Lilin Ye

Follicular CXCR5-expressing CD8 + T cells curtail chronic viral infection

During chronic viral infection, virus-specific CD8+ T cells become exhausted, exhibit poor effector function and lose memory potential. However, exhausted CD8+ T cells can still contain viral replication in chronic infections, although the mechanism of this containment is largely unknown. Here we show that a subset of exhausted CD8+ T cells expressing the chemokine receptor CXCR5 has a critical role in the control of viral replication in mice that were chronically infected with lymphocytic choriomeningitis virus (LCMV). These CXCR5+ CD8+ T cells were able to migrate into B-cell follicles, expressed lower levels of inhibitory receptors and exhibited more potent cytotoxicity than the CXCR5− subset. Furthermore, we identified the Id2–E2A signalling axis as an important regulator of the generation of this subset. In patients with HIV, we also identified a virus-specific CXCR5+ CD8+ T-cell subset, and its number was inversely correlated with viral load. The CXCR5+ subset showed greater therapeutic potential than the CXCR5− subset when adoptively transferred to chronically infected mice, and exhibited synergistic reduction of viral load when combined with anti-PD-L1 treatment. This study defines a unique subset of exhausted CD8+ T cells that has a pivotal role in the control of viral replication during chronic viral infection.

The transcription factor TCF-1 initiates the differentiation of T FH cells during acute viral infection

Induction of the transcriptional repressor Bcl-6 in CD4+ T cells is critical for the differentiation of follicular helper T cells (TFH cells), which are essential for B cell–mediated immunity. In contrast, the transcription factor Blimp1 (encoded by Prdm1) inhibits TFH differentiation by antagonizing Bcl-6. Here we found that the transcription factor TCF-1 was essential for both the initiation of TFH differentiation and the effector function of differentiated TFH cells during acute viral infection. Mechanistically, TCF-1 bound directly to the Bcl6 promoter and Prdm1 5′ regulatory regions, which promoted Bcl-6 expression but repressed Blimp1 expression. TCF-1-null TFH cells upregulated genes associated with non-TFH cell lineages. Thus, TCF-1 functions as an important hub upstream of the Bcl-6–Blimp1 axis to initiate and secure the differentiation of TFH cells during acute viral infection.

ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

ABSTRACT Autophagy critically contributes to metabolic reprogramming and chromosomal stability. It has been reported that monoallelic loss of the essential autophagy gene BECN1 (encoding BECN1/Beclin 1) promotes cancer development and progression. However, the mechanism by which BECN1 is inactivated in malignancy remains largely elusive. We have previously reported a tumor suppressor role of ABHD5 (abhydrolase domain containing 5), a co-activator of PNPLA2 (patatin like phospholipase domain containing 2) in colorectal carcinoma (CRC). Here we report a noncanonical role of ABHD5 in regulating autophagy and CRC tumorigenesis. ABHD5 directly competes with CASP3 for binding to the cleavage sites of BECN1, and consequently prevents BECN1 from being cleaved by CASP3. ABHD5 deficiency provides CASP3 an advantage to cleave and inactivate BECN1, thus impairing BECN1-induced autophagic flux and augmenting genomic instability, which subsequently promotes tumorigenesis. Notably, clinical data also confirm that ABHD5 proficiency is significantly correlated with the expression levels of BECN1, LC3-II and CASP3 in human CRC tissues. Our findings suggest that ABHD5 possesses a PNPLA2-independent function in regulating autophagy and tumorigenesis, further establishing the tumor suppressor role of ABHD5, and offering an opportunity to develop new approaches aimed at preventing CRC carcinogenesis.

Oral administration of visceral adipose tissue antigens ameliorates metabolic disorders in mice and elevates visceral adipose tissue-resident CD4+CD25+Foxp3+ regulatory T cells

Obesity and type 2 diabetes are linked with chronic, low-grade inflammation in visceral adipose tissue (VAT). A unique population of VAT-resident CD4+Foxp3+ Tregs plays a crucial role in regulating VAT inflammation and metabolic homeostasis. VAT-resident Tregs display a highly restricted TCR repertoire, suggesting they recognize certain autoantigen(s) in VAT. A dramatic reduction of VAT-resident Tregs has been shown to closely correlate with obesity-related VAT chronic inflammation and metabolic disorders. Oral tolerance strategy may modulate inflammatory response to autoantigens by several mechanisms including induction of autoantigen-specific Tregs. Here, we explored the effects and cellular mechanism of oral administration of VAT pooled antigens on high-fat diet (HFD)-induced metabolic disorders in mice. Indeed, we found that oral treatment of VAT mixture antigens effectively inhibited gain in body weight and fat mass, ameliorated serum lipid parameters, and improved insulin sensitivity in HFD mice. This strategy was shown to significantly restore HFD-induced decrease of VAT-resident Tregs, accompanied by a hampered M2-type to M1-type macrophages phenotypic switch as well as decreased CD8+ T cells infiltration in VAT. Thus, oral administration of VAT antigens may be a novel and safe strategy against obesity and its related metabolic disorders.

Mammalian target of rapamycin complex 1 signalling is essential for germinal centre reaction

The mammalian target of rapamycin (mTOR) is a serine‐threonine kinase that has been shown to be essential for the differentiation and function of various immune cells. Earlier in vitro studies showed that mTOR signalling regulates B‐cell biology by supporting their activation and proliferation. However, how mTOR signalling temporally regulates in vivo germinal centre B (GCB) cell development and differentiation into short‐lived plasma cells, long‐lived plasma cells and memory cells is still not well understood. In this study, we used a combined conditional/inducible knock‐out system to investigate the temporal regulation of mTOR complex 1 (mTORC1) in the GCB cell response to acute lymphocytic choriomeningitis virus infection by deleting Raptor, a main component of mTORC1, specifically in B cells in pre‐ and late GC phase. Early Raptor deficiency strongly inhibited GCB cell proliferation and differentiation and plasma cell differentiation. Nevertheless, late GC Raptor deficiency caused only decreases in the size of memory B cells and long‐lived plasma cells through poor maintenance of GCB cells, but it did not change their differentiation. Collectively, our data revealed that mTORC1 signalling supports GCB cell responses at both early and late GC phases during viral infection but does not regulate GCB cell differentiation into memory B cells and plasma cells at the late GC stage.

Analyzing Mouse B Cell Responses Specific to LCMV Infection

B cell responses play a central role in humoral immunity, which protects an individual from invading pathogens by antigen-specific antibodies. Understanding the basic principles of the B cell responses during viral infection is of substantial importance for anti-viral vaccine development. In inbred mice, lymphocytic choriomeningitis virus (LCMV) infection elicits robust and typical T cell-dependent B cell responses, including germinal center reaction, memory B cell formation, and a long-lived plasma cell pool in bone marrow. Therefore, this system represents an ideal model to investigate anti-viral B cell responses. In this protocol, we describe how to propagate and quantify LCMV and successfully establish an acute LCMV infection in mice. This protocol also provides three different techniques to analyze B cell responses specific to an acute LCMV infection: the identification of germinal center (GC) B cells and follicular helper CD4 T (TFH) cells from the spleens and lymph nodes via flow cytometry, titration of LCMV-specific IgG in the serum after LCMV infection using an enzyme-linked immunosorbent assay (ELISA) analysis, and detection of LCMV-IgG secreted plasma cells from bone marrow with an enzyme-linked immunospot (ELISPOT) assay.

Efficient control of chronic LCMV infection by a CD4 T cell epitope-based heterologous prime-boost vaccination in a murine model

CD4+ T cells are essential for sustaining CD8+ T cell responses during a chronic infection. The adoptive transfer of virus-specific CD4+ T cells has been shown to efficiently rescue exhausted CD8+ T cells. However, the question of whether endogenous virus-specific CD4+ T cell responses can be enhanced by certain vaccination strategies and subsequently reinvigorate exhausted CD8+ T cells remains unexplored. In this study, we developed a CD4+ T cell epitope-based heterologous prime-boost immunization strategy and examined the efficacy of this strategy using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We primed chronically LCMV-infected mice with a Listeria monocytogenes vector that expressed the LCMV glycoprotein-specific I-Ab-restricted CD4+ T cell epitope GP61–80 (LM-GP61) and subsequently boosted the primed mice with an influenza virus A (PR8 strain) vector that expressed the same CD4+ T cell epitope (IAV-GP61). This heterologous prime-boost vaccination strategy elicited strong anti-viral CD4+ T cell responses, which further improved both the quantity and quality of the virus-specific CD8+ T cells and led to better control of the viral loads. The combination of this strategy and the blockade of the programmed cell death-1 (PD-1) inhibitory pathway further enhanced the anti-viral CD8+ T cell responses and viral clearance. Thus, a heterologous prime-boost immunization that selectively induces virus-specific CD4+ T cell responses in conjunction with blockade of the inhibitory pathway may represent a promising therapeutic approach to treating patients with chronic viral infections.

Flow Cytometry Analysis of mTOR Signaling in Antigen-Specific B Cells

B lymphocytes and their differentiated daughter cells are charged with responding to invading pathogens and producing protective antibodies against these pathogens. The physiology of B cells is intimately connected with the function of the B cell antigen receptor (BCR). Upon activation of BCR, transmembrane signals are generated, and several downstream pathways are activated, which provide a primary directive for the cells subsequent response. mTOR is a serine/threonine kinase that controls cell proliferation and metabolism in response to a diverse range of extracellular stimuli. The activation of mTOR signaling downstream of PI3K/Akt activity by B cell receptor (BCR) engagement has been generally assumed to be essential for B cell responses. This chapter seeks to present two protocols to evaluate mTOR activity in B cells bearing BCR specific to 4-hydroxy-3-nitrophenylacetyl (NP)-hapten.

Late-stage tumors induce anemia and immunosuppressive extramedullary erythroid progenitor cells

Impaired immunity in patients with late-stage cancer is not limited to antitumor responses, as demonstrated by poor vaccination protection and high susceptibility to infection1–3. This has been largely attributed to chemotherapy-induced impairment of innate immunity, such as neutropenia2, whereas systemic effects of tumors on hematopoiesis and adoptive immunity remain incompletely understood. Here we observed anemia associated with severe deficiency of CD8+ T cell responses against pathogens in treatment-naive mice bearing large tumors. Specifically, we identify CD45+ erythroid progenitor cells (CD71+TER119+; EPCs) as robust immunosuppressors. CD45+ EPCs, induced by tumor growth–associated extramedullary hematopoiesis, accumulate in the spleen to become a major population, outnumbering regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). The CD45+ EPC transcriptome closely resembles that of MDSCs, and, like MDSCs, reactive oxygen species production is a major mechanism underlying CD45+ EPC–mediated immunosuppression. Similarly, an immunosuppressive CD45+ EPC population was detected in patients with cancer who have anemia. These findings identify a major population of immunosuppressive cells that likely contributes to the impaired T cell responses commonly observed in patients with advanced cancer.Large tumors induce anemia and expansion of CD45+ immature erythroid cells, which represent a major immunosuppressive population in the spleen, contributing to systemic suppression of T cell immunity in late-stage cancer.

Differentiation and Function of Follicular CD8 T Cells During Human Immunodeficiency Virus Infection

The combination antiretroviral therapeutic (cART) regime effectively suppresses human immunodeficiency virus (HIV) replication and prevents progression to acquired immunodeficiency diseases. However, cART is not a cure, and viral rebound will occur immediately after treatment is interrupted largely due to the long-term presence of an HIV reservoir that is composed of latently infected target cells that maintain a quiescent state or persistently produce infectious viruses. CD4 T cells that reside in B-cell follicles within lymphoid tissues, called follicular helper T cells (TFH), have been identified as a major HIV reservoir. Due to their specialized anatomical structure, HIV-specific CD8 T cells are largely insulated from this TFH reservoir. It is increasingly clear that the elimination of TFH reservoirs is a key step toward a functional cure for HIV infection. Recently, several studies have suggested that a fraction of HIV-specific CD8 T cells can differentiate into a CXCR5-expressing subset, which are able to migrate into B-cell follicles and inhibit viral replication. In this review, we discuss the differentiation and functions of this newly identified CD8 T-cell subset and propose potential strategies for purging TFH HIV reservoirs by utilizing this unique population.