Previous studies have established a significant correlation between the dysregulation of the intestinal immune environment and the aberrant intestinal inflammatory response observed in patients with CD. Most prior studies have primarily emphasized the role of conventional T cells in the context of CD. Recently, studies have found that unconventional γδT cells also play a role in the pathogenesis and progression of CD [22]. Moreover, CD8+ γδT is the main population of intestinal γδT cells, and its immune characteristics in the intestinal tract of CD patients and its correlation with disease activity are still unclear. Therefore, we focused on the immune characteristics of CD8+ γδT cells in the intestinal tract of CD patients and their correlation with the degree of disease activity in this study.
We first assessed the absolute count and frequency of CD8+ γδ T cells in the gut of active CD patients. The results showed that the absolute count and frequency of CD8+ γδ T cells in the gut of active CD patients were reduced, with a more pronounced reduction as the disease activity degrees increased. Previous research has also shown that the frequency of intestinal CD8+ γδ T cells in active IBD patients was significantly lower compared with HCs, correlated negatively with the degree of disease activity, and increased to normal levels as a result of anti-TNF-α therapy [23]. The γδ T cells express the CXCR3 receptor on their surface, which binds to local cell-produced chemokines in inflammatory lesions, thereby recruiting CXCR3+ γδ T cells to inflammatory lesions [24]. Similarly, our research found that CD8+ γδ T cells express CXCR3 receptors on the surface. To comprehend the underlying cause of the decrease in intestinal CD8+ γδ T cells among patients with active CD, we investigated the expression of the chemokine receptor CXCR3 on CD8+ γδ T cells. This analysis aimed to assess the migratory potential of intestinal CD8+ γδ T cells. We found that the migratory potential of intestinal CD8+ γδ T cells was not altered in patients with active CD regardless of disease activity, so the decrease in CD8+ γδ T cells was not due to migratory behavior but could be due to other causes. For example, as the intestinal inflammatory environment worsened, cell death increased and cell proliferation decreased.
We then assessed whether the immune status of intestinal CD8+ γδ T cells in active CD patients was activated or suppressed by examining the activation marker HLA-DR and the immunosuppressive molecule PD-1. We found intestinal CD8+ γδ T cells were in a state of immune activation in active CD patients. Additionally, we found that although intestinal CD8+ γδ T cells were highly activated in mildly and moderately active CD patients compared with HCs, intestinal CD8+ γδ T cell activation was relatively attenuated in moderately active CD patients compared with mildly active CD patients. The reason may be that in patients with low disease activity, the intestinal immune environment is not yet imbalanced, CD8+ γδ T cells can still exert anti-inflammatory effects, and the cells remain active. As disease activity continues to increase, the patient’s intestinal inflammatory environment becomes more and more serious. Imbalance in the intestinal immune environment leads to weakened activation of CD8+ γδ T cells.
One study reported that aggravation of intestinal inflammation by depletion/deficiency of gammadelta T cells in different types of IBD animal models [25]. Another study reported CD8+ γδ T cells showed negative correlation with disease activity [23]. These data collectively suggest that CD8+ γδ T cells play an anti-inflammatory role in the human intestinal mucosa. Conventional CD8+ αβ T cells constitute the major cytotoxic T cell population in vivo, whereas γδ T cells have been proven cytotoxic [26]. Recent discoveries have demonstrated the cytotoxic nature of CD8+ γδ T cells, implying their potential anti-inflammatory role in eliminating infected cells, tumor cells, or cells experiencing stress due to various factors, including inflammation [23]. Furthermore, lymphocytes may upregulate their cytotoxic potential in situations requiring greater cytotoxicity in the gut, i.e., infection, tumor, or inflammation. Cellular cytotoxicity plays a role in inducing epithelial cell apoptosis and maintaining homeostasis. So how exactly do highly activated CD8+ γδ T cells in the intestine of active CD patients play an “anti-inflammatory” role? We further investigated the cytotoxicity of intestinal CD8+ γδ T cells in active CD patients by examining the expression of cytotoxic molecules (Perforin, Granzyme B, and TRAIL) in intestinal CD8+ γδ T cells. We found intestinal CD8+ γδ T cells in active CD patients had enhanced cytotoxicity compared with HCs. We further studied the relationship between the cytotoxicity of intestinal CD8+ γδ T cells in active CD patients and the different degrees of disease activity. We found that intestinal CD8+ γδ T cells had enhanced cytotoxicity only in mildly active CD patients. As disease activity increased, the cytotoxicity of intestinal CD8+ γδ T cells in moderately active CD patients decreased relatively compared with mildly active CD patients. These findings have allowed us to identify the disparities in immune characteristics of intestinal CD8+ γδ T cells between healthy individuals and those with CD. Furthermore, we have also delineated the variations in immune characteristics of intestinal CD8+ γδ T cells among CD patients with varying degrees of disease activity. These insights offer a valuable theoretical foundation for the precise diagnosis of active CD and the timely and effective prediction of disease activity levels.
At present, clinicians cannot accurately diagnose based on existing laboratory indicators. Although pathological diagnosis is the gold standard, diagnosis takes too long (about one week). We still need to find diagnostic methods that are accurate, convenient and time-consuming to gain critical treatment time for patients. It only takes about 6 h to obtain results using flow cytometry detection of biological markers in cells in this study. In addition, it does not require a separate invasion to obtain the specimen, but a small piece of biopsy tissue can be obtained during the necessary pathological biopsy, so it is feasible. Therefore, we further analyzed the ROC curve and found that HLA-DR+ CD8+ γδT cell ratio (AUC, 0.886; Sp, 95.24%; Se, 66.67%), CD8+ γδT ratio (AUC, 0.819; Sp, 100%; Se, 66.67%) and CD8+ γδT count (AUC, 0.776; Sp, 95.24%; Se, 66.67%) exhibited a good value for assisting diagnosis of active CD. In addition, we found that Granzyme B+ CD8+ γδT cell ratio (AUC, 0.846; Sp, 80%; Se, 90.91%) and Perforin+ CD8+ γδT cell ratio (AUC, 0.755; Sp, 70%; Se, 90.91%) are valuable indicators to help distinguish mildly-moderately active CD cases.
In conclusion, this study found that intestinal CD8+ γδT was reduced in active CD patients, but their activation and cytotoxicity were enhanced. Further evaluation revealed that the intestinal CD8+ γδT cells in mildly active CD patients decreased, and their activation and cytotoxicity were enhanced, which might demonstrate the positive anti-inflammatory effects of CD8+ γδT cells in mildly active CD patients. However, as the disease activity levels increased, we observed a more pronounced reduction in intestinal CD8+ γδ T cells among moderately active CD patients compared to those with mild disease activity. Meanwhile, their activation and cytotoxicity were relatively attenuated. This characterization reveals that the anti-inflammatory effects of CD8+ γδT cells might be impaired with increasing disease activity of CD patients. In addition, we found that HLA-DR+ CD8+ γδT cell ratio, CD8+ γδT ratio, and CD8+ γδT count had a good value for assisting diagnosis of active CD, whereas the ratios of Granzyme B+ CD8+ γδT cell and Perforin+ CD8+ γδT cell were valuable indicators to help distinguish mildly-moderately active CD cases. These findings may provide a new perspective and theoretical basis for CD patients’ clinical diagnosis and immunotherapy.
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