Mechanisms of Leukocyte Migration Following Cytokine Administration to Mice

不同器官微环境中不同白细胞亚群和基质细胞之间的相互作用对于优化器官特异性免疫应答至关重要，这些免疫应答可以限制转移形成并控制疾病诱导的炎症。IL-12和IL-18是自然杀伤（NK）细胞、NKT细胞和T细胞的有效免疫调节细胞因子，并且对不同器官中的NKT/NK亚群具有非常不同的作用。肝脏是转移瘤形成的主要靶点。我们使用小鼠肝转移模型来研究IL-18和/或IL-12细胞因子的抗肿瘤活性以及NK和NKT细胞对这些应答的作用。用IL-18加IL-12治疗小鼠比单独使用任何一种细胞因子更大程度地减少了肝脏中肿瘤结节的数量。如所预期的，IL-18加IL-12刺激了荷瘤小鼠中全身IFN-γ的协同增加。IL-18加IL-12治疗的抗肿瘤活性在IFN-γ（-/-）小鼠中被消除。使用细胞内染色，NK和NKT细胞被鉴定为用IL-18和/或IL-12处理的小鼠肝脏中IFN-γ的主要产生者。每天用IL-18加IL-12治疗小鼠，肝脏NK细胞增加，而肝脏NKT细胞通过这种治疗减少。用抗asGM 1优先消耗NK细胞导致IL-18加IL-12治疗的抗肿瘤活性部分丧失，并揭示NK细胞是肿瘤消退机制的重要组成部分。相比之下，用β GalCer优先消耗NKT细胞减少了用载体对照或单独IL-18处理的小鼠中的肝肿瘤结节的数量。同样，所有治疗方法在缺乏NKT细胞的CD1d（-/-）小鼠中显示出增加的抗肿瘤活性。因此，我们的数据表明，IL-18加IL-12诱导的小鼠抗肿瘤活性是NK和IFN-γ依赖性的，并且能够克服NKT细胞的内源性免疫抑制作用。因此，这些结果表明，增强NK细胞数量和功能，同时优先消耗NKT细胞的免疫方法可以有效治疗肝癌。由于IL-18加IL-12治疗荷瘤小鼠引起显著的抗肿瘤活性，同时可检测到的不变NKT（iNKT）细胞数量减少，我们比较了α-GalCer（一种已知调节iNKT细胞的糖脂）和IL-18加IL-12的iNKT细胞调节机制。我们发现用IL-18 + IL-12的急性处理诱导iNKT细胞的NK1.1（+）亚群的损失，而用aGalCer的急性处理在24小时耗尽所有肝iNKT细胞。然而，其他淋巴组织几乎不显示这些细胞的损失，表明肝脏微环境是iNKT细胞的关键调节因子。在小鼠的aGalCer和IL-18加IL-12处理后，在72小时，iNKT细胞在肝脏中重新增殖并扩增。为了研究这种扩增，我们在用IL-18加IL-12或aGalCer急性治疗后24小时向小鼠施用BrdU，并发现高百分比的增殖iNKT细胞。我们进一步发现脾对于iNKT再增殖和aGalCer后的扩增是需要的，但对于IL-18加IL-12治疗不是需要的，因为在脾切除的小鼠中没有观察到扩增。我们接下来检查了用aGalCer或IL-18加IL-12对小鼠的长期治疗，发现aGalCer诱导肝脏iNKT细胞的严重损失，而仅IL-18加IL-12引起iNKT细胞的全身性损失。在两种处理后，需要长期（60天）胸腺发育以重新填充肝脏。>这些数据揭示了肝脏微环境在调节iNKT细胞对炎症信号的反应中的重要作用。基于我们的数据，我们对该项目的假设是，通过细胞因子受体与TCR刺激NKT细胞导致NKT细胞亚群和功能的质的不同变化，从而改变它们在炎症和/或抗肿瘤免疫中的作用。迄今为止，我们已经对比了IL-12和IL-18的作用，这两种IFNg的有效诱导剂已知显著影响NKT细胞的功能，其具有通过连接在这些细胞上表达的不变TCR诱导的生物学上不同的作用。主要发现包括证明IL-12和IL-18协同作用优先消耗/不存在NKT细胞，并且NKT细胞的损失增加了对转移形成的抗性，这揭示了NKT细胞在肝脏中的先前未被认识到的调节作用。在用IL-12 + IL-18或NKT特异性抗原α-半乳糖基神经酰胺（aGalCer）急性或慢性处理后对NKT细胞群体的详细检查也揭示了活化诱导的消耗和恢复的不同机制，并显示NKT细胞表型和功能可以通过TCR与细胞因子信号传导深刻且差异性地重塑。

Interaction between different leukocyte subsets and stromal cells in different organ microenvironments is critical for optimizing organ-specific immune responses that could limit metastasis formation and control disease-induced inflammation. IL-12 and IL-18 are potent immunoregulatory cytokines for natural killer (NK), NKT, and T cells, and have very distinctive effects on the NKT/NK subsets in different organs. The liver is a major target for the formation of metastases. We used a mouse model of liver metastasis to study the anti-tumor activity of IL-18 and/or IL-12 cytokines and the roles of NK and NKT cells to these responses. Treating mice with IL-18 plus IL-12 significantly reduced the number of tumor nodules in the liver to a greater degree than did either of the cytokines alone. As expected, IL-18 plus IL-12 stimulated a synergistic increase in systemic IFN-gamma in tumor bearing mice. The anti-tumor activity of IL-18 plus IL-12 therapy was abolished in IFN-gamma(-/-) mice. Using intracellular staining, NK and NKT cells were identified as the major producers of IFN-gamma in the livers of mice treated with IL-18 and/or IL-12. Liver NK cells were increased with daily treatment of mice with IL-18 plus IL-12 whereas liver NKT cells were diminished by this treatment. Preferential depletion of NK cells with anti-asGM1 resulted in a partial loss of the anti-tumor activity of IL-18 plus IL-12 therapy and revealed NK cells to be an important component of the mechanism for tumor regression. In contrast, the preferential depletion of NKT cells with betaGalCer decreased the number of liver tumor nodules in mice treated with vehicle control or IL-18 alone. Similarly, all treatment approaches showed increased anti-tumor activity in CD1d(-/-) mice, which lack NKT cells. Our data therefore shows that the IL-18 plus IL-12 induced anti-tumor activity in mice is NK and IFN-gamma dependent, and is able to overcome an endogenous immunosuppressive effect of NKT cells. These results thereby suggest that immunotherapeutic approaches that enhance NK cell numbers and function while preferentially depleting NKT cells could be effective in the treatment of cancer in the liver. Because IL-18 plus IL-12 treatment of tumor bearing mice elicited significant anti-tumor activity concurrent with a decrease in the detectable number of invariant NKT (iNKT) cells, we compared iNKT cell modulation mechanisms of alpha-GalCer, a glycolipid known to modulate iNKT cells, and IL-18 plus IL-12. We found acute treatment with IL-18 + IL-12 induced a loss of NK1.1(+) subset of iNKT cells, while acute treatment with aGalCer depleted all liver iNKT cells at 24hrs. However, other lymphoid tissues showed little to know loss of these cells suggesting the liver microenviroment is a critical regulator of iNKT cells. At 72hrs iNKT cells repopulated and expanded in the liver following aGalCer and IL-18 plus IL-12 treatment of mice. To study this expansion, we administered BrdU to mice 24hrs after acute treatment with IL-18 plus IL-12 or aGalCer and found a high percentage of proliferating iNKT cells. We further found the spleen was needed for iNKT repopulation and expansion following aGalCer, but not for IL-18 plus IL-12 treatment, as no expansion was observed in splenectomized mice. We next examined chronic treatment of mice with aGalCer or IL-18 plus IL-12 and found aGalCer induced a severe loss of liver iNKT cells, while only IL-18 plus IL-12 caused a systemic loss of iNKT cells. Long term (>60 days) thymic development was needed to repopulate the liver following both treatments. These data reveal an important role for the liver microenvironment in regulating iNKT cell response to inflammatory signals. Based on our data, our hypothesis for this project is that stimulation of NKT cells via cytokine receptors vs the TCR results in qualitatively different changes in NKT cell subsets and functions, and thereby alter their role in inflammation and/or anti-tumor immunity. To date, we have contrasted the effects of IL-12 and IL-18, two potent inducers of IFNg, which are known to dramatically influence functions of NKT cells with the biologically distinct effects induced by ligation of the invariant TCR expressed on these cells. Major findings include the demonstration that IL-12 and IL-18 synergize for the preferential depletion/absence of NKT cells, and the loss of NKT cells increases resistance to metastasis formation which has revealed a previously unappreciated regulatory role for NKT cells in the liver. Detailed examination of NKT cell populations following acute or chronic treatment with IL-12 + IL-18 or the NKT- specific antigen a-galactosylceramide (aGalCer) has also revealed different mechanisms for activation- induced depletion and recovery, and show that NKT cell phenotypes and functions can be profoundly and differentially reshaped by TCR vs cytokine signaling.