1α, but not HIF-2α, was essential for the effector state in CD8 T cells. Furthermore, loss of HIF-1 α in CD8 T cells reduced tumor infiltration and tumor cell killing, and altered tumor vascularization. Deletion of VEGF-A, an HIF target gene, in CD8 T cells accelerated tumorigenesis while also altering vascularization. Analyses of human breast cancer showed inverse correlations between VEGF-A expression and CD8 T cell infiltration, and a link between T cell infiltration and vascularization. These data demonstrate that the HIF-1α/VEGF-A axis is an essential aspect of tumor immunity.
主要內容：細胞毒性 T 淋細胞（CTL）又稱殺傷性 T 淋巴細胞，是機體抗腫瘤機制的重要環節，也是腫瘤免疫過繼療法主要效應細胞之一。CD8+T 細胞中低氧誘導因子 HIF-1α的缺失減少了腫瘤浸潤和腫瘤細胞殺傷，改變了腫瘤血管生成。CD8+T 細胞中 HIF 靶基因 VEGF-A 的缺失加速了腫瘤的發生，同時也改變了血管生成。人乳腺癌 VEGF-A 表達與 CD8+T 細胞浸潤呈負相關，T 細胞浸潤與血管生成呈正相關。這些數據表明，HIF-1α/VEGF-A 軸是腫瘤免疫的一個重要方面。
Figure 1. Hypoxia Promotes CD8+ T Cell Glycolytic Metabolism in an HIF-1a- but Not HIF-2a-Dependent Fashion(F) CD8+ T cells were isolated from spleens and activated with aCD3/CD28 for 48 hr, and then expanded for 3 days in IL-2 and subjected to 21% or 1% O2 for24 hr. Western blotting was performed on nuclear fractions; densitometric analyses are shown.(G) CD8+ T cells from HIF-1afl/fldlckCRE (maroon), HIF-2afl/fldlckCRE (green), and littermate controls (black for HIF-1afl/fl, gray for HIF-2afl/fl) were isolated, activated, expanded for 5 days in the presence of IL-2, and cultured for 24 hr under 21% versus 1% O2. qRT-PCR was performed for Hk2, Pdk1, Mct4, and Pgk (n = 3, error bars represent SD). (H) Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of CD8+ T cells prepared as in (G) were measured by flux analysis, under basal conditions and after injection (dashed line) of oligomycin and FCCP (stressed) (n = 4 per genotype, error bars represent SEM).
在常氧(21%O2)和低氧(1%O2)條件下培養突變型和對照型CTL 細胞(圖F)，當CD8+T 細胞分化為效應細胞時，缺乏HIF-1a 的CD8+T 細胞，表現出糖酵解代謝相關基因的表達受損(圖G)。HIF-1a 在維持糖酵解(圖H)、葡萄糖攝取(圖I)和乳酸產生(圖J)方面起著至關重要的作用。如流式細胞儀分析所示，缺氧和HIF-1a 的缺失，降低了CD8+CTL 的存活率(圖K)。
Phenotypic switch of CD8+ T cells reactivated under hypoxia toward IL‐10 secreting,poorly proliferative effector cellsCD8+ T
低氧條件下CD8+T 細胞向分泌IL-10、增殖能力差的效應細胞的表型轉換
文章出處：Eur. J. Immunol. 2015 Aug;458(8) ；10.1002/eji.201445284；日內瓦大學醫學院
工作站使用情況：In Vivo2 300
Abstract
CD8+ T cells controlling pathogens or tumors must function at sites where oxygen tension is frequently low, and never as high as under atmospheric culture conditions. However, T ‐cell function in vivo is generally analyzed indirectly, or is extrapolated from in vitro studies under nonphysiologic oxygen tensions. In this study, we delineate the role of physiologic and pathologic oxygen tension in vitro during reactivation and differentiation of tumor ‐specific CD8+ T cells. Using CD8+ T cells from pmel‐1 mice, we observed that the generation of CTLs under 5% O2, which corresponds to physioxia in lymph nodes, gave rise to a higher effector signature than those generated under atmospheric oxygen fractions (21% O2). Hypoxia (1% O2) did not modify cytotoxicity, but decreasing O2 tensions during CTL and CD8+ tumor ‐infiltrating lymphocyte reactivation dose ‐ dependently decreased proliferation, induced secretion of the immunosuppressive cytokine IL‐10, and upregulated the expression of CD137 (4‐1BB) and CD25. Overall, our data indicate that oxygen tension is a key regulator of CD8+ T‐cell function and fate and suggest that IL‐10 release may be an unanticipated component of CD8+ T cell‐mediated immune responses in most in vivo microenvironments.
主要內容：在體內 CD8+T 細胞在氧分壓較低的地方發揮作用，故了解缺氧對 CD8+T 細胞影響是預測體內免疫功能的重要一步。分別在體外模擬生理氧和病理氧分壓對 CD8+T 細胞重新激活和分化過程中的作用研究發現，與在大氣氧含量(21% O2)下產生的 CTL 相比，在 5% O2(對應於淋巴結中的生理氧）下產生的效應信號更高；低氧(1% O2)不會改變細胞毒性，但隨著 O2 降低會依賴性地降低細胞增殖，誘導免疫抑制細胞因子 IL‐10 的分泌，並上調 CD137 和 CD25 的表達。
Figure 1. CTLs generated under physioxia have a higher effector profile than those generated under atmospheric oxygen fraction. (A and B) CTLs from Pmel-1 splenocytes were generated under 21% (black bars) or 5% O2 (gray bars) and were analyzed for RNA expression. Results represent (A) the mean relative gene expression or (B) the RNA absolute count + SEM of five independent experiments (n = 5). The checkerboard represents the 42 genes analyzed (only genes modulated by more than 30% with a p < 0.05 are colored in red or blue).
對一組42 個基因與低氧、免疫功能或參與細胞生存的基因進行研究發現，一些與CD8+ T細胞存活和擴張正相關的基因被上調，包括gata3、il2ra 和cd28；與細胞凋亡增加和免疫調節5相關的maf 和ctla4 也被上調。在空氣中氧濃度狀態下產生的CLT 表現出更高的效應，blimp1 和fasl 的上調和tcf7 的下調可以證明這一點。
Figure 2. Hypoxia modulates expansion and RNA profile of reactivated CTLs. CTLs generated under 21%(squares) or 5% (circles) O2 from Pmel-1 splenocytes were reactivated for indicated times under 21% O2 (closed squares with solid line), 5% O2 (closed circles with solid line) or 1% O2 (open squares or open circles with dashed line). Results show (A) mean cell number, (B) mean cell division number, (C) mean cell viability, and (D) mean apoptotic cells ± SEM out of at least three independent experiments (n≥3). CTLs generated under (E) 21% or (F) 5% O2 from Pmel-1 splenocytes were reactivated for two days under indicated oxygen fractions. (E) RNA profile from CTLs reactivated under 21% (black bars) or 1% O2(gray bars). (F) RNA profile from CTLs reactivated under 5% (black histograms) or 1% O2 (gray histograms). Results represent the mean relative gene expression+ SEM out of four independent experiments (n = 4). The checkerboard represents the 42 genes analyzed (only genes modulated by more than 30% with ap < 0.05 are colored in red or blue). To display common genes modulated under each condition, genes composing the checkerboard are organized identically (in an arbitrary fashion). ns: not statistically significant, *p < 0.05, **p < 0.01, *** p <0.001 (A–D: Student’s t-test; E, F: Three-way ANOVA)
通過平均細胞數、平均細胞分裂數、平均細胞存活率及平均凋亡細胞數可知，缺氧減少了重新激活的CTL 的擴增。
Hypoxia Induced Impairment of NK Cell Cytotoxicity against Multiple Myeloma Can Be Overcome by IL-2 Activation of the NK Cells
NK 細胞IL-2 活化可克服缺氧性NK 細胞對多發性骨髓瘤的殺傷作用
文章出處：PLOS ONE. 2013；10.1371/journal.pone.0064835；荷蘭馬斯垂克大學醫學中心
工作站使用情況：In Vivo2 1000
BACKGROUND:Multiple Myeloma (MM) is an incurable plasma cell malignancy residing within the bone marrow (BM). We aim to develop allogeneic Natural Killer (NK) cell immunotherapy for MM. As the BM contains hypoxic regions and the tumor environment can be immunosuppressive, we hypothesized that hypoxia inhibits NK cell anti-MM responses.
METHODS:NK cells were isolated from healthy donors by negative selection and NK cell function and phenotype were examined at oxygen levels representative of hypoxic BM using flowcytometry. Additionally, NK cells were activated with IL-2 to enhance NK cell cytotoxicity under hypoxia.
RESULTS:Hypoxia reduced NK cell killing of MM cell lines in an oxygen dependent manner. Under hypoxia, NK cells maintained their ability to degranulate in response to target cells, though, the percentage of degranulating NK cells was slightly reduced. Adaptation of NK- or MM cells to hypoxia was not required, hence, the oxygen level during the killing process was critical. Hypoxia did not alter surface expression of NK cell ligands (HLA-ABC, -E, MICA/B and ULBP1-2) and receptors (KIR, NKG2A/C, DNAM-1, NCRs and 2B4). It did, however, decrease expression of the activating NKG2D receptor and of intracellular perforin and granzyme B. Pre-activation of NK cells by IL-2 abrogated the detrimental effects of hypoxia and increased NKG2D expression. This emphasized that activated NK cells can mediate anti-MM effects, even under hypoxic conditions.
CONCLUSIONS:Hypoxia abolishes the killing potential of NK cells against multiple myeloma, which can be restored by IL-2 activation. Our study shows that for the design of NK cell-based immunotherapy it is necessary to study biological interactions between NK- and tumor cells also under hypoxic conditions.
主要內容：缺氧是大多數腫瘤組織的特徵，可以改變不同免疫細胞的功能，研究發現缺氧可消除 NK細胞對多發性骨髓瘤的殺傷作用，故為了設計基於 NK 細胞的免疫治療，也有必要研究 NK 細胞與腫瘤細胞在低氧條件下的相互作用。
Figure 4. Oxygen concentration during NK cell killing is the key regulating parameter determining the cytotoxic potential. (A) Preincubation (16 hours) of MM- and NK cells followed by 4.5 hour assessment of cytotoxicity were performed at the O2 concentration depicted on the x-axis. Each dot represents mean of triplicate cultures of individual donors. Statistics were performed with one-way repeated measures ANOVA with Bonferroni correction * p,0.05, ** p,0.01, *** p,0.001.
將NK 細胞、MM 細胞或兩者都預先孵育在21%或0%的O2 中，並將它們組合在21%或0% O2中進行細胞毒性評估。在0% O2 中預孵育的NK 細胞不影響其殺傷活性(圖中第3 組和第5 組)。相反，在0% O2 條件下進行細胞毒試驗(第2、4、6 和8 組)，與在21% O2(第1、3、5 和7 組)中進行細胞毒試驗相比，均顯示NK 細胞的細胞毒性降低。
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