ID Proteins Regulate Exhausted Cd8+ T Cells During Chronic Viral Infection

Chronic viral infections, such as HIV, HBV, HCV, are serious threats to human health,especially the functional cure for HIV infection is the current challenge and hot spot. The current clinical treatment for HIV mainly relys on HAART, which is unable to completely remove all the latent HIV virus. In the early phase of infection, TFH cells within lymphatic follicles can be infected by HIV which lurk in infected cells and escape from HAART or immune cells. The persistent infection in these cells represents a major obstacle in the pursuit of functional cure for HIV patients. To enhance the function of the immune system and clear the HIV virus, it is necessary to have a better understanding of the mechanisms that influence immune system in the context of chronic viral infection.In the immune system, CD8+ T cells become the primary cell subpopulation that controls viral infection by killing the target cells infected by the virus. After acute viral infection, activated CD8+ T cells proliferate to a great expand and remove the virus in a short time, which is followed by the contraction phase that most of the CD8+ T cells go through apoptosis without antigen stimulation. But there are still a few cells that survived and differentiated into antigen specific memory CD8+ T cells. These memory T cells have high proliferative potential and are maintained by antigen independent homeostatic proliferation, thus they can rapidly reactivate to a second infection and confer life-long protective immunity. In contrast, during chronic viral infection, the virus could not be removed completely and persist in the host. Continuously receiving antigen stimulation from the virus, the effector CD8+ T cells upregulate multiple inhibitory molecules and gradually lose their poly-function, and ultimately step into exhaustion.Exhausted CD8+ T cells generated in chronic viral infection are recognized with a progressive increase in the amount and diversity of inhibitory receptors expressed as infection goes on. At the same time, these cells gradually lose their functions in a hierarchical manner, and finally the severely exhausted antigen specific CD8+ T cells step to physical deletion. In the course of chronic viral infection, higher viral load, longer duration of infection or less help from CD4+ T cells leads to more severe exhaustion. In human chronic viral infection, such as HIV, HBV, HCV and HTLV infection, antigen specific CD8+ T cells also undergo exhaustion.However, exhausted CD8+ T cells still partially retain effector function and control the viral replication to a certain extent. Recent studies have found that there is a group of CXCR5+ CD8+ T cells among exhausted CD8+ T cells in the murine LCMV-C113 infection model. Compared to CXCR5- CD8+ T cells, these cells express less surface inhibitory molecules such as PD-1, Tim3, 2B4, Lag3 and so on. And CXCR5+ CD8+ T cells could secrete more cytokines and de-granulation marker CD 107 after in vitro peptide stimulation,and higher proportion CXCR5+ CD8+ T cells can secrete multiple cytokines. These cells also represent stronger killing ability and better control of viral replication, and express higher levels of memory markers CD 127 and CD62L with better ability to proliferate and resemble stem cells. Thus, these CXCR5+ CD8+ T cells may play a major antiviral effect in exhausted CD8+ T cells. And recently, based on LCMV, SIV, HIV chronic viral infection, a number of studies found the phenomenon that CXCR5+ antigen specific CD8+ T cells migrate into the B cell follicles in lymphoid tissues. The CXCR5+ CD8+ T cells were found be able to kill infected TFH cells in follicles and control the viral replication. The discovery of this cell group provided new important clues for the reduction of HIV virus reservoir and functional cure for HIV infection.During acute and chronic viral infections, the differentiation and function of CD8+ T cells is regulated by a series of molecular pathways and transcription factors, among which ID proteins are important for the regulation of CD8+ T cell differentiation in acute viral infection. During acute viral infection, Id3 promotes memory CD8+ T cell formation and maintain its survival by enhancing expression of genes related to DNA replication and repair. In addition, Id2 is also involved in the regulation of CD8+ T cell differentiation playing a role opposite to Id3. And Id2 may inhibit the expression of IM3 through their downstream molecule E2A. however, during chronic viral infection, E2A binds directly to the regulatory region of gene Cxcr5 and promote the expression of CXCR5 in CD8+ T cells,while Id2 inhibit the binding of E2A to the gene Cxcr5. Id2 knockout or E2A overexpression could promote the generation of CXCR5+ CD8+ T cells and enhance their cytotoxicity and cytokine secretion. In the LCMV-Docile chronic infection model, Id3+2B4- antigen specific CD8+ T cells can also differentiate into Id3-2B4- and Id3-2B4+ CD8+T cells, but not reverse. While overexpression of Id3 inhibited the expression of Fas and reduced the Fas/FasL-mediated apoptosis of CD8+ T cells. Therefore, the pattern in transcription factors, surface phenotype, cytokine expression, killing ability and stem cell like differentiation are showing a high degree of consistency between Id3+ CD8 T cells and CXCR5+ CDS T cells in chronic viral infection. However, the relationship between Id3 and CXCR5 expression in exhausted CD8+ T cells and the effect of Id3 on the phenotype and function of CXCR5+ CD8+ T cells remain unclear.Therefore, we further studied how Id3 regulate the phenotype and function of CXCR5+CD8+ T cells during chronic viral infection. The experimental design, results and conclusions of this study are listed as follows:1. At day 8, 24 and 32 post infection of LCMV-C113 in B6 mice, splenocytes were collected and enriched for CD8+ T cells. After staining with antibodies, CD44hi CXCR5-and CD44hi CXCR5+ CD8+ T cells were sorted by flow cytometry for total RNA extraction.These RNA were reverse transcribed into cDNA, and the mRNA levels of Id2 and Id3 in the two cell groups was measured by RT-qPCR. The results showed that the expression of Id3 in CD44hi CXCR5+ CD8+ T cells was higher than that in CD44hi CXCR5-CD8+ T cells during the chronic infection course, while Id2 expression was lower in CD44hi CXCR5+CD8+ T cells. And the expression levels of Id2 and Id3 in these two cell groups remained stable during the infection.2. Cd4Cre Id2flox/flox" mice were infected with LCMV-C113 virus and splenocytes were obtained in exhaustion phase and analyzed by flow cytometry. Results indicated that the percentage and the number of CD44hi CXCR5+ CD8+ T cells from Id2-/- mice increased in activated CD8+ T cells, while the expression of surface inhibitory molecules PD-1 and Tim3 decreased. After in vitro viral peptide stimulation, CD44hi CXCR5+ CD8+ T cells from Id2-/- mice secrete more cytokine TNFa and express more CD107a/b. In addition, the viral titer in the spleen and lung of Id2 knockout mice significantly reduced.3. The number and function of CD44hi CXCR5+ and CD44hi CXCR5- CD8+ T cells in the spleens of Cd4Cre Id3flox/flox mice infected with LCMV-C113 were analyzed by flow cytometry. The results showed that the expression of inhibitory molecules in CD44hi CXCR5+ CD8+ T cells was lower than that of CXCR5- CD8+ T cells,in accordance with previous data. However, the frequency of CD44hi CXCR5+ CD8+ T cells were similar between Id3 knockout and control group, while the number of both CD44hi CXCR5+ and CD44hi CXCR5- CD8+ T cells increased in Id3 knockout mice. And total CD44hi CD8+ T cells secreted more TNFa and expressed higher levels of CD 107 in Id3 knockout mice after in vitro peptide stimulation. The viral titer in the tissues of the two groups were measured by RT-qPCR method, and it was significantly reduced by nearly 10 times in Id3 knockout mice compared with that in the control mice.4. We then constructed Id3 over-expression plasmid and eventually transduced into P14 cells. These cells were then transfered into recipient mice followed by LCMV-C113 infection. We measured the expression of CXCR5 in P14 cells overexpressing Id3 and the cytokine secretion at day 8 post infection. The results showed that the levels of CXCR5 expression in P14 cells was similar between Id3 over-expression and control group. While the Id3 over-expression did not significantly alter the cytokine secretion of CD8+ T cells,either.5. Immunofluorescent staining of lymph nodes from HIV patients revealed that a few of CD8+ T cells migrated into B cell follicles. Flow cytometry analysis of cells from lymph nodes showed that CXCR5+ CD8+ T cells expressed higher Id3 and had stronger effect function. In peptide stimulated PBMCs, the expression levels of Id3 in CD107a/b- and TNFα- IFNγ+ CD8+ T cell population was slightly higher than that in CD107a/b+ and TNFα+IFNγ+ CD8+ T cell populations, respectively.