Dynamics of hematopoietic differentiation

我们介绍了一种新的方法，结合了细胞免疫分析、IdU脉冲追踪和数学建模，以获得对免疫稳态的系统理解。CyTOF是最近获得的一种仪器（在美国国立卫生研究院人类免疫学中心内），用于进行大规模细胞术：它类似于经典的流式细胞仪，除了它的检测能力依赖于重金属标记抗体的大规模细胞分辨率。我们已经在NIH内实施了CyTOF，并证明了其使用40多种抗体（例如针对造血分化的关键标记物）实现免疫分析的能力。我们还实施了IdU脉冲追踪，以标记增殖细胞（通过在DNA复制过程中加入金属标记的核苷酸），并通过细胞增殖或分化（依靠CyTOF的大规模细胞计数能力）检测其稀释度：IdU脉冲追踪将BrdU脉冲追踪的非扰动高信号噪声与CyTOF的深度免疫谱结合起来。最后，我们建立了一个数学框架来模拟CyTOF IdU脉冲追踪的实验结果：我们的定量分析使我们能够“激活”我们的免疫细胞网络，并量化细胞分化的速度。我们已经应用我们的方法来描绘胸腺中NK T细胞的分化（与EIB-NCI的Hyun Park博士合作），单核细胞的分化（与Memorial Sloan-Kettering的Frederic Geissmann博士合作）和其他系统正在考虑中。我们正在展示我们的新方法的灵活性和分辨率，以更好地分析肿瘤发展过程中发生的系统性造血扰动（与CIP-NCI的Romina Goldzmid博士合作）。迄今为止的一个主要发现是，小鼠免疫系统的单核细胞不仅在这些动物的血液中分化，而且在骨髓和淋巴器官中也遵循平行的分化动态。这意味着血液单核细胞的主要来源可能不是血液本身，而是来自骨髓。这样的结果（在不受干扰的情况下，在稳态下获得）挑战了对血液单核细胞分化的普遍理解，即从遗传扰动中获得。第二个发现是NKT2细胞（即2型自然杀伤T细胞）不是来自NKT1细胞，而是直接（但效率低下）从未成熟NKT细胞分化而来。再一次，我们对脉冲追逐实验的动力学分析揭示了分化的另一种途径，这种途径部分被遗传扰动所揭示。总而言之，这个项目提供了新的工具来剖析免疫系统的分化和稳态的动态。

We have introduced a new methodology using a combination of CyTOF immune profiling, IdU pulse-chasing and mathematical modeling to derive a systemic understanding of immune homeostasis. CyTOF is a recently-acquired instrument (within the NIH-wide Center for Human Immunology) that performs mass cytometry: it is similar to the classical flow cytometer, except that its detection capacity relies on the mass-cytometric resolution of heavy metals tagging antibodies. We have implemented CyTOF within NIH, and demonstrated its capacity to achieve immune profiling with a 40+ panel of antibodies (e.g. targeted against key markers of hematopoietic differentiation). We have also implemented IdU Pulse-chasing, to mark proliferating cells (by incorporation of the metal-tagged nucleotide during DNA replication), and to detect its dilution by cell proliferation or differentiation (by relying on the mass-cytometric capacity of CyTOF): IdU pulse-chasing combines the non-perturbative high-signal noise of BrdU pulse-chasing with the deep immunological profiling of CyTOF. Finally, we developed a mathematical framework to model the experimental results obtained from CyTOF IdU pulse-chasing: our quantitative analysis enables us to "animate" our network of immune cells, and to quantitate the rates at which cells differentiate We have applied our methodology to delineate the differentiation of NK T cells in the thymus (in collaboration with Dr. Hyun Park from EIB-NCI), of monocytes (in collaboration with Dr. Frederic Geissmann from Memorial Sloan-Kettering) and other systems are under consideration. We are demonstrating the flexibility and resolution power of our new methodology to better analyze the systemic perturbation of hematopoiesis occurring during tumor development (in collaboration with Dr. Romina Goldzmid from CIP-NCI). One major finding so far has been that monocytes of the mouse immune system do not differentiate only in the blood of these animals, but follow a parallel dynamic of differentiation in the bone marrow and in the lymphoid organs. This implies that the main source of blood monocytes may not reside in the blood itself, but rather come from the bone marrow. Such result (obtained without perturbation, at homeostasis) challenges the common understanding of blood monocyte differentiation as obtained from genetic perturbations. A second finding was that NKT2 cells (i.e. Type 2 natural-killer T cells) do not derive from NKT1 cells but rather have a direct (yet inefficient) path of differentiation from immature NK T cells. Again, our kinetic analysis of pulse-chase experiments revealed an alternative path of differentiation that was partially uncovered by genetic perturbation. All in all, this project offers new tools to dissect the dynamics of differentiation and homeostasis in the immune system.