Chemokine Transfer by Liver Sinusoidal Endothelial Cells Contributes to the Recruitment of CD4+ T Cells into the Murine Liver

Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4⁺ T cells into the liver. In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4⁺ T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin-coated vesicles to CD4⁺ T cells leading to enhanced transmigration of CXCR4⁺ total CD4⁺ T cells and CXCR3⁺ effector/memory CD4⁺ T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4⁺ T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4⁺ T-cell transmigration in vitro as well as migration of CD4⁺ T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3⁺ CD4⁺ T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4- and clathrin-dependent intracellular transport mechanisms thereby contributing to the hepatic recruitment of CD4⁺ T-cell populations during immune surveillance and liver inflammation.     

IL-4 Induced Innate CD8+ T Cells Control Persistent Viral Infection

Memory-like CD8⁺ T cells expressing eomesodermin are a subset of innate T cells initially identified in a number of genetically modified mice, and also exist in wild mice and human. The acquisition of memory phenotype and function by these T cells is dependent on IL–4 produced by PLZF⁺ innate T cells; however, their physiologic function is still not known. Here we found that these IL-4-induced innate CD8⁺ T cells are critical for accelerating the control of chronic virus infection. In CIITA-transgenic mice, which have a substantial population of IL-4-induced innate CD8⁺ T cells, this population facilitated rapid control of viremia and induction of functional anti-viral T-cell responses during infection with chronic form of lymphocytic choriomeningitis virus. Characteristically, anti-viral innate CD8⁺ T cells accumulated sufficiently during early phase of infection. They produced a robust amount of IFN-γ and TNF-α with enhanced expression of a degranulation marker. Furthermore, this finding was confirmed in wild-type mice. Taken together, the results from our study show that innate CD8⁺ T cells works as an early defense mechanism against chronic viral infection.     

Memory CD4 T Cells Direct Protective Responses to Influenza Virus in the Lungs through Helper-Independent Mechanisms

Memory CD4 T cells specific for influenza virus are generated from natural infection and vaccination, persist long-term, and recognize determinants in seasonal and pandemic influenza virus strains. However, the protective potential of these long-lived influenza virus-specific memory CD4 T cells is not clear, including whether CD4 T-cell helper or effector functions are important in secondary antiviral responses. Here we demonstrate that memory CD4 T cells specific for H1N1 influenza virus directed protective responses to influenza virus challenge through intrinsic effector mechanisms, resulting in enhanced viral clearance, recovery from sublethal infection, and full protection from lethal challenge. Mice with influenza virus hemagglutinin (HA)-specific memory CD4 T cells or polyclonal influenza virus-specific memory CD4 T cells exhibited protection from influenza virus challenge that occurred in the presence of CD8-depleting antibodies in B-cell-deficient mice and when CD4 T cells were transferred into lymphocyte-deficient RAG2^−/− mice. Moreover, the presence of memory CD4 T cells mobilized enhanced T-cell recruitment and immune responses in the lung. Neutralization of gamma interferon (IFN-γ) production in vivo abrogated memory CD4 T-cell-mediated protection from influenza virus challenge by HA-specific memory T cells and heterosubtypic protection by polyclonal memory CD4 T cells. Our results indicate that memory CD4 T cells can direct enhanced protection from influenza virus infection through mobilization of immune effectors in the lung, independent of their helper functions. These findings have important implications for the generation of universal influenza vaccines by promoting long-lived protective CD4 T-cell responses.Influenza virus poses substantial threats to world health due to the emergence of new pandemic strains through viral mutation and reassortment, including the 2009 H1N1 pandemic strain. Developing effective vaccines that can provide immune-mediated protection to multiple influenza virus strains remains a major challenge, as current vaccines generate neutralizing antibodies directed against the highly variable hemagglutinin (HA) and neuraminidase (NA) surface viral glycoproteins (18). These vaccines are only partially effective at protecting individuals from succumbing to seasonal strains and are largely ineffective at protecting individuals from new pandemics. In contrast, T lymphocytes have the potential to provide long-term cross-strain protection, through their recognition of invariant viral determinants (3, 9), generation of effector responses to coordinate both cellular and humoral immunity, and development of memory populations that persist for decades (34). In humans, influenza virus-specific CD4 and CD8 T cells recognize internal polymerase, matrix, and nucleoprotein components of influenza virus which are conserved in multiple strains (3). Influenza virus-specific memory T cells generated from virus exposure and vaccines can be detected readily in the peripheral blood of healthy older children and adults (16, 30). Elucidating the protective capacities of memory T cells in antiviral immunity and their underlying mechanisms is therefore crucial to understanding clinical responses to influenza and to developing strategies to boost T-cell-mediated immunity for the next emerging pandemic.The potent cytolytic responses of virus-specific CD8 T cells and their roles in antiviral primary and secondary responses have been well established (58); however, considerably less is known about the function of memory CD4 T cells in antiviral immunity. Memory CD4 T cells have the potential to play more diverse roles in coordinating secondary responses than those of memory CD8 T cells via their ability to “help” or promote cellular and humoral immunity, and also through direct effector functions. Compared to CD8 T-cell responses, memory CD4 T-cell responses in humans were found to recognize a more diverse array of influenza virus-specific epitopes (46-48) and to exhibit cross-reactivities with new pandemic strains, including avian H5N1 and 2009 H1N1 “swine flu” strains (23, 28, 36, 48). In addition, antiviral memory CD4 T cells generated as a result of influenza vaccination (22) were found to persist longer than CD8 T cells in vivo following smallpox vaccination (29). These findings suggest that memory CD4 T-cell responses could be potential targets for boosting long-term cellular immunity following vaccination, although their protective capacity remains undefined.The role of CD4 T cells in anti-influenza virus immunity has been elucidated mainly for primary responses, and less is known about the protective potential and mechanisms underlying memory CD4 T-cell-directed secondary responses. In primary influenza virus infection, CD4 T cells promote antibody production by B cells necessary for complete viral clearance (2, 17, 19, 39, 40, 57) and also promote the generation of memory CD8 T cells (4). Whether memory CD4 T cells have a similar helper-intensive role in promoting B cells and CD8 T cells in secondary influenza responses or whether effector responses predominate is not known. In this study, we investigated the mechanisms by which memory CD4 T cells mediate secondary responses and promote recovery from influenza virus infection in the clinically relevant scenario of a persisting CD4 T-cell response but no preexisting antibody response to a new influenza virus strain. We demonstrate that both influenza virus HA-specific and polyclonal influenza virus-specific memory CD4 T cells direct rapid lung viral clearance and protect from lethality via secondary antiviral responses in the absence of CD8 T cells, B cells, or any lymphocytes. Unlike primary responses to influenza virus, which can mediate protection independent of gamma interferon (IFN-γ), memory CD4 T-cell-mediated protection in the lung is dependent on secreted IFN-γ and is associated with localized interactions with lung airways and foci of T-cell-directed responses. Our findings reveal that memory CD4 T cells drive antiviral protection in the lung through a qualitatively distinct mechanism and have important implications for exploiting the protective role of persisting memory CD4 T cells in vaccines and immunotherapies.     

CD73 Is Dispensable for the Regulation of Inflationary CD8+ T-Cells after Murine Cytomegalovirus Infection and Adenovirus Immunisation

The ecto-5''-nucleotidase (CD73) is expressed by T-cell subsets, myeloid derived suppressive cells and endothelial cells. It works in conjunction with CD39 to regulate the formation and degradation of adenosine in vivo. Adenosine has previously been shown to suppress the proliferation and cytokine secretion of T-cells and recent evidence suggests that inhibition of CD73 has the potential to enhance T-cell directed therapies. Here we utilised a CD73 knockout mouse model to assess the suppressive ability of CD73 on CD8⁺ T-cell classical memory and memory “inflation”, induced by murine cytomegalovirus (MCMV) infection and adenovirus immunisation. We show that CD73 is dispensable for normal CD8⁺ T-cell differentiation and function in both models. Thus CD73 as a suppressor of CD8⁺ T-cells is unlikely to play a deterministic role in the generation and functional characteristics of antiviral memory in these settings.     

Priming of CD8+ T Cells during Central Nervous System Infection with a Murine Coronavirus Is Strain Dependent

Virus-specific CD8⁺ T cells are critical for protection against neurotropic coronaviruses; however, central nervous system (CNS) infection with the recombinant JHM (RJHM) strain of mouse hepatitis virus (MHV) elicits a weak CD8⁺ T-cell response in the brain and causes lethal encephalomyelitis. An adoptive transfer model was used to elucidate the kinetics of CD8⁺ T-cell priming during CNS infection with RJHM as well as with two MHV strains that induce a robust CD8⁺ T-cell response (RA59 and SJHM/RA59, a recombinant A59 virus expressing the JHM spike). While RA59 and SJHM/RA59 infections resulted in CD8⁺ T-cell priming within the first 2 days postinfection, RJHM infection did not lead to proliferation of naïve CD8⁺ T cells. While all three viruses replicated efficiently in the brain, only RA59 and SJHM/RA59 replicated to appreciable levels in the cervical lymph nodes (CLN), the site of T-cell priming during acute CNS infection. RJHM was unable to suppress the CD8⁺ T-cell response elicited by RA59 in mice simultaneously infected with both strains, suggesting that RJHM does not cause generalized immunosuppression. RJHM was also unable to elicit a secondary CD8⁺ T-cell response in the brain following peripheral immunization against a viral epitope. Notably, the weak CD8⁺ T-cell response elicited by RJHM was unique to CNS infection, since peripheral inoculation induced a robust CD8⁺ T-cell response in the spleen. These findings suggest that the failure of RJHM to prime a robust CD8⁺ T-cell response during CNS infection is likely due to its failure to replicate in the CLN.Members of the family Coronaviridae infect a wide range of mammalian species, including humans, and induce mild to severe disease of the respiratory tract, gastrointestinal tract, and central nervous system (CNS). Mouse hepatitis virus (MHV) infection provides a useful model for the study of acute and chronic CNS disease and specifically the process of demyelination, the hallmark of the human disease multiple sclerosis. Different strains of MHV induce disease with various degrees of severity. For example, CNS infection with the recombinant wild-type A59 (RA59) strain causes acute encephalitis during the first week of infection; a strong CD8⁺ T-cell response is observed in the brain, coinciding with viral clearance. However, despite clearance of infectious RA59 virus, demyelination develops, peaking at approximately 4 weeks postinfection (p.i.) (17, 20). In contrast, infection with the recombinant wild-type JHM (RJHM) strain (derived from the JHM isolate referred to as MHV-4 or JHM.SD [7, 28]) causes severe encephalomyelitis; the virus is not cleared, and mice typically succumb to disease by the end of the first week of infection. Furthermore, RJHM infection of the CNS elicits a very weak virus-specific CD8⁺ T-cell response in the brain (7, 20, 34). However, we have examined only the most virulent strain of JHM. It should be noted that there are other strains of JHM that have deletions and mutations within the spike glycoprotein, rendering them less virulent and sometimes resulting in a change in cell tropism. The ability of neurotropic strains of MHV to replicate within cells of the CNS and cause disease of various degrees is ideal for allowing the dissection of both viral and host determinants of neuropathogenesis.The spike glycoprotein of MHV is a major determinant of neurovirulence (32). It controls virus tropism and spread as it both binds the cellular receptor and induces fusion with target cells. In addition, it encodes neutralizing antibody epitopes and the H-2^b-restricted CD8⁺ T-cell epitopes recognized in C57BL/6 (B6) mice. The A59 spike differs from the JHM spike in that it contains a deletion of 52 amino acids within the hypervariable region. The hypervariable region has been well documented to tolerate mutation, but with attenuating effects on virulence (5, 7). RA59 and RJHM both encode an H-2K^b epitope at positions S598 to S605 (S598); however, due to the deletion, the A59 spike lacks the immunodominant H-2D^b epitope at positions S510 to S519 (S510). We previously selected isogenic recombinant viruses expressing the JHM spike in which all other genes are derived from the A59 strain of MHV (SJHM/RA59). The isogenic SJHM/RA59 virus has a 50% lethal dose (LD₅₀) similar to that of RJHM, demonstrating that the JHM spike is sufficient to generate a highly neurovirulent phenotype and an increased ability to spread within the CNS (32, 33). However, SJHM/RA59-infected mice exhibit slower kinetics of death than RJHM-infected mice, and notably, unlike RJHM, the chimeric SJHM/RA59 virus induces a strong CD8⁺ T-cell response in the brain (14, 34).In addition to the spike, there is increasing evidence that other viral genes play important roles in pathogenesis. We (14, 21) and others (34, 35) have noted that the low CD8⁺ T-cell response observed during RJHM infection is not dependent on the spike, since the SJHM/RA59 recombinant induces a robust virus-specific CD8⁺ T-cell response. The difference between the CD8⁺ T-cell responses elicited by SJHM/RA59 and RJHM may explain why SJHM/RA59 kills mice more slowly than RJHM. Furthermore, the reverse chimeric recombinant virus expressing the A59 spike in the JHM background (SA59/RJHM) is unable to replicate in the liver despite the fact that it expresses the spike from the hepatotropic RA59 strain (27), suggesting that background genes play a significant role in viral tropism.It is well established that virus-specific CD8⁺ T cells play a protective role against MHV and are essential for clearance of infectious virus from the CNS (6, 20, 40, 41). The effector mechanisms exerted by activated, virus-specific CD8⁺ T cells include the ability to secrete cytokines and the ability to lyse target cells. Gamma interferon (IFN-γ) expression is essential for clearance of MHV from the brain (3, 22, 29), and perforin-mediated lysis of infected cells also appears to play a role in viral clearance (6, 31). In contrast to infection with RA59 or the relatively neuroattenuated glial-cell-tropic strains of JHM, CNS infection with the highly neurovirulent RJHM strain results in very low levels of activated, virus-specific CD8⁺ T cells in the spleen and brain (14, 34). Furthermore, RJHM infection induces a different profile of cytokines and chemokines in the brains of infected mice than infection with RA59 (34, 35, 38). One dramatic difference is that RA59 infection results in a robust IFN-γ response whereas RJHM infection results in higher, sustained levels of IFN-β (34). These observations prompted us to address the following questions. (i) Does RJHM elicit a CD8⁺ T-cell response in the brain following intranasal (i.n.) inoculation, a route that requires more virus and results in slower infection than intracranial (i.c.) inoculation? (ii) What are the kinetics of CD8⁺ T-cell priming during CNS infections with RA59, SJHM/RA59, and RJHM? (iii) Is CNS infection with RJHM generally immunosuppressive? (iv) Do RA59, SJHM/RA59, and RJHM replicate efficiently in the draining cervical lymph nodes (CLN)? (v) Can RJHM elicit a secondary CD8⁺ T-cell response in the brain following peripheral immunization against a viral epitope? (vi) Is the low CD8⁺ T-cell response elicited during RJHM infection an inherent characteristic of the viral strain or specific to RJHM infection of the CNS? Our results suggest that RJHM fails to prime a CD8⁺ T-cell response specifically during infection of the CNS without causing generalized immunosuppression and that this lack of priming correlates with a low level of RJHM replication in the draining CLN, the site of CD8⁺ T-cell priming during acute CNS infection.     

Influenza-Infected Neutrophils within the Infected Lungs Act as Antigen Presenting Cells for Anti-Viral CD8+ T Cells

Influenza A virus (IAV) is a leading cause of respiratory tract disease worldwide. Anti-viral CD8⁺ T lymphocytes responding to IAV infection are believed to eliminate virally infected cells by direct cytolysis but may also contribute to pulmonary inflammation and tissue damage via the release of pro-inflammatory mediators following recognition of viral antigen displaying cells. We have previously demonstrated that IAV antigen expressing inflammatory cells of hematopoietic origin within the infected lung interstitium serve as antigen presenting cells (APC) for infiltrating effector CD8⁺ T lymphocytes; however, the spectrum of inflammatory cell types capable of serving as APC was not determined. Here, we demonstrate that viral antigen displaying neutrophils infiltrating the IAV infected lungs are an important cell type capable of acting as APC for effector CD8⁺ T lymphocytes in the infected lungs and that neutrophils expressing viral antigen as a result of direct infection by IAV exhibit the most potent APC activity. Our findings suggest that in addition to their suggested role in induction of the innate immune responses to IAV, virus clearance, and the development of pulmonary injury, neutrophils can serve as APCs to anti-viral effector CD8⁺ T cells within the infected lung interstitium.     

CD8+ T Cells Restrict Yersinia pseudotuberculosis Infection: Bypass of Anti-Phagocytosis by Targeting Antigen-Presenting Cells

All Yersinia species target and bind to phagocytic cells, but uptake and destruction of bacteria are prevented by injection of anti-phagocytic Yop proteins into the host cell. Here we provide evidence that CD8⁺ T cells, which canonically eliminate intracellular pathogens, are important for restricting Yersinia, even though bacteria are primarily found in an extracellular locale during the course of disease. In a model of infection with attenuated Y. pseudotuberculosis, mice deficient for CD8⁺ T cells were more susceptible to infection than immunocompetent mice. Although exposure to attenuated Y. pseudotuberculosis generated T_H1-type antibody responses and conferred protection against challenge with fully virulent bacteria, depletion of CD8⁺ T cells during challenge severely compromised protective immunity. Strikingly, mice lacking the T cell effector molecule perforin also succumbed to Y. pseudotuberculosis infection. Given that the function of perforin is to kill antigen-presenting cells, we reasoned that cell death marks bacteria-associated host cells for internalization by neighboring phagocytes, thus allowing ingestion and clearance of the attached bacteria. Supportive of this model, cytolytic T cell killing of Y. pseudotuberculosis–associated host cells results in engulfment by neighboring phagocytes of both bacteria and target cells, bypassing anti-phagocytosis. Our findings are consistent with a novel function for cell-mediated immune responses protecting against extracellular pathogens like Yersinia: perforin and CD8⁺ T cells are critical for hosts to overcome the anti-phagocytic action of Yops.     

iNKT Cells Suppress the CD8(+) T Cell Response to a Murine Burkitt's-Like B Cell Lymphoma

The T cell response to B cell lymphomas differs from the majority of solid tumors in that the malignant cells themselves are derived from B lymphocytes, key players in immune response. B cell lymphomas are therefore well situated to manipulate their surrounding microenvironment to enhance tumor growth and minimize anti-tumor T cell responses. We analyzed the effect of T cells on the growth of a transplantable B cell lymphoma and found that iNKT cells suppressed the anti-tumor CD8(+) T cell response. Lymphoma cells transplanted into syngeneic wild type (WT) mice or Jalpha18(-/-) mice that specifically lack iNKT cells grew initially at the same rate, but only the mice lacking iNKT cells were able to reject the lymphoma. This effect was due to the enhanced activity of tumor-specific CD8(+) T cells in the absence of iNKT cells, and could be partially reversed by reconstitution of iNKT cells in Jalpha 18(-/-) mice. Treatment of tumor-bearing WT mice with alpha -galactosyl ceramide, an activating ligand for iNKT cells, reduced the number of tumor-specific CD8(+) T cells. In contrast, lymphoma growth in CD1d1(-/-) mice that lack both iNKT and type II NKT cells was similar to that in WT mice, suggesting that type II NKT cells are required for full activation of the anti-tumor immune response. This study reveals a tumor-promoting role for iNKT cells and suggests their capacity to inhibit the CD8(+) T cell response to B cell lymphoma by opposing the effects of type II NKT cells.     

Host Defense and Recruitment of Foxp3+ T Regulatory Cells to the Lungs in Chronic Mycobacterium tuberculosis Infection Requires Toll-like Receptor 2

Acute resistance to low dose M. tuberculosis (Mtb) infection is not dependent on Toll-like receptor (TLR) 2. However, whether TLR2 contributes to resistance in chronic Mtb infection has remained uncertain. Here we report that, following low dose aerosol infection with Mtb, mice lacking TLR2 (TLR2KO), in comparison with wild type (WT) mice, exhibit enhanced cellular infiltration and inflammation in the lungs, and fail to stably control bacterial burden during chronic infection. IFNγ and IL-17 was expressed at equivalent levels in the two groups; however, the characteristic accumulation of Foxp3⁺ T regulatory cells (Tregs) in pulmonary granulomas was significantly reduced in TLR2KO mice. Nonetheless, this reduction in Tregs was independent of whether Tregs expressed TLR2 or not. To directly link the reduced number of Tregs to the increased inflammation present in the TLR2KO mice, we used a macrophage adoptive transfer model. At seven weeks post-Mtb infection, TLR2KO mice, which were adoptively transferred with WT macrophages, displayed enhanced accumulation of Tregs in the lungs and a concomitant reduction in inflammation in contrast with control mice that received TLR2KO macrophages. However, the pulmonary bacterial burden between the two groups remained similar indicating that TLR2''s role in modulating immunopathology is functionally distinct from its role in restricting Mtb growth in chronic infection. Together, these findings unequivocally demonstrate that TLR2 contributes to host resistance against chronic Mtb infection and reveal a novel role for TLR2 in mediating the recruitment of Foxp3⁺ Tregs to the lungs to control inflammation.