| Hepatitis B virus (HBV) infection is a public health problem. Most neonates and imunocompromised adults are much more likely to develop into persistent infection. Previous findings have shown that polyclonal and multi-specific CD4+and CD8+T cell activity against HBV can be detected in patients with acute self-limiting HBV infection. In contrast, it is difficult to detect HBV-specific response in HBV chronic infected patients. It has been verified that acute HBV patients who have HBcAg-specific cytokine (IFN-y and TNF-a) producing capacity are more prone to exert HBV clearance. The magnitude of HBcAg/HBeAg-specific CD4+T cell response is lower in chronic HBV patients than in acute HBV patients. However, the function of HBV-specific CD4+T cell is restored in patients with HBeAg seroconversion. It implied that HBV-specific CD4+and CD8+T cells play an important role in HBV elimination. Nevertheless, whether the cells equipped with CD4and CD8simultaneously also have HBV-specific response is still unknown.CD4+CD8+double positive T (DPT) cells are the precursor cell of peripheral CD4single positive (sp) and CD8sp T cell. In the thymus, DPT cells develop into mature single positive T cells via positive and negative selection. Therefore, normally, DPT cells are considered to reside in the thymus. Interestingly, in1980s Blue and Patel reported that a small proportion of DPT cells appeared in peripheral blood. But for more than two decades, this particular T cell subset was not paid enough attention from investigators. Recently, peripheral DPT cells were considered as antigen-specific T cells in humans, which implied that peripheral DPT cells may be an important T cell subset involved in HBV-specific immune response. As the information about peripheral DPT cells in HBV patients is still poorly understood, a comprehensive analysis of these cells in HBV patients is needed.Firstly, in our cohort, we found HBV infection can cause significant up-regulation of peripheral DPT cells, in regardless of infection status. While in healthy people, the overall DPT cells account for0.5%(median value) of CD3+T cells. There were1.0%and1.5%DPT cells among the CD3+T cells from chronic and acute HBV infected patients, respectively. Next we studied the phenotypes of these cells exhibited in HBV patients. CCR7and CD45RO were used to identify naive (CCR7+CD45RO-), effector (CCR7-CD45RO-), central memory (CCR7+CD45RO+) and effector memory (CCR7-CD45RO+) T cells with freshly isolated PBMCs from HBV patients. Results showed that the majority (70%) of DPT cells were CD45RO+memory T cell. Interestingly, more than70%of memory DPT cells were central memory T cell with high expression of CCR7. As some of T cell subsets also express CD4or CD3on their membrane, such as regulated T cell (CD4+CD25+Foxp3+), NKT cell (CD3+CD56+) and T follicular cell (CD4+CXCR5+PD-1+), further study was undertaken to investigate whether DPT cells express these markers. The data showed that DPT cells hardly expressed regulatory T cell or T follicular helper cell markers. However, only a small fraction of DPT cells expressed CD56(NKT or NK marker).To determine the possible mechanisms which contribute to the up-regulation of peripheral DPT cells in HBV patients, we detected whether the frequency of peripheral DPT cells was up-regulated by stimulation of HBV peptides for ten days in vitro. As we expected, after10days’culture, the proportion of DPT cell was up-regulated from1.2%to4.1%. Furthermore, we adopted CESF staining to detect proliferation of DPT cells in presence of HBV peptides. There was a higher percentage of CFSEdim DPT cells than single positive T cells in presence of HBV peptides (37.4%CFSEdim DPT,6.22%CFSEdim CD8sp,2.5%CFSEdim CD4sp). It indicated that peripheral DPT cells possessed a higher proliferative capacity than single positive T cells in response to HBV antigens. However, previous study showed that single positive T cells can be transformed into DPT (sp-DPT) cell by activation-induced de novo synthesis. Whether the high percentage of CFSEdim DPT cells was caused by proliferation of sp-DPT cells in response to HBV stimulation is still a problem. To verify this possibility, CD4+or CD8+T cells depleted-PBMCs fromacute HBV patients were used to culture for ten days in presence of HBV peptides or anti-CD3antibody(as positive control). In consistent with previous report, we found that activation by anti-CD3but not HBV peptides can generate sp-DPT cells in vitro. Thus, we confirmed that the high percentage of CFSEdim DPT cells was caused by high proliferation of peripheral DPT cells but not sp-DPT cells in our system.To further elucidate the function of these cells in HBV patients, we analyzed cytokine (Cytolytic granule:CD107a; Noncytolytic cytokine:IFN-y, TNF-a) producing capacity of DPT cells in comparison with CD8sp or CD4sp T cells stimulating with HBV peptides for10days. Interestingly, we found that circulating DPT cells in HBV-infected individuals had stronger cytokine secreting capacity than both CD8sp and CD4sp T cells after stimulation. It indicated that peripheral DPT cells in acute patients have HBV-specific response. Given T cells recognize target cells through the interaction with vial epitopes presented on the cell surface in context with HLA molecular, we next want to know whether the response of peripheral DPT cells can be induced by HLA-A*02restricted HBV immunedominate epitope. As we expected, the peripheral DPT cells can produce higher percentage of HBV-specific response than single positive T cells. It implied that peripheral DPT cells can recognize HBV epitope presented by HLA-A*02.Whether dynamic change of circulating DPT cells was correlated with kinetics of HBV infection is another important issue. To address this question, we used hydrodynamic-based HBV mouse model to mimic HBV acute infection. Before injection, we collected the blood of each mouse to monitor the background of peripheral DPT cells. The proportion of circulating DPT cells in healthy mouse was extremely rare (<0.5%). Furthermore, we found that the majority (80%) of these DPT cells in mouse was effector memory T cell:CD44+CD62L+. These data showed that the characteristics of peripheral DPT cells in mouse were the same as in human. We did not observe obvious changes of DPT cells in the first week post HBV replication in the mice. However, these DPT cells were significantly increased from9to16dpi. Although these cells were slowly back to normal level from day16, the proportion of DPT cells still maintained at a high level for28days. Additionally, the activating condition of circulating DPT cells was significantly correlated with HBV kinetics. To address whether these peripheral DPT cells played an effector role,we isolated PBMCs from resolved mice and then used HBV peptides pool (percore/core region, GenotypeA) to stimulate so as to trigger HBV-specific immune responses. As we expected, these DPT cells can produce a large amount of IFN-y on HBV specific stimulation. These data implied that circulating DPT cells may be involved in host cellular immune response against viral infection. Collectively, our data showed that HBV infection can cause up-regulation of peripheral DPT cells. These DPT cells have stronger cytokine producing and proliferative capacity than single positive T cells when they encountered HBV antigen in vitro. Importantly, we found that dynamic changes of peripheral DPT cells were correlated with the course of acute HBV infection in mice. Thus, peripheral DPT cells in HBV patients may represent an important T cell subset with HBV-specific response. |
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