Functional anatomy of T cell activation and synapse formation.

T cell activation and function require a structured engagement of antigen-presenting cells. These cell contacts are characterized by two distinct dynamics in vivo: transient contacts resulting from promigratory junctions called immunological kinapses or prolonged contacts from stable junctions called immunological synapses. Kinapses operate in the steady state to allow referencing to self-peptide-MHC (pMHC) and searching for pathogen-derived pMHC. Synapses are induced by T cell receptor (TCR) interactions with agonist pMHC under specific conditions and correlate with robust immune responses that generate effector and memory T cells. High-resolution imaging has revealed that the synapse is highly coordinated, integrating cell adhesion, TCR recognition of pMHC complexes, and an array of activating and inhibitory ligands to promote or prevent T cell signaling. In this review, we examine the molecular components, geometry, and timing underlying kinapses and synapses. We integrate recent molecular and physiological data to provide a synthesis and suggest ways forward.

Transcriptional insights into the CD8(+) T cell response to infection and memory T cell formation.

After infection, many factors coordinate the population expansion and differentiation of CD8+ effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8+ T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8+ effector cells. Long-lived memory CD8+ cells ultimately expressed a small subset of genes shared by natural killer T and γδ T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8+ effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8+ T cell immunity.

The transcriptional landscape of αß T cell differentiation.

The differentiation of αßT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

Identification of transcriptional regulators in the mouse immune system.

The differentiation of hematopoietic stem cells into cells of the immune system has been studied extensively in mammals, but the transcriptional circuitry that controls it is still only partially understood. Here, the Immunological Genome Project gene-expression profiles across mouse immune lineages allowed us to systematically analyze these circuits. To analyze this data set we developed Ontogenet, an algorithm for reconstructing lineage-specific regulation from gene-expression profiles across lineages. Using Ontogenet, we found differentiation stage-specific regulators of mouse hematopoiesis and identified many known hematopoietic regulators and 175 previously unknown candidate regulators, as well as their target genes and the cell types in which they act. Among the previously unknown regulators, we emphasize the role of ETV5 in the differentiation of γδ T cells. As the transcriptional programs of human and mouse cells are highly conserved, it is likely that many lessons learned from the mouse model apply to humans.

Actin polymerization-dependent activation of Cas-L promotes immunological synapse stability.

The immunological synapse formed between a T-cell and an antigen-presenting cell is important for cell-cell communication during T-cell-mediated immune responses. Immunological synapse formation begins with stimulation of the T-cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization-dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte-specific Crk-associated substrate (Cas-L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas-L is phosphorylated at TCR microclusters in an actin polymerization-dependent fashion. Furthermore, Cas-L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside-out integrin activation, and actomyosin contraction. We propose a new role for Cas-L in T-cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin-dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T-cell-mediated immune responses.

Thyroid hormones and deiodinase activity in plasma and tissues in relation to high levels of organohalogen contaminants in East Greenland polar bears (Ursus maritimus).

Previous studies have shown relationships between organohalogen contaminants (OHCs) and circulating levels of thyroid hormones (THs) in arctic wildlife. However, there is a lack of knowledge concerning the possible functional effects of OHCs on TH status in target tissues for TH-dependent activity. The relationships between circulating (plasma) levels of OHCs and various TH variables in plasma as well as in liver, muscle and kidney tissues from East Greenland sub-adult polar bears (Ursus maritimus) sampled in 2011 (n=7) were therefore investigated. The TH variables included 3.3',5.5'-tetraiodothyronine or thyroxine (T4), 3.3',5-triiodothyronine (T3) and type 1 (D1) and type 2 (D2) deiodinase activities. Principal component analysis (PCA) combined with correlation analyses demonstrated negative relationships between individual polychlorinated biphenyls (PCBs) and their hydroxylated (OH-) metabolites and T4 in both plasma and muscle. There were both positive and negative relationships between individual OHCs and D1 and D2 activities in muscle, liver and kidney tissues. In general, PCBs, OH-PCBs and polybrominated dipehenyl ethers (PBDEs) were positively correlated to D1 and D2 activities, whereas organochlorine pesticides and byproducts (OCPs) were negatively associated with D1 and D2 activities. These results support the hypothesis that OHCs can affect TH status and action in the target tissues of polar bears. TH levels and deiodinase activities in target tissues can be sensitive endpoints for exposure of TH-disrupting compounds in arctic wildlife, and thus, tissue-specific responses in target organs should be further considered when assessing TH disruption in wildlife studies.

Distinct mechanisms mediate naive and memory CD8 T-cell tolerance.

Peripheral tolerance to developmentally regulated antigens is necessary to sustain tissue homeostasis. We have now devised an inducible and reversible system that allows interrogation of T-cell tolerance induction in endogenous naïve and memory CD8 T cells. Our data show that peripheral CD8 T-cell tolerance can be preserved through two distinct mechanisms, antigen addiction leading to anergy for naïve T cells and ignorance for memory T cells. Induction of antigen in dendritic cells resulted in substantial expansion and maintenance of endogenous antigen-specific CD8 T cells. The self-reactive cells initially exhibited effector activity but eventually became unresponsive. Upon antigen removal, the antigen-specific population waned, resulting in development of a self-specific memory subset that recalled to subsequent challenge. In striking contrast to naïve CD8 T cells, preexisting antigen-specific memory CD8 T cells failed to expand after antigen induction and essentially ignored the antigen despite widespread expression by dendritic cells. The inclusion of inflammatory signals partially overcame memory CD8 T-cell ignorance of self-antigen. Thus, peripheral CD8 T-cell tolerance for naïve CD8 T cells depended on the continuous presence of antigen, whereas memory CD8 T cells were prohibited from autoreactivity in the absence of inflammation.

Pathogen-induced inflammatory environment controls effector and memory CD8+ T cell differentiation.

In response to infection, CD8(+) T cells integrate multiple signals and undergo an exponential increase in cell numbers. Simultaneously, a dynamic differentiation process occurs, resulting in the formation of short-lived effector cells (SLECs; CD127(low)KLRG1(high)) and memory precursor effector cells (CD127(high)KLRG1(low)) from an early effector cell that is CD127(low)KLRG1(low) in phenotype. CD8(+) T cell differentiation during vesicular stomatitis virus infection differed significantly than during Listeria monocytogenes infection with a substantial reduction in early effector cell differentiation into SLECs. SLEC generation was dependent on Ebi3 expression. Furthermore, SLEC differentiation during vesicular stomatitis virus infection was enhanced by administration of CpG-DNA, through an IL-12-dependent mechanism. Moreover, CpG-DNA treatment enhanced effector CD8(+) T cell functionality and memory subset distribution, but in an IL-12-independent manner. Population dynamics were dramatically different during secondary CD8(+) T cell responses, with a much greater accumulation of SLECs and the appearance of a significant number of CD127(high)KLRG1(high) memory cells, both of which were intrinsic to the memory CD8(+) T cell. These subsets persisted for several months but were less effective in recall than memory precursor effector cells. Thus, our data shed light on how varying the context of T cell priming alters downstream effector and memory CD8(+) T cell differentiation.

Duration of antigen availability influences the expansion and memory differentiation of T cells.

The initial engagement of the TCR through interaction with cognate peptide-MHC is a requisite for T cell activation and confers Ag specificity. Although this is a key event in T cell activation, the duration of these interactions may affect the proliferative capacity and differentiation of the activated cells. In this study, we developed a system to evaluate the temporal requirements for antigenic stimulation during an immune response in vivo. Using Abs that target specific Ags in the context of MHC, we were able to manipulate the duration of Ag availability to both CD4 and CD8 T cells during an active infection. During the primary immune response, the magnitude of the CD4 and CD8 T cell response was dependent on the duration of Ag availability. Both CD4 and CD8 T cells required sustained antigenic stimulation for maximal expansion. Memory cell differentiation was also dependent on the duration of Ag exposure, albeit to a lesser extent. However, memory development did not correlate with the magnitude of the primary response, suggesting that the requirements for continued expansion of T cells and memory differentiation are distinct. Finally, a shortened period of Ag exposure was sufficient to achieve optimal expansion of both CD4 and CD8 T cells during a recall response. It was also revealed that limiting exposure to Ag late during the response may enhance the CD4 T cell memory pool. Collectively, these data indicated that Ag remains a critical component of the T cell response after the initial APC-T cell interaction.

T cell and APC dynamics in situ control the outcome of vaccination.

The factors controlling the progression of an immune response to generation of protective memory are poorly understood. We compared the in situ and ex vivo characteristics of CD8 T cells responding to different forms of the same immunogen. Immunization with live Listeria monocytogenes, irradiated L. monocytogenes (IRL), or heat-killed L. monocytogenes (HKL) induced rapid activation of CD8 T cells. However, only IRL and live L. monocytogenes inoculation induced sustained proliferation and supported memory development. Gene and protein expression analysis revealed that the three forms of immunization led to three distinct transcriptional and translational programs. Prior to cell division, CD8 T cell-dendritic cell clusters formed in the spleen after live L. monocytogenes and IRL but not after HKL immunization. Furthermore, HKL immunization induced rapid remodeling of splenic architecture, including loss of marginal zone macrophages, which resulted in impaired bacterial clearance. These results identify initial characteristics of a protective T cell response that have implications for the development of more effective vaccination strategies.

In situ biosensing with a surface plasmon resonance fiber grating aptasensor.

Surface plasmon resonance (SPR) biosensors prepared using optical fibers can be used as a cost-effective and relatively simple-to-implement alternative to well established biosensor platforms for monitoring biomolecular interactions in situ or possibly in vivo. The fiber biosensor presented in this study utilizes an in-fiber tilted Bragg grating to excite the SPR on the surface of the sensor over a large range of external medium refractive indices, with minimal cross-sensitivity to temperature and without compromising the structural integrity of the fiber. The label-free biorecognition scheme used demonstrates that the sensor relies on the functionalization of the gold-coated fiber with aptamers, synthetic DNA sequences that bind with high specificity to a given target. In addition to monitoring the functionalization of the fiber by the aptamers in real-time, the results also show how the fiber biosensor can detect the presence of the aptamer's target, in various concentrations of thrombin in buffer and serum solutions. The findings also show how the SPR biosensor can be used to evaluate the dissociation constant (K(d)), as the binding constant agrees with values already reported in the literature.

Durable Interactions of T Cells with T Cell Receptor Stimuli in the Absence of a Stable Immunological Synapse.

T cells engage in two modes of interaction with antigen-presenting surfaces: stable synapses and motile kinapses. Although it is surmised that durable interactions of T cells with antigen-presenting cells involve synapses, in situ 3D imaging cannot resolve the mode of interaction. We have established in vitro 2D platforms and quantitative metrics to determine cell-intrinsic modes of interaction when T cells are faced with spatially continuous or restricted stimulation. All major resting human T cell subsets, except memory CD8 T cells, spend more time in the kinapse mode on continuous stimulatory surfaces. Surprisingly, we did not observe any concordant relationship between the mode and durability of interaction on cell-sized stimulatory spots. Naive CD8 T cells maintain kinapses for more than 3 hr before leaving stimulatory spots, whereas their memory counterparts maintain synapses for only an hour before leaving. Thus, durable interactions do not require stable synapses.

Legacy and emerging organic pollutants in liver and plasma of long-finned pilot whales (Globicephala melas) from waters surrounding the Faroe Islands.

Concentrations of PCBs, organochlorine pesticides (OCPs), brominated flame retardants and a suite of relevant metabolites of these POPs, in all 175 different compounds, were determined in liver and plasma of traditionally hunted pilot whales (n=14 males and n=13 females of different age groups) from the Faroe Islands. The main objectives of this study were to determine differences in the presence and concentrations of the compounds in the liver and plasma, how they depend on developmental stage (calves, sub adults, and adult females), and to assess maternal transfer of the compounds to suckling calves. Generally, the lipid weight (lw) concentrations of quantified POPs in the liver and plasma of pilot whales were positively correlated, and lw concentrations of most POPs did not differ between these matrices. However, concentrations of some individual POPs differed significantly (p<0.05) between plasma and liver; CB-153 (p=0.044), CB-174 (p=0.027) and BDE-47 (p=0.017) were higher in plasma than in liver, whereas p,p'-DDE (p=0.004) and HCB (p<0.001) were higher in liver than in plasma. POP concentrations differed between age/gender groups with lower levels in adult females than in juveniles. The relative distribution of compounds also differed between the age groups, due to the influence of the maternal transfer of the compounds. The results indicated that larger, more hydrophobic POPs were transferred to the offspring less efficiently than smaller or less lipid soluble compounds. Very low levels of both OH- and/or MeSO2-PCB and PBDE metabolites were found in all age groups, with no significant (p>0.05) differences between the groups, strongly suggesting a very low metabolic capacity for their formation in pilot whales. The lack of difference in the metabolite concentrations between the age groups also indicates less maternal transfer of these contaminant groups compared to the precursor compounds.

Increased competition for antigen during priming negatively impacts the generation of memory CD4 T cells.

The factors involved in the differentiation of memory CD4 T cells from naïve precursors are poorly understood. We developed a system to examine the effect of increased competition for antigen by CD4 T cells on the generation of memory in response to infection with a recombinant vesicular stomatitis virus. Competition was initially regulated by increasing the precursor frequency of adoptively transferred naïve T cell antigen receptor transgenic CD4 T cells. Despite robust proliferation at high precursor frequencies, memory CD4 T cells did not develop, whereas decreasing the input number of naïve CD4 T cells promoted memory development after infection. The lack of memory development was linked to reduced blastogenesis and poor effector cell induction, but not to initial recruitment or proliferation of antigen-specific CD4 T cells. To prove that availability of antigen alone could regulate memory CD4 T cell development, we used treatment with an mAb specific for the epitope recognized by the transferred CD4 T cells. At high doses, this mAb effectively inhibited the antigen-specific CD4 T cell response. However, at a very low dose of mAb, primary CD4 T cell expansion was unaffected, although memory development was dramatically reduced. Moreover, the induction of effector function was concomitantly inhibited. Thus, competition for antigen during CD4 T cell priming is a major contributing factor to the development of the memory CD4 T cell pool.

Trend of depression and the use of psychiatric medications in U.S. Veterans with hepatitis C during interferon-based therapy.

OBJECTIVES: Interferon-based therapy in patients with HCV infection may cause new psychiatric symptoms or worsening of existing psychiatric conditions. The aim of this study was to evaluate the trend of depression, and the use and the effect of psychiatric medications during interferon therapy. METHODS: Patients with HCV were evaluated at our clinic following a standardized template to collect clinical and psychiatric data at baseline, weeks 2, 4, 8, 12, and every 6 wk thereafter. Depression was measured at each visit by obtaining the CES-D (Center for Epidemiologic Studies-Depression) score. A PMI was defined as the addition or increase in the dose of a psychiatric medication. RESULTS: During interferon therapy, patients with psychiatric history (N = 46) had minor fluctuation in the CES-D score compared to baseline, while patients without psychiatric history (N = 33) had significant increase in their CES-D score, were more likely to require PMIs with antidepressants (63.6%vs 39.1%, respectively; P= 0.04), and they required the first PMI with sedatives/hypnotics earlier (4.1 wk vs 8.9 wk after starting interferon, respectively; P= 0.01). PMIs in the two groups resulted in significant decrease in CES-D score at 4-6 wk and 8-12 wk post-PMIs, but it continued to be higher than baseline. The overall sustained viral response was 47%. CONCLUSION: Hepatitis C patients with stable psychiatric history can be successfully treated with interferon-based therapy if followed closely by a multidisciplinary team that includes specialists in hepatitis C and behavioral health.

Dynamics of blood-borne CD8 memory T cell migration in vivo.

Memory T cells are distributed throughout the body following infection, but the migratory dynamics of the memory pool in vivo is unknown. The ability of circulating microbe-specific memory T cells to populate lymphoid and nonlymphoid tissues was examined using adoptive transfer and parabiosis systems. While migration of memory CD8 T cells to lymph nodes and peritoneal cavity required G(i)-coupled receptor signaling, migration to the spleen, bone marrow, lung, and liver was independent of this pathway. Following parabiosis, memory T cells rapidly equilibrated into the lymphoid tissues, lung, and liver of each parabiont, implying most memory cells were not obligately tissue resident. Equilibration of memory cell populations was delayed in the brain, peritoneal cavity, and intestinal lamina propria, indicating controlled gating for entry into these tissues. In addition, memory cell migration to the lamina propria required beta7 integrins. Thus, the blood-borne T cell pool serves to maintain the homeostasis of tissue-based memory populations.