Prevention of respiratory virus transmission by resident memory CD8+ T cells.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies1. One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections2-6. However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8+ T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8+ T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8+ T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population.

Persistent Antigen Harbored by Alveolar Macrophages Enhances the Maintenance of Lung-Resident Memory CD8+ T Cells.

Lung tissue-resident memory T cells are crucial mediators of cellular immunity against respiratory viruses; however, their gradual decline hinders the development of T cell-based vaccines against respiratory pathogens. Recently, studies using adenovirus (Ad)-based vaccine vectors have shown that the number of protective lung-resident CD8+ TRMs can be maintained long term. In this article, we show that immunization of mice with a replication-deficient Ad serotype 5 expressing influenza (A/Puerto Rico/8/34) nucleoprotein (AdNP) generates a long-lived lung TRM pool that is transcriptionally indistinct from those generated during a primary influenza infection. In addition, we demonstrate that CD4+ T cells contribute to the long-term maintenance of AdNP-induced CD8+ TRMs. Using a lineage tracing approach, we identify alveolar macrophages as a cell source of persistent NP Ag after immunization with AdNP. Importantly, depletion of alveolar macrophages after AdNP immunization resulted in significantly reduced numbers of NP-specific CD8+ TRMs in the lungs and airways. Combined, our results provide further insight to the mechanisms governing the enhanced longevity of Ag-specific CD8+ lung TRMs observed after immunization with recombinant Ad.

Broadening CD4+ and CD8+ T Cell Responses against Hepatitis C Virus by Vaccination with NS3 Overlapping Peptide Panels in Cross-Priming Liposomes.

Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4+ and CD8+ T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4+ T cells, whereas the NS3 pepmix induced a far more vigorous CD4+ T cell response and was as potent a CD8+ T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-γ+) tumor necrosis factor alpha-positive (TNF-α+) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity.IMPORTANCE With at least 700,000 annual deaths, development of a vaccine against hepatitis C virus (HCV) has high priority, but the tremendous ability of the virus to dodge the human immune system poses great challenges. Furthermore, many chronic infections, including HCV infection, have a remarkable ability to drive initially strong CD4+ and CD8+ T cell responses against dominant epitopes toward an exhausted, dysfunctional state. Thus, new and innovative vaccine approaches to control HCV should be evaluated. Here, we report on a novel peptide-based nanoparticle vaccine strategy (NS3 pepmix) aimed at generating T cell immunity against potential subdominant T cell epitopes that are not efficiently targeted by vaccination with full-length recombinant protein (rNS3) or infection with HCV. As proof of concept, we found that NS3 pepmix excels in broadening the repertoire of epitope-specific, multifunctional, and cytotoxic CD4+ and CD8+ T cells compared to vaccination with rNS3, which generated only CD4+ T cell responses.

Vaccine Targeting of Subdominant CD8+ T Cell Epitopes Increases the Breadth of the T Cell Response upon Viral Challenge, but May Impair Immediate Virus Control.

As a result of the difficulties in making efficient vaccines against genetically unstable viruses such as HIV, it has been suggested that future vaccines should preferentially target subdominant epitopes, the idea being that this should allow a greater breadth of the induced T cell response and, hence, a greater efficiency in controlling escape variants. However, to our knowledge the evidence supporting this concept is limited at best. To improve upon this, we used the murine lymphocytic choriomeningitis virus model and adenoviral vectors to compare a vaccine expressing unmodified Ag to a vaccine expressing the same Ag without its immunodominant epitope. We found that removal of the dominant epitope allowed the induction of CD8(+) T cell responses targeting at least two otherwise subdominant epitopes. Importantly, the overall magnitude of the induced T cell responses was similar, allowing us to directly compare the efficiency of these vaccines. Doing this, we observed that mice vaccinated with the vaccine expressing unmodified Ag more efficiently controlled an acute viral challenge. In the course of a more chronic viral infection, mice vaccinated using the vaccine targeting subdominant epitopes caught up with the conventionally vaccinated mice, and analysis of the breadth of the CD8(+) T cell response revealed that this was notably greater in the former mice. However, under the conditions of our studies, we never saw any functional advantage of this. This may represent a limitation of our model, but clearly our findings underscore the importance of carefully weighing the pros and cons of changes in epitope targeting before any implementation.

Adaptive Immunity against Streptococcus pyogenes in Adults Involves Increased IFN-γ and IgG3 Responses Compared with Children.

Each year, millions of people are infected with Streptococcus pyogenes, leading to an estimated 500,000 annual deaths worldwide. For unknown reasons, school-aged children have substantially higher infection rates than adults. The goal for this study was to provide, to our knowledge, the first detailed characterization of the human adaptive immune response against S. pyogenes in both children and adults. We report that all adults in our study, as well as most children, showed immunity against the two conserved group A streptococci (GAS) Ags, streptococcal C5a peptidase and immunogenic secreted protein. The response primarily consisted of three subsets of Th1 T cells, in which the TNF-α(+) and IL-2(+)TNF-α(+) subsets were most frequent. Humoral immunity was dominated by IgG1 and IgG3, whereas the Th2-associated IgG4 isotype was only detected at very low amounts. IgG3 levels correlated significantly with IFN-γ, but not with IL-5, IL-13, IL-17, or TNF-α. Interestingly, children showed a similar pattern of Ag-specific cytokine release, but displayed significantly lower levels of IgG3 and IFN-γ compared with adults. Thus, human immune responses against S. pyogenes consist of a robust Th1 cellular memory response in combination with IgG1/IgG3-dominated humoral immunity that increase with age. The significance of these data regarding both the increased GAS infection rate in children and the development of protective GAS vaccines is discussed.

CD8+ T cells complement antibodies in protecting against yellow fever virus.

The attenuated yellow fever (YF) vaccine (YF-17D) was developed in the 1930s, yet little is known about the protective mechanisms underlying its efficiency. In this study, we analyzed the relative contribution of cell-mediated and humoral immunity to the vaccine-induced protection in a murine model of YF-17D infection. Using different strains of knockout mice, we found that CD4(+) T cells, B cells, and Abs are required for full clinical protection of vaccinated mice, whereas CD8(+) T cells are dispensable for long-term survival after intracerebral challenge. However, by analyzing the immune response inside the infected CNS, we observed an accelerated T cell influx into the brain after intracerebral challenge of vaccinated mice, and this T cell recruitment correlated with improved virus control in the brain. Using mice deficient in B cells we found that, in the absence of Abs, YF vaccination can still induce some antiviral protection, and in vivo depletion of CD8(+) T cells from these animals revealed a pivotal role for CD8(+) T cells in controlling virus replication in the absence of a humoral response. Finally, we demonstrated that effector CD8(+) T cells also contribute to viral control in the presence of circulating YF-specific Abs. To our knowledge, this is the first time that YF-specific CD8(+) T cells have been demonstrated to possess antiviral activity in vivo.

Detection of local inflammation induced by repeated exposure to contact allergens by use of IVIS SpectrumCT analyses.

BACKGROUND: Contact allergy is characterized by local skin inflammation that, in some cases, can result in systemic immune activation. OBJECTIVES: To investigate whether IVIS SpectrumCT analyses can be used to detect the immune response induced by contact allergens. METHODS: Mice were repeatedly exposed to vehicle or allergens on the ears. The local and systemic responses were analysed at different times with the ProSense 750 FAST probe in IVIS SpectrumCT measurements. In addition, changes in ear thickness, cytokine profile in the skin and immunological phenotype in the draining lymph nodes and spleen were determined. RESULTS: Local inflammation was detected by ProSense 750 FAST and correlated with changes in ear thickness, cytokine profile and immunological phenotype following exposure to the strong contact allergen 2,4-dinitrofluorobenzene. Analysis of the systemic response with ProSense 750 FAST did not show any difference between allergen-exposed and control mice, although fluorescence-activated cell sorting analysis of the spleen showed increased numbers of γδ T cells and CD11b+ CD11c+ MHCII+ cells in allergen-treated mice. CONCLUSIONS: IVIS SpectrumCT analyses with ProSense 750 FAST as the probe can be used to detect local immune responses induced by contact allergens.

Priming of CD8 T cells by adenoviral vectors is critically dependent on B7 and dendritic cells but only partially dependent on CD28 ligation on CD8 T cells.

Adenoviral vectors have long been forerunners in the development of effective CD8 T cell-based vaccines; therefore, it is imperative that we understand the factors controlling the induction of robust and long-lasting transgene-specific immune responses by these vectors. In this study, we investigated the organ sites, molecules, and cell subsets that play a critical role in the priming of transgene-specific CD8 T cells after vaccination with a replication-deficient adenoviral vector. Using a human adenovirus serotype 5 (Ad5) vector and genetically engineered mice, we found that CD8(+) and/or CD103(+) dendritic cells in the draining lymph node played a critical role in the priming of Ad5-induced CD8 T cell responses. Moreover, we found that CD80/86, but not CD28, was essential for efficient generation of both primary effectors and memory CD8 T cells. Interestingly, the lack of CD28 expression resulted in a delayed primary response, whereas memory CD8 T cells generated in CD28-deficient mice appeared almost normal in terms of both phenotype and effector cytokine profile, but they exhibited a significantly reduced proliferative capacity upon secondary challenge while retaining immediate in vivo effector capabilities: in vivo cytotoxicity and short-term in vivo protective capacity. Overall, our data point to an absolute requirement for professional APCs and the expression of the costimulatory molecules CD80/86 for efficient CD8 T cell priming by adenoviral vectors. Additionally, our results suggest the existence of an alternative receptor for CD80/86, which may substitute, in part, for CD28.

Protein energy malnutrition during vaccination has limited influence on vaccine efficacy but abolishes immunity if administered during Mycobacterium tuberculosis infection.

Protein energy malnutrition (PEM) increases susceptibility to infectious diseases, including tuberculosis (TB), but it is not clear how PEM influences vaccine-promoted immunity to TB. We demonstrate that PEM during low-level steady-state TB infection in a mouse model results in rapid relapse of Mycobacterium tuberculosis, as well as increased pathology, in both Mycobacterium bovis BCG-vaccinated and unvaccinated animals. PEM did not change the overall numbers of CD4 T cells in BCG-vaccinated animals but resulted in an almost complete loss of antigen-specific cytokine production. Furthermore, there was a change in cytokine expression characterized by a gradual loss of multifunctional antigen-specific CD4 T cells and an increased proportion of effector cells expressing gamma interferon and tumor necrosis factor alpha (IFN-γ(+) TNF-α(+) and IFN-γ(+) cells). PEM during M. tuberculosis infection completely blocked the protection afforded by the H56-CAF01 subunit vaccine, and this was associated with a very substantial loss of the interleukin-2-positive memory CD4 T cells promoted by this vaccine. Similarly, PEM during the vaccination phase markedly reduced the H56-CAF01 vaccine response, influencing all cytokine-producing CD4 T cell subsets, with the exception of CD4 T cells positive for TNF-α only. Importantly, this impairment was reversible and resupplementation of protein during infection rescued both the vaccine-promoted T cell response and the protective effect of the vaccine against M. tuberculosis infection.

Comparison of vaccine-induced effector CD8 T cell responses directed against self- and non-self-tumor antigens: implications for cancer immunotherapy.

It is generally accepted that CD8 T cells play a major role in tumor control, yet vaccination aimed at eliciting potent CD8 T cell responses are rarely efficient in clinical trials. To try and understand why this is so, we have generated potent adenoviral vectors encoding the endogenous tumor Ags (TA) tyrosinase-related protein-2 (TRP-2) and glycoprotein 100 (GP100) tethered to the invariant chain (Ii). Using these vectors, we sought to characterize the self-TA-specific CD8 T cell response and compare it to that induced against non-self-Ags expressed from a similar vector platform. Prophylactic vaccination with adenoviral vectors expressing either TRP-2 (Ad-Ii-TRP-2) or GP100 (Ad-Ii-GP100) had little or no effect on the growth of s.c. B16 melanomas, and only Ad-Ii-TRP-2 was able to induce a marginal reduction of B16 lung metastasis. In contrast, vaccination with a similar vector construct expressing a foreign (viral) TA induced efficient tumor control. Analyzing the self-TA-specific CD8 T cells, we observed that these could be activated to produce IFN-γ and TNF-α. In addition, surface expression of phenotypic markers and inhibitory receptors, as well as in vivo cytotoxicity and degranulation capacity matched that of non-self-Ag-specific CD8 T cells. However, the CD8 T cells specific for self-TAs had a lower functional avidity, and this impacted on their in vivo performance. On the basis of these results and a low expression of the targeted TA epitopes on the tumor cells, we suggest that low avidity of the self-TA-specific CD8 T cells may represent a major obstacle for efficient immunotherapy of cancer.

Molecular consequences of peripheral Influenza A infection on cell populations in the murine hypothalamus.

Infection with Influenza A virus (IAV) causes the well-known symptoms of the flu, including fever, loss of appetite, and excessive sleepiness. These responses, mediated by the brain, will normally disappear once the virus is cleared from the system, but a severe respiratory virus infection may cause long-lasting neurological disturbances. These include encephalitis lethargica and narcolepsy. The mechanisms behind such long lasting changes are unknown. The hypothalamus is a central regulator of the homeostatic response during a viral challenge. To gain insight into the neuronal and non-neuronal molecular changes during an IAV infection, we intranasally infected mice with an H1N1 virus and extracted the brain at different time points. Using single-nucleus RNA sequencing (snRNA-seq) of the hypothalamus, we identify transcriptional effects in all identified cell populations. The snRNA-seq data showed the most pronounced transcriptional response at 3 days past infection, with a strong downregulation of genes across all cell types. General immune processes were mainly impacted in microglia, the brain resident immune cells, where we found increased numbers of cells expressing pro-inflammatory gene networks. In addition, we found that most neuronal cell populations downregulated genes contributing to the energy homeostasis in mitochondria and protein translation in the cytosol, indicating potential reduced cellular and neuronal activity. This might be a preventive mechanism in neuronal cells to avoid intracellular viral replication and attack by phagocytosing cells. The change of microglia gene activity suggest that this is complemented by a shift in microglia activity to provide increased surveillance of their surroundings.

When you are ill, your behaviour changes. You sleep more, eat less and are less likely to go out and be active. This behavioural change is called the 'sickness response' and is believed to help the immune system fight infection. An area of the brain called the hypothalamus helps to regulate sleep and appetite. Previous research has shown that when humans are ill, the immune system sends signals to the hypothalamus, likely initiating the sickness response. However, it was not clear which brain cells in the hypothalamus are involved in the response and how long after infection the brain returns to its normal state. To better understand the sickness response, Lemcke et al. infected mice with influenza then extracted and analysed brain tissue at different timepoints. The experiments showed that the major changes to gene expression in the hypothalamus early during an influenza infection are not happening in neurons ­ the cells in the brain that transmit electrical signals and usually control behaviour. Instead, it is cells called glia ­ which provide support and immune protection to the neurons ­ that change during infection. The findings suggest that these cells prepare to protect the neurons from influenza should the virus enter the brain. Lemcke et al. also found that the brain takes a long time to go back to normal after an influenza infection. In infected mice, molecular changes in brain cells could be detected even after the influenza infection had been cleared from the respiratory system. In the future, these findings may help to explain why some people take longer than others to fully recover from viral infections such as influenza and aid development of medications that speed up recovery.

Bispecific T cell-engager targeting oncofetal chondroitin sulfate induces complete tumor regression and protective immune memory in mice.

BACKGROUND: The malaria protein VAR2CSA binds oncofetal chondroitin sulfate (ofCS), a unique chondroitin sulfate, expressed on almost all mammalian cancer cells. Previously, we produced a bispecific construct targeting ofCS and human T cells based on VAR2CSA and anti-CD3 (V-aCD3Hu). V-aCD3Hu showed efficacy against xenografted tumors in immunocompromised mice injected with human immune cells at the tumor site. However, the complex effects potentially exerted by the immune system as a result of the treatment cannot occur in mice without an immune system. Here we investigate the efficacy of V-aCD3Mu as a monotherapy and combined with immune checkpoint inhibitors in mice with a fully functional immune system. METHODS: We produced a bispecific construct consisting of a recombinant version of VAR2CSA coupled to an anti-murine CD3 single-chain variable fragment. Flow cytometry and ELISA were used to check cell binding capabilities and the therapeutic effect was evaluated in vitro in a killing assay. The in vivo efficacy of V-aCD3Mu was then investigated in mice with a functional immune system and established or primary syngeneic tumors in the immunologically "cold" 4T1 mammary carcinoma, B16-F10 malignant melanoma, the pancreatic KPC mouse model, and in the immunologically "hot" CT26 colon carcinoma model. RESULTS: V-aCD3Mu had efficacy as a monotherapy, and the combined treatment of V-aCD3Mu and an immune checkpoint inhibitor showed enhanced effects resulting in the complete elimination of solid tumors in the 4T1, B16-F10, and CT26 models. This anti-tumor effect was abscopal and accompanied by a systemic increase in memory and activated cytotoxic and helper T cells. The combined treatment also led to a higher percentage of memory T cells in the tumor without an increase in regulatory T cells. In addition, we observed partial protection against re-challenge in a melanoma model and full protection in a breast cancer model. CONCLUSIONS: Our findings suggest that V-aCD3Mu combined with an immune checkpoint inhibitor renders immunologically "cold" tumors "hot" and results in tumor elimination. Taken together, these data provide proof of concept for the further clinical development of V-aCD3 as a broad cancer therapy in combination with an immune checkpoint inhibitor.

The role of CD80/CD86 in generation and maintenance of functional virus-specific CD8+ T cells in mice infected with lymphocytic choriomeningitis virus.

Lymphocytic choriomeningitis virus (LCMV)-specific CD8(+) T cell responses are considered to be independent of CD28-B7 costimulation. However, the LCMV-specific response has never been evaluated in B7.1/B7.2(-/-) mice. For this reason, we decided to study the T cell response in B7.1/B7.2(-/-) mice infected with two different strains of LCMV, one (Traub strain) typically causing low-grade chronic infection, and another (Armstrong clone 53b) displaying very limited capacity for establishing chronic infection. Using Traub virus we found that most B7.1/B7.2(-/-) mice were unable to rid themselves of the infection. Chronic infection was associated with a perturbed CD8(+) T cell epitope hierarchy, as well as with the accumulation of cells expressing markers of terminal differentiation and being unable to respond optimally to Ag restimulation. Examination of matched CD28(-/-) mice revealed a similar albeit less pronounced pattern of CD8(+) T cell dysfunction despite lack of virus persistence. Finally, analysis of B7.1/B7.2(-/-) mice infected with Armstrong virus revealed a scenario quite similar to that in Traub infected CD28(-/-) mice; that is, the mice displayed evidence of T cell dysfunction, but no chronic infection. Taken together, these results indicate that B7 costimulation is required for induction and maintenance of LCMV-specific CD8(+) T cell memory, irrespective of the LCMV strain used for priming. However, the erosion of CD8(+) T cell memory in B7.1/B7.2(-/-) mice was more pronounced in association with chronic infection. Finally, virus-specific T cell memory was more impaired in the absence of B7 molecules than in the absence of the CD28 receptor, supporting earlier data suggesting the existence of additional stimulatory receptors for B7.

A Novel H-2d Epitope for Influenza A Polymerase Acidic Protein.

Understanding the complexity of the T-cell epitope hierarchy in humans through mouse models can be difficult. In particular, using only one murine strain, the C57BL/6 mouse, to investigate the immune response to influenza virus infection limits our understanding. In the present study, by immunizing C57BL/6 mice with an adenoviral vector encoding the polymerase acidic (AdIiPA) protein of influenza A virus, we were able to induce a high number of PA-specific T cells. However, upon challenge, these cells were only partly protective. When instead immunizing BALB/c mice with AdIiPA, we found that the immunized mice were fully protected against challenge. We found that this protection was dependent on CD8 T cells, and we identified a novel H-2Dd-restricted epitope, PA33. These findings provide a new tool for researchers to study PA-specific immunity in mice with an H-2d haplotype. Additionally, our findings underscore the importance of critically evaluating important limitations of using a single inbred mouse strain in vaccine studies.

Difference in TB10.4 T-cell epitope recognition following immunization with recombinant TB10.4, BCG or infection with Mycobacterium tuberculosis.

Most novel vaccines against infectious diseases are based on recombinant Ag; however, only few studies have compared Ag-specific immune responses induced by natural infection with that induced by the same Ag in a recombinant form. Here, we studied the epitope recognition pattern of the tuberculosis vaccine Ag, TB10.4, in a recombinant form, or when expressed by the pathogen Mycobacterium tuberculosis (M.tb), or by the current anti-tuberculosis vaccine, Mycobacterium bovis BCG. We showed that BCG and M.tb induced a similar CD4+ T-cell specific TB10.4 epitope-pattern, which differed completely from that induced by recombinant TB10.4. This difference was not due to post-translational modifications of TB10.4 or because TB10.4 is secreted from BCG and M.tb as a complex with Rv0287. In addition, BCG and TB10.4/CAF01 were both taken up by DC and macrophages in vivo, and in vitro uptake experiments revealed that both TB10.4 and BCG were transported to Lamp+-compartments. BCG and TB10.4 however, were directed to different types of Lamp+-compartments in the same APC, which may lead to different epitope recognition patterns. In conclusion, we show that different vectors can induce completely different recognition of the same protein.

Fulminant lymphocytic choriomeningitis virus-induced inflammation of the CNS involves a cytokine-chemokine-cytokine-chemokine cascade.

Intracerebral inoculation of immunocompetent mice with lymphocytic choriomeningitis virus (LCMV) normally results in fatal CD8+ T cell mediated meningoencephalitis. However, in CXCL10-deficient mice, the virus-induced CD8+ T cell accumulation in the neural parenchyma is impaired, and only 30-50% of the mice succumb to the infection. Similar results are obtained in mice deficient in the matching chemokine receptor, CXCR3. Together, these findings point to a key role for CXCL10 in regulating the severity of the LCMV-induced inflammatory process. For this reason, we now address the mechanisms regulating the expression of CXCL10 in the CNS of LCMV-infected mice. Using mice deficient in type I IFN receptor, type II IFN receptor, or type II IFN, as well as bone marrow chimeras expressing CXCL10 only in resident cells or only in bone marrow-derived cells, we analyzed the up-stream regulation as well as the cellular source of CXCL10. We found that expression of CXCL10 initially depends on signaling through the type I IFN receptor, while late expression and up-regulation requires type II IFN produced by the recruited CD8+ T cells. Throughout the infection, the producers of CXCL10 are exclusively resident cells of the CNS, and astrocytes are the dominant expressors in the neural parenchyma, not microglial cells or recruited bone marrow-derived cell types. These results are consistent with a model suggesting a bidirectional interplay between resident cells of the CNS and the recruited virus-specific T cells with astrocytes as active participants in the local antiviral host response.

CXCR3 directs antigen-specific effector CD4+ T cell migration to the lung during parainfluenza virus infection.

Effector T cells are a crucial component of the adaptive immune response to respiratory virus infections. Although it was previously reported that the chemokine receptors CCR5 and CXCR3 affect trafficking of respiratory virus-specific CD8+ T cells, it is unclear whether these receptors govern effector CD4+ T cell migration to the lungs. To assess the role of CCR5 and CXCR3 in vivo, we directly compared the migration of Ag-specific wild-type and chemokine receptor-deficient effector T cells in mixed bone marrow chimeric mice during a parainfluenza virus infection. CXCR3-deficient effector CD4+ T cells were 5- to 10-fold less efficient at migrating to the lung compared with wild-type cells, whereas CCR5-deficient effector T cells were not impaired in their migration to the lung. In contrast to its role in trafficking, CXCR3 had no impact on effector CD4+ T cell proliferation, phenotype, or function in any of the tissues examined. These findings demonstrate that CXCR3 controls virus-specific effector CD4+ T cell migration in vivo, and suggest that blocking CXCR3-mediated recruitment may limit T cell-induced immunopathology during respiratory virus infections.

Long-term maintenance of lung resident memory T cells is mediated by persistent antigen.

Tissue-resident memory T cells (TRM) in the lungs are pivotal for protection against repeated infection with respiratory viruses. However, the gradual loss of these cells over time and the associated decline in clinical protection represent a serious limit in the development of efficient T cell based vaccines against respiratory pathogens. Here, using an adenovirus expressing influenza nucleoprotein (AdNP), we show that CD8 TRM in the lungs can be maintained for at least 1 year post vaccination. Our results reveal that lung TRM continued to proliferate in situ 8 months after AdNP vaccination. Importantly, this required airway vaccination and antigen persistence in the lung, as non-respiratory routes of vaccination failed to support long-term lung TRM maintenance. In addition, parabiosis experiments show that in AdNP vaccinated mice, the lung TRM pool is also sustained by continual replenishment from circulating memory CD8 T cells that differentiate into lung TRM, a phenomenon not observed in influenza-infected parabiont partners. Concluding, these results demonstrate key requirements for long-lived cellular immunity to influenza virus, knowledge that could be utilized in future vaccine design.

Vaccination with an adenoviral vector encoding the tumor antigen directly linked to invariant chain induces potent CD4(+) T-cell-independent CD8(+) T-cell-mediated tumor control.

Antigen-specific immunotherapy is an attractive strategy for cancer control. In the context of antiviral vaccines, adenoviral vectors have emerged as a favorable means for immunization. Therefore, we chose a strategy combining use of these vectors with another successful approach, namely linkage of the vaccine antigen to invariant chain (Ii). To evaluate this strategy we used a mouse model, in which an immunodominant epitope (GP33) of the LCMV glycoprotein (GP) represents the tumor-associated neoantigen. Prophylactic vaccination of C57BL/6 mice with a replication-deficient human adenovirus 5 vector encoding GP linked to Ii (Ad-Ii-GP) resulted in complete protection against GP33-expressing B16.F10 tumors. Therapeutic vaccination with Ad-Ii-GP delayed tumor growth by more than 2 wk compared with sham vaccination. Notably, therapeutic vaccination with the linked vaccine was significantly better than vaccination with adenovirus expressing GP alone (Ad-GP), or GP and Ii unlinked (Ad-GP+Ii). Ad-Ii-GP- induced tumor control depended on an improved generation of the tumor-associated neoantigen-specific CD8(+) T-cell response and was independent of CD4(+) T cells. IFN-gamma was shown to be a key player during the tumor degradation. Finally, Ad-Ii-GP but not Ad-GP vaccination can break the immunological non-reactivity in GP transgenic mice indicating that our vaccine strategy will prove efficient also against endogenous tumor antigens.

Delayed contraction of the CD8+ T cell response toward lymphocytic choriomeningitis virus infection in mice lacking serglycin.

We previously reported that the lack of serglycin proteoglycan affects secretory granule morphology and granzyme B (GrB) storage in in vitro generated CTLs. In this study, the role of serglycin during viral infection was studied by infecting wild-type (wt) mice and serglycin-deficient (SG(-/-)) mice with lymphocytic choriomeningitis virus (LCMV). Wt and SG(-/-) mice cleared 10(3) PFU of highly invasive LCMV with the same kinetics, and the CD8(+) T lymphocytes from wt and SG(-/-) animals did not differ in GrB, perforin, IFN-gamma, or TNF-alpha content. However, when a less invasive LCMV strain was used, SG(-/-) GrB(+) CD8(+) T cells contained approximately 30% less GrB than wt GrB(+) CD8(+) T cells. Interestingly, the contraction of the antiviral CD8(+) T cell response to highly invasive LCMV was markedly delayed in SG(-/-) mice, and a delayed contraction of the virus-specific CD8(+) T cell response was also seen after infection with vesicular stomatitis virus. BrdU labeling of cells in vivo revealed that the delayed contraction was associated with sustained proliferation of Ag-specific CD8(+) T cells in SG(-/-) mice. Moreover, wt LCMV-specific CD8(+) T cells from TCR318 transgenic mice expanded much more extensively in virus-infected SG(-/-) mice than in matched wt mice, indicating that the delayed contraction represents a T cell extrinsic phenomenon. In summary, the present report points to a novel, previously unrecognized role for serglycin proteoglycan in regulating the kinetics of antiviral CD8(+) T cell responses.