Microfluidic Squeezing Enables MHC Class I Antigen Presentation by Diverse Immune Cells to Elicit CD8+ T Cell Responses with Antitumor Activity.

CD8+ T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8+ T cell responses, therapeutic approaches to generate Ag-specific CD8+ T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8+ T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ∼1000-fold more potent at eliciting CD8+ T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8+ T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8+ T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8+ T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8+ T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients.

Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis.

Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

IL-21 signaling is essential for optimal host resistance against Mycobacterium tuberculosis infection.

IL-21 is produced predominantly by activated CD4+ T cells and has pleiotropic effects on immunity via the IL-21 receptor (IL-21R), a member of the common gamma chain (γc) cytokine receptor family. We show that IL-21 signaling plays a crucial role in T cell responses during Mycobacterium tuberculosis infection by augmenting CD8+ T cell priming, promoting T cell accumulation in the lungs, and enhancing T cell cytokine production. In the absence of IL-21 signaling, more CD4+ and CD8+ T cells in chronically infected mice express the T cell inhibitory molecules PD-1 and TIM-3. We correlate these immune alterations with increased susceptibility of IL-21R-/- mice, which have increased lung bacterial burden and earlier mortality compared to WT mice. Finally, to causally link the immune defects with host susceptibility, we use an adoptive transfer model to show that IL-21R-/- T cells transfer less protection than WT T cells. These results prove that IL-21 signaling has an intrinsic role in promoting the protective capacity of T cells. Thus, the net effect of IL-21 signaling is to enhance host resistance to M. tuberculosis. These data position IL-21 as a candidate biomarker of resistance to tuberculosis.

Inflammatory signaling in human tuberculosis granulomas is spatially organized.

Granulomas are the pathological hallmark of tuberculosis (TB). However, their function and mechanisms of formation remain poorly understood. To understand the role of granulomas in TB, we analyzed the proteomes of granulomas from subjects with tuberculosis in an unbiased manner. Using laser-capture microdissection, mass spectrometry and confocal microscopy, we generated detailed molecular maps of human granulomas. We found that the centers of granulomas have a pro-inflammatory environment that is characterized by the presence of antimicrobial peptides, reactive oxygen species and pro-inflammatory eicosanoids. Conversely, the tissue surrounding the caseum has a comparatively anti-inflammatory signature. These findings are consistent across a set of six human subjects and in rabbits. Although the balance between systemic pro- and anti-inflammatory signals is crucial to TB disease outcome, here we find that these signals are physically segregated within each granuloma. From the protein and lipid snapshots of human and rabbit lesions analyzed here, we hypothesize that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during the development of the granuloma.

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis.

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRß deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3ß sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

Multiple Inflammatory Cytokines Converge To Regulate CD8+ T Cell Expansion and Function during Tuberculosis.

The differentiation of effector CD8(+) T cells is a dynamically regulated process that varies during different infections and is influenced by the inflammatory milieu of the host. In this study, we define three signals regulating CD8(+) T cell responses during tuberculosis by focusing on cytokines known to affect disease outcome: IL-12, type I IFN, and IL-27. Using mixed bone marrow chimeras, we compared wild-type and cytokine receptor knockout CD8(+) T cells within the same mouse following aerosol infection with Mycobacterium tuberculosis. Four weeks postinfection, IL-12, type 1 IFN, and IL-27 were all required for efficient CD8(+) T cell expansion in the lungs. We next determined if these cytokines directly promote CD8(+) T cell priming or are required only for expansion in the lungs. Using retrogenic CD8(+) T cells specific for the M. tuberculosis Ag TB10.4 (EsxH), we observed that IL-12 is the dominant cytokine driving both CD8(+) T cell priming in the lymph node and expansion in the lungs; however, type I IFN and IL-27 have nonredundant roles supporting pulmonary CD8(+) T cell expansion. Thus, IL-12 is a major signal promoting priming in the lymph node, but a multitude of inflammatory signals converge in the lung to promote continued expansion. Furthermore, these cytokines regulate the differentiation and function of CD8(+) T cells during tuberculosis. These data demonstrate distinct and overlapping roles for each of the cytokines examined and underscore the complexity of CD8(+) T cell regulation during tuberculosis.

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection.

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

Orchestration of pulmonary T cell immunity during Mycobacterium tuberculosis infection: immunity interruptus.

Despite the introduction almost a century ago of Mycobacterium bovis BCG (BCG), an attenuated form of M. bovis that is used as a vaccine against Mycobacterium tuberculosis, tuberculosis remains a global health threat and kills more than 1.5 million people each year. This is mostly because BCG fails to prevent pulmonary disease--the contagious form of tuberculosis. Although there have been significant advances in understanding how the immune system responds to infection, the qualities that define protective immunity against M. tuberculosis remain poorly characterized. The ability to predict who will maintain control over the infection and who will succumb to clinical disease would revolutionize our approach to surveillance, control, and treatment. Here we review the current understanding of pulmonary T cell responses following M. tuberculosis infection. While infection elicits a strong immune response that contains infection, M. tuberculosis evades eradication. Traditionally, its intracellular lifestyle and alteration of macrophage function are viewed as the dominant mechanisms of evasion. Now we appreciate that chronic inflammation leads to T cell dysfunction. While this may arise as the host balances the goals of bacterial sterilization and avoidance of tissue damage, it is becoming clear that T cell dysfunction impairs host resistance. Defining the mechanisms that lead to T cell dysfunction is crucial as memory T cell responses are likely to be subject to the same subject to the same pressures. Thus, success of T cell based vaccines is predicated on memory T cells avoiding exhaustion while at the same time not promoting overt tissue damage.

In search of a new paradigm for protective immunity to TB.

Clinical trials of vaccines against Mycobacterium tuberculosis are well under way and results are starting to come in. Some of these results are not so encouraging, as exemplified by the latest Aeras-422 and MVA85A trials. Other than empirically determining whether a vaccine reduces the number of cases of active tuberculosis, which is a daunting prospect given the chronic nature of the disease, we have no way of assessing vaccine efficacy. Therefore, investigators seek to identify biomarkers that predict vaccine efficacy. Historically, focus has been on the production of interferon-γ by CD4(+) T cells, but this has not been a useful correlate of vaccine-induced protection. In this Opinion article, we discuss recent advances in our understanding of the immune control of M. tuberculosis and how this knowledge could be used for vaccine design and evaluation.

Microarray-based gene expression profiling in patients with cryopyrin-associated periodic syndromes defines a disease-related signature and IL-1-responsive transcripts.

OBJECTIVE: To analyse gene expression patterns and to define a specific gene expression signature in patients with the severe end of the spectrum of cryopyrin-associated periodic syndromes (CAPS). The molecular consequences of interleukin 1 inhibition were examined by comparing gene expression patterns in 16 CAPS patients before and after treatment with anakinra. METHODS: We collected peripheral blood mononuclear cells from 22 CAPS patients with active disease and from 14 healthy children. Transcripts that passed stringent filtering criteria (p values≤false discovery rate 1%) were considered as differentially expressed genes (DEG). A set of DEG was validated by quantitative reverse transcription PCR and functional studies with primary cells from CAPS patients and healthy controls. We used 17 CAPS and 66 non-CAPS patient samples to create a set of gene expression models that differentiates CAPS patients from controls and from patients with other autoinflammatory conditions. RESULTS: Many DEG include transcripts related to the regulation of innate and adaptive immune responses, oxidative stress, cell death, cell adhesion and motility. A set of gene expression-based models comprising the CAPS-specific gene expression signature correctly classified all 17 samples from an independent dataset. This classifier also correctly identified 15 of 16 post-anakinra CAPS samples despite the fact that these CAPS patients were in clinical remission. CONCLUSIONS: We identified a gene expression signature that clearly distinguished CAPS patients from controls. A number of DEG were in common with other systemic inflammatory diseases such as systemic onset juvenile idiopathic arthritis. The CAPS-specific gene expression classifiers also suggest incomplete suppression of inflammation at low doses of anakinra.

Efferocytosis is an innate antibacterial mechanism.

Mycobacterium tuberculosis persists within macrophages in an arrested phagosome and depends upon necrosis to elude immunity and disseminate. Although apoptosis of M. tuberculosis-infected macrophages is associated with reduced bacterial growth, the bacteria are relatively resistant to other forms of death, leaving the mechanism underlying this observation unresolved. We find that after apoptosis, M. tuberculosis-infected macrophages are rapidly taken up by uninfected macrophages through efferocytosis, a dedicated apoptotic cell engulfment process. Efferocytosis of M. tuberculosis sequestered within an apoptotic macrophage further compartmentalizes the bacterium and delivers it along with the apoptotic cell debris to the lysosomal compartment. M. tuberculosis is killed only after efferocytosis, indicating that apoptosis itself is not intrinsically bactericidal but requires subsequent phagocytic uptake and lysosomal fusion of the apoptotic body harboring the bacterium. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, these data indicate that it can also function as an antimicrobial effector mechanism.

Familial Mediterranean fever with a single MEFV mutation: where is the second hit?

OBJECTIVE: Familial Mediterranean fever (FMF) has traditionally been considered an autosomal-recessive disease; however, it has been observed that a substantial number of patients with clinical FMF possess only 1 demonstrable MEFV mutation. The purpose of this study was to perform an extensive search for a second MEFV mutation in 46 patients diagnosed clinically as having FMF and carrying only 1 high-penetrance FMF mutation. METHODS: MEFV and other candidate genes were sequenced by standard capillary electrophoresis. In 10 patients, the entire 15-kb MEFV genomic region was resequenced using hybridization-based chip technology. MEFV gene expression levels were determined by quantitative reverse transcription-polymerase chain reaction. Pyrin protein levels were examined by Western blotting. RESULTS: A second MEFV mutation was not identified in any of the patients who were screened. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between patients with a single mutation and those with a double mutation; however, FMF patients of both types showed higher protein expression as compared with controls and with non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare mutations in a small number of patients, suggesting the possibility of digenic inheritance. CONCLUSION: Our data underscore the existence of a significant subset of FMF patients who are carriers of only 1 MEFV mutation and demonstrate that complete MEFV sequencing is not likely to yield a second mutation. Screening for the set of the most common mutations and detection of a single mutation appears to be sufficient in the presence of clinical symptoms for the diagnosis of FMF and the initiation of a trial of colchicine.

STAT4 and the risk of rheumatoid arthritis and systemic lupus erythematosus.

BACKGROUND: Rheumatoid arthritis is a chronic inflammatory disease with a substantial genetic component. Susceptibility to disease has been linked with a region on chromosome 2q. METHODS: We tested single-nucleotide polymorphisms (SNPs) in and around 13 candidate genes within the previously linked chromosome 2q region for association with rheumatoid arthritis. We then performed fine mapping of the STAT1-STAT4 region in a total of 1620 case patients with established rheumatoid arthritis and 2635 controls, all from North America. Implicated SNPs were further tested in an independent case-control series of 1529 patients with early rheumatoid arthritis and 881 controls, all from Sweden, and in a total of 1039 case patients and 1248 controls from three series of patients with systemic lupus erythematosus. RESULTS: A SNP haplotype in the third intron of STAT4 was associated with susceptibility to both rheumatoid arthritis and systemic lupus erythematosus. The minor alleles of the haplotype-defining SNPs were present in 27% of chromosomes of patients with established rheumatoid arthritis, as compared with 22% of those of controls (for the SNP rs7574865, P=2.81x10(-7); odds ratio for having the risk allele in chromosomes of patients vs. those of controls, 1.32). The association was replicated in Swedish patients with recent-onset rheumatoid arthritis (P=0.02) and matched controls. The haplotype marked by rs7574865 was strongly associated with lupus, being present on 31% of chromosomes of case patients and 22% of those of controls (P=1.87x10(-9); odds ratio for having the risk allele in chromosomes of patients vs. those of controls, 1.55). Homozygosity of the risk allele, as compared with absence of the allele, was associated with a more than doubled risk for lupus and a 60% increased risk for rheumatoid arthritis. CONCLUSIONS: A haplotype of STAT4 is associated with increased risk for both rheumatoid arthritis and systemic lupus erythematosus, suggesting a shared pathway for these illnesses.