Dok-3 deficient mice display different immune clustering and Tim-3 expression

Background Dok-3 has been shown to play an important role in immune system. Tim-3 also has been recognized as an important immune regulator which involves in many diseases. The relationship of them is still unclear. Methods We detected the expression of Tim-3 on spleen immune cells from Dok-3 deficient mice and control mice by flow cytometry. Results In this article, we found that Dok-3−/− mice display almost entirely different immune clustering characteristics compared with wild type 129 mice. The CD4 T cells and CD8 T cells decreased and DC cells, macrophages, MDSCs increased when the Dok-3 gene knocked-out. The Tim-3 expression on CD4 T cells, CD8 T cells, NK cells, DC cells increased when the Dok-3 gene knocked-out. The macrophages and MDSCs just display the opposite results. Conclusions Although Dok-3−/− mice display different immune clustering and Tim-3 expression, the mechanism still needs further study. Electronic supplementary material The online version of this article (10.1186/s40001-019-0384-7) contains supplementary material, which is available to authorized users.


Background
The Dok (downstream of kinase) family consists of seven members, with each possessing an NH2-terminal pleckstrin homology (PH), a central phosphotyrosine-binding (PTB) and a C-terminal tyrosine-rich domain [1]. Dok-1, 2 and 3 are expressed in hematopoietic cells, while the other Dok members are preferentially expressed in cells of the nervous or muscular system [2]. Dok proteins function mainly as adaptors to facilitate protein-protein interactions since they have no catalytic activity [1]. Given Dok-1 to Dok-3 are also expressed in the immune cells, they play an important role in the development of the tumor progression. Recent researches showed that they have been identified as not only tumor suppressors for lung cancer in human and mice [3] but also the depressors in the development of aggressive histiocytic sarcoma [4].
Dok-3 has been shown to play an important role in immune system. First, it can inhibit the Ca 2+ [5] and JNK [6] activation in B cell receptor signaling. Secondly, macrophages are more sensitive to LPS-induced ERK activation with subsequent upregulation of a gene expression profile that promotes endotoxin tolerance in the absence of Dok-3 [7]. Finally, Dok-3 plays a critical and positive role in TLR3 signaling by enabling TRAF3/TBK1 complex formation and facilitating TBK1 and IFN regulatory factor 3 activation and the induction of IFN-β production [8].
T-cell immunoglobulin and mucin-domain-containing molecule 3 (Tim-3) are a membrane protein initially identified as a negative regulator of Th1 immunity [9,10]. A strong correlation between Tim-3 expression and the immune cell function has been confirmed. Recently, Tim-3 was shown to play important roles on activated Th17 [11], Tc1 [12], macrophages/monocytes [13,14], dendritic cells (DCs) [15] and natural killer (NK) cells  [16]. However, the relationship between the Dok-3 and Tim-3 in the immune cells is still unclear. Here, we will focus on the immune cell proportion and Tim-3 expression in the Dok-3 deficient mice.

Methods
Mice 129 S1/SvImJ mice (129 mice) were obtained from Huafukang (Beijing, China). DOK3-deficient 129 S1/ SvImJ mice were kindly provided by Mary Beth Humphrey from University of Oklahoma. The mice were housed in a pathogen-free room which could keep appropriate temperature and humidity. The mice were killed before the experiment. All animal experimental procedures were approved by the Shandong University Animal Care Committee and performed in accordance with the Animal Management Rules of the Chinese Ministry of Health.

Statistical analyses
All the data were analyzed by the GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA). The unpaired t test was used for comparison between groups. Data were reported as mean values ± SEM. Value of p < 0.05 was considered as significant difference.

Discussion
In this work, we present a comprehensive analysis of the immune cells proportion difference between the 129 mice and Dok-3 −/− mice. We find that the CD4 T cells and CD8 T cells decreased and DC cells, macrophages, MDSCs increased when the Dok-3 gene knocked-out. NK cells display no difference between the two group mice.
Dok-3 has been demonstrated to be the key molecule in the immunity especially in the function of macrophages [5][6][7][8]. Here, we report the immune clustering alteration caused by Dok-3. The main reason why immune cells proportion changed is the proliferation, differentiation and apoptosis of these cells. But there is no evidence to prove that Dok-3 plays important role in the proliferation, differentiation and apoptosis of the immune cells. So, we still need more work to find the mechanism why Dok-3 affects the changed immune cells proportion.
Tim-3 has been recognized as an important immune regulator which expresses in both innate and adaptive immune cells. Increased evidence has shown that dysregulation of Tim-3 expression on peripheral CD4, CD8 T cells and monocytes is closely related to many diseases [11,12,17]. However, the roles of Dok-3 in Tim-3 expression on immune cells remain largely unknown. Here, for the first time, we demonstrated the knocked-out of Dok-3 gene which affects the Tim-3 expression on immune cells. Our data provide a previously unrecognized link between Dok-3 gene and the Tim-3 expression.
There is evidence to prove that Tim-3 is related to the apoptosis of some immune cells, especially the CD8 T cells [18]. In this study, the Dok-3 −/− mice display the decreased CD8 T cells and increased Tim-3 expression (Fig. 1b). Maybe the high Tim-3 expression leads to the apoptosis of CD8 T cells which is the main reason why CD8 T cells decrease.  Figure S1. *p < 0.05, **p < 0.01, ***p < 0.001