Cellular architecture shapes the naïve T cell response.

After antigen stimulation, naïve T cells display reproducible population-level responses, which arise from individual T cells pursuing specific differentiation trajectories. However, cell-intrinsic predeterminants controlling these single-cell decisions remain enigmatic. We found that the subcellular architectures of naïve CD8 T cells, defined by the presence (TØ) or absence (TO) of nuclear envelope invaginations, changed with maturation, activation, and differentiation. Upon T cell receptor (TCR) stimulation, naïve TØ cells displayed increased expression of the early-response gene Nr4a1, dependent upon heightened calcium entry. Subsequently, in vitro differentiation revealed that TØ cells generated effector-like cells more so compared with TO cells, which proliferated less and preferentially adopted a memory-precursor phenotype. These data suggest that cellular architecture may be a predeterminant of naïve CD8 T cell fate.

Role and Function of T Cell-Derived Exosomes and Their Therapeutic Value.

Exosomes are membrane-bound extracellular vesicles that are produced in the endosomal compartment of most eukaryotic cells. Containing proteins, RNA, and DNA, exosomes mediate intercellular communication between different cell types by transferring their contents and thus are involved in numerous physiological and pathological processes. T cells are an indispensable part of adaptive immunity, and the functions of T cell-derived exosomes have been widely studied. In the more than three decades since the discovery of exosomes, several studies have revealed that T cell-derived exosomes play a novel role in cell-to-cell signaling, especially in inflammatory responses, autoimmunity, and infectious diseases. In this review, we will summarize the function of T cell-derived exosomes and their therapeutic potential.

Fever supports CD8+ effector T cell responses by promoting mitochondrial translation.

Fever can provide a survival advantage during infection. Metabolic processes are sensitive to environmental conditions, but the effect of fever on T cell metabolism is not well characterized. We show that in activated CD8+ T cells, exposure to febrile temperature (39 °C) augmented metabolic activity and T cell effector functions, despite having a limited effect on proliferation or activation marker expression. Transcriptional profiling revealed an up-regulation of mitochondrial pathways, which was consistent with increased mass and metabolism observed in T cells exposed to 39 °C. Through in vitro and in vivo models, we determined that mitochondrial translation is integral to the enhanced metabolic activity and function of CD8+ T cells exposed to febrile temperature. Transiently exposing donor lymphocytes to 39 °C prior to infusion in a myeloid leukemia mouse model conferred enhanced therapeutic efficacy, raising the possibility that exposure of T cells to febrile temperatures could have clinical potential.

HTNV infection of CD8+ T cells is associated with disease progression in HFRS patients.

Hantaan viruses (HTNVs) are zoonotic pathogens transmitted mainly by rodents and capable of infecting humans. Increasing knowledge of the human response to HTNV infection can guide the development of new preventative vaccines and therapeutic strategies. Here, we show that HTNV can infect CD8+ T cells in vivo in patients diagnosed with hemorrhagic fever with renal syndrome (HFRS). Electron microscopy-mediated tracking of the life cycle and ultrastructure of HTNV-infected CD8+ T cells in vitro showed an association between notable increases in cytoplasmic multivesicular bodies and virus production. Notably, based on a clinical cohort of 280 patients, we found that circulating HTNV-infected CD8+ T cell numbers in blood were proportional to disease severity. These results demonstrate that viral infected CD8+ T cells may be used as an adjunct marker for monitoring HFRS disease progression and that modulating T cell functions may be explored for new treatment strategies.

A CD8+ NK cell transcriptomic signature associated with clinical outcome in relapsing remitting multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) with the majority of cases characterised by relapsing/remitting (RRMS) attacks of neurologic dysfunction followed by variable resolution. Improving clinical outcomes in RRMS requires both a better understanding of the immunological mechanisms driving recurrent demyelination and better means of predicting future disease course to facilitate early targeted therapy. Here, we apply hypothesis-generating network transcriptomics to CD8+ cells isolated from patients in RRMS, identifying a signature reflecting expansion of a subset of CD8+ natural killer cells (NK8+) associated with favourable outcome. NK8+ are capable of regulating CD4+ T cell activation and proliferation in vitro, with reduced expression of HLA-G binding inhibitory receptors and consequent reduced sensitivity to HLA-G-mediated suppression. We identify surrogate markers of the NK8+ signature in peripheral blood leucocytes and validate their association with clinical outcome in an independent cohort, suggesting their measurement may facilitate early, targeted therapy in RRMS.

Human CD8+ T Cells Release Extracellular Traps Co-Localized With Cytotoxic Vesicles That Are Associated With Lesion Progression and Severity in Human Leishmaniasis.

Cell death plays a fundamental role in mounting protective and pathogenic immunity. Etosis is a cell death mechanism defined by the release of extracellular traps (ETs), which can foster inflammation and exert microbicidal activity. While etosis is often associated with innate cells, recent studies showed that B cells and CD4+ T cells can release ETs. Here we investigate whether CD8+ T cells can also release ETs, which might be related to cytotoxicity and tissue pathology. To these ends, we first employed an in vitro system stimulating human CD8+ T cells isolated from healthy volunteers with anti-CD3/anti-CD28. Using time-frame video, confocal and electron microscopy, we demonstrate that human CD8+ T cells release ETs upon stimulation (herein LETs - lymphocyte extracellular traps), which display unique morphology and functional characteristics. CD8+ T cell-derived LETs form long strands that co-localize with CD107a, a marker of vesicles containing cytotoxic granules. In addition, these structures connect the LET-releasing cell to other neighboring cells, often resulting in cell death. After demonstrating the release of LETs by human CD8+ T cells in vitro, we went on to study the occurrence of CD8-derived LETs in a human disease setting. Thus, we evaluated the occurrence of CD8-derived LETs in lesions from patients with human tegumentary leishmaniasis, where CD8+ T cells play a key role in mediating pathology. In addition, we evaluated the association of these structures with the intensity of the inflammatory infiltrate in early and late cutaneous, as well as in mucosal leishmaniasis lesions. We demonstrated that progression and severity of debilitating and mutilating forms of human tegumentary leishmaniasis are associated with the frequency of CD8+ T cells in etosis, as well as the occurrence of CD8-derived LETs carrying CD107a+ vesicles in the lesions. We propose that CD8+ T cell derived LETs may serve as a tool for delivering cytotoxic vesicles to distant target cells, providing insights into mechanisms of CD8+ T cell mediated pathology.

Metabolic characteristics of CD8+ T cell subsets in young and aged individuals are not predictive of functionality.

Virtual memory T (TVM) cells are antigen-naïve CD8+ T cells that exist in a semi-differentiated state and exhibit marked proliferative dysfunction in advanced age. High spare respiratory capacity (SRC) has been proposed as a defining metabolic characteristic of antigen-experienced memory T (TMEM) cells, facilitating rapid functionality and survival. Given the semi-differentiated state of TVM cells and their altered functionality with age, here we investigate TVM cell metabolism and its association with longevity and functionality. Elevated SRC is a feature of TVM, but not TMEM, cells and it increases with age in both subsets. The elevated SRC observed in aged mouse TVM cells and human CD8+ T cells from older individuals is associated with a heightened sensitivity to IL-15. We conclude that elevated SRC is a feature of TVM, but not TMEM, cells, is driven by physiological levels of IL-15, and is not indicative of enhanced functionality in CD8+ T cells.

Some Aspects of CD8+ T-Cell Exhaustion Are Associated With Altered T-Cell Mitochondrial Features and ROS Content in HIV Infection.

BACKGROUND: Reversing or preventing T-cell exhaustion is an important treatment goal in the context of HIV disease; however, the mechanisms that regulate HIV-specific CD8 T-cell exhaustion are incompletely understood. Since mitochondrial mass (MM), mitochondrial membrane potential (MMP), and cellular reactive oxygen species (ROS) content are altered in exhausted CD8 T cells in other settings, we hypothesized that similar lesions may arise in HIV infection. METHODS: We sampled cryopreserved peripheral blood mononuclear cells from HIV-uninfected (n = 10) and HIV-infected participants with varying levels and mechanisms of viral control: viremic (VL > 2000 copies/mL; n = 8) or aviremic (VL < 40 copies/mL) due to antiretroviral therapy (n = 11) or natural control (n = 9). We characterized the MM, MMP, and ROS content of bulk CD8 T cells and MHC class I tetramer+ HIV-specific CD8 T cells by flow cytometry. RESULTS: We observed higher MM, MMP, and ROS content across bulk effector-memory CD8 T-cell subsets in HIV-infected compared with HIV-uninfected participants. Among HIV-specific CD8 T cells, these features did not vary by the extent or mechanism of viral control but were significantly altered in cells displaying characteristics associated with exhaustion (eg, high PD-1 expression, low CD127 expression, and impaired proliferative capacity). CONCLUSIONS: While we did not find that control of HIV replication in vivo correlates with the CD8 T-cell MM, MMP, or ROS content, we did find that some features of CD8 T-cell exhaustion are associated with alterations in mitochondrial state. Our findings support further studies to probe the relationship between mitochondrial dynamics and CD8 T-cell functionality in HIV infection.

Ovalbumin Epitope SIINFEKL Self-Assembles into a Supramolecular Hydrogel.

Here we show that the well-known ovalbumin epitope SIINFEKL that is routinely used to stimulate ovalbumin-specific T cells and to test new vaccine adjuvants can form a stable hydrogel. We investigate properties of this hydrogel by a range of spectroscopic and imaging techniques demonstrating that the hydrogel is stabilized by self-assembly of the peptide into nanofibres via stacking of ß-sheets. As peptide hydrogels are known to stimulate an immune response as adjuvants, the immunoactive properties of the SIINFEKL peptide may also originate from its propensity to self-assemble into a hydrogel. This finding requires a re-evaluation of this epitope in adjuvant testing.

RORγt limits the amount of the cytokine receptor γc through the prosurvival factor Bcl-xL in developing thymocytes.

The cytokine receptor subunit γc provides critical signals for T cell survival and differentiation. We investigated the molecular mechanism that controls the cell surface abundance of γc during T cell development in the thymus. We found that the amount of γc was low on CD4+CD8+ double-positive (DP) thymocytes before their positive selection to become mature T cells. The transcription factor RORγt was abundant in immature DP thymocytes, and its loss resulted in an increase in the abundance of surface γc, specifically on preselection DP cells. Rather than directly repressing expression of the gene encoding γc, RORγt acted through the antiapoptotic protein Bcl-xL to reduce the abundance of surface γc, which resulted in decreased cytokine signaling and was associated with inhibition of cell metabolism and mitochondrial biogenesis. Accordingly, overexpression of Bcl-xL in RORγt-deficient thymocytes restored the amount of surface γc to that present on normal preselection DP cells. Together, these data highlight a previously unappreciated role for RORγt and Bcl-xL in limiting γc abundance at the cell surface and reveal a signaling circuit in which survival factors control cytokine signaling by limiting the abundance and surface distribution of a receptor subunit shared by several cytokines.

A transcriptionally and functionally distinct PD-1+ CD8+ T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade.

Evidence from mouse chronic viral infection models suggests that CD8+ T cell subsets characterized by distinct expression levels of the receptor PD-1 diverge in their state of exhaustion and potential for reinvigoration by PD-1 blockade. However, it remains unknown whether T cells in human cancer adopt a similar spectrum of exhausted states based on PD-1 expression levels. We compared transcriptional, metabolic and functional signatures of intratumoral CD8+ T lymphocyte populations with high (PD-1T), intermediate (PD-1N) and no PD-1 expression (PD-1-) from non-small-cell lung cancer patients. PD-1T T cells showed a markedly different transcriptional and metabolic profile from PD-1N and PD-1- lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1T lymphocytes were impaired in classical effector cytokine production, they produced CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures. Strikingly, the presence of PD-1T cells was strongly predictive for both response and survival in a small cohort of non-small-cell lung cancer patients treated with PD-1 blockade. The characterization of a distinct state of tumor-reactive, PD-1-bright lymphocytes in human cancer, which only partially resembles that seen in chronic infection, provides potential avenues for therapeutic intervention.

Enhancing CD8+ T Cell Fatty Acid Catabolism within a Metabolically Challenging Tumor Microenvironment Increases the Efficacy of Melanoma Immunotherapy.

How tumor-infiltrating T lymphocytes (TILs) adapt to the metabolic constrains within the tumor microenvironment (TME) and to what degree this affects their ability to combat tumor progression remain poorly understood. Using mouse melanoma models, we report that CD8+ TILs enhance peroxisome proliferator-activated receptor (PPAR)-α signaling and catabolism of fatty acids (FAs) when simultaneously subjected to hypoglycemia and hypoxia. This metabolic switch partially preserves CD8+ TILs' effector functions, although co-inhibitor expression increases during tumor progression regardless of CD8+ TILs' antigen specificity. Further promoting FA catabolism improves the CD8+ TILs' ability to slow tumor progression. PD-1 blockade delays tumor growth without changing TIL metabolism or functions. It synergizes with metabolic reprogramming of T cells to achieve superior antitumor efficacy and even complete cures.

Myristoylation increases the CD8+T-cell response to a GFP prototype antigen delivered by modified vaccinia virus Ankara.

Activation of CD8(+)T-cells is an essential part of immune responses elicited by recombinant modified vaccinia virus Ankara (MVA). Strategies to enhance T-cell responses to antigens may be particularly necessary for broadly protective immunization against influenza A virus infections or for candidate vaccines targeting chronic infections and cancer. Here, we tested recombinant MVAs that targeted a model antigen, GFP, to different localizations in infected cells. In vitro characterization demonstrated that GFP accumulated in the nucleus (MVA-nls-GFP), associated with cellular membranes (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). On vaccination, we found significantly higher levels of GFP-specific CD8(+)T-cells in MVA-myr-GFP-vaccinated BALB/c mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8(+)T-cell responses to MVA-delivered target antigens.

Uremia-associated immunological aging is stably imprinted in the T-cell system and not reversed by kidney transplantation.

The uremia-induced inflammatory environment in end-stage renal disease (ESRD) patients is associated with premature T-cell aging resulting in a defective T-cell immunity. As kidney transplantation (KTx) reduces the pro-inflammatory environment, we hypothesized that KTx would rejuvenate the aged T-cell system. As aging parameters, we determined in 70 KTx recipients the differentiation status by immunophenotyping, thymic output by the T-cell receptor excision circle (TREC) content together with CD31(+) naïve T-cell numbers and the relative telomere length (RTL) as a measure for proliferative history at pre-KTx, 3, 6 and 12 months post-KTx. In addition, T-cell function was determined by measuring the proliferative capacity and percentages of cytokine-producing cells. Directly post-KTx, memory T-cell numbers were diminished but restored to pre-KTx values at 12 months, except for CD4(+) EM T cells. The RTL of (memory) CD4(+) and CD8(+) T cells did not change. In contrast, TREC content and CD31(+) naïve T-cell numbers were stable post-KTx although the RTL of naïve CD4(+) and CD8(+) T cells decreased implying homeostatic proliferation of naïve cells, in response to a temporary decrease in memory cells. The T-cell function was not improved post-KTx. Our findings demonstrate that the uremia-associated aged phenotype is stably imprinted in the T-cell system and not reversed by KTx.

In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages.

CD8(+) T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8(+) T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8(+) T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8(+) T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8(+) T cells and signaling by G protein-coupled receptors, although CD8(+) T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8(+) T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8(+) T cells.

Augmented telomerase activity and reduced telomere length in parthenium-induced contact dermatitis.

BACKGROUND: Parthenium dermatitis is a common chronic inflammatory disease with activated T lymphocytes that recognize the antigens, which leads to proliferation and differentiation. Telomeres and telomerase play an important role in the regulation of life span of the cell. Telomere length maintained by telomerase, are specialized repeats present at the end of chromosomes which protect it from degradation, end-to-end fusion and are important for integrity of chromosomes. OBJECTIVES: The aim of this study was to measure telomerase activity and telomere length in Peripheral blood mononuclear cell (PBMC), CD4(+) and CD8(+) T lymphocytes from parthenium dermatitis patients. METHODS: The study includes 50 patients of parthenium dermatitis confirmed by patch testing and 50 healthy controls. Telomerase activity was measured using the telomere repeat amplification protocol using PCR-ELISA kit. Telomere length was measured by using Telo TAGGG Telomere Length Assay Kit. RESULTS: Significantly elevated levels of telomerase activity was observed in PBMC, CD4(+) and CD8(+) T cells of parthenium dermatitis patients as compared with healthy controls. However, significantly reduced telomere length in PBMC, CD4(+) and CD8(+) T cells have been found in patients than healthy subjects, but there was no difference between CD4(+) and CD8(+) T cells in patients. CONCLUSION: This study might have provided insight into the role of telomerase in parthenium dermatitis that is characterized by the recruitment of T lymphocytes, which play an important role in this inflammatory disease. The augmented telomerase activity and reduced terminal restriction fragment length might be explored as a potential diagnostic/prognostic marker for parthenium dermatitis in future.

Analysis of stem-cell-like properties of human CD161++IL-18Rα+ memory CD8+ T cells.

CD161(++)IL-18Rα(+)CD8(+) human T cells have recently been identified as a new subset of memory cells but their exact role remains unclear. CD161(++)IL-18Rα(+)CD8(+), mucosal-associated invariant T cells express a semi-invariant TCR Vα7.2-Jα33, which recognizes the MHC-related protein 1. On the basis of properties including the expression of the ABC-B1 transporter, cKit expression and survival after chemotherapy, CD161(++)IL-18Rα(+)CD8(+) T cells have been designated as 'stem' cells. Here we analyse location and functional properties of CD161(++)IL-18Rα(+) CD8(+) T cells and question whether they have other traits that would mark them as genuine 'stem' cells. CD161(++)IL-18Rα(+)CD8(+) T cells were found in peripheral blood, spleen and bone marrow but interestingly hardly at all in lymph nodes (LNs), which may possibly be explained by the finding that these cells express a specific set of chemokine receptors that allows migration to inflamed tissue rather than to LNs. In addition to TCR ligation and co-stimulation, CD161(++)IL-18Rα(+) CD8(+) T cells require cytokines for proliferation. The CD161(++)IL-18Rα(+) CD8(+) pool contains cells reactive towards peptides, derived from both persisting and cleared viruses. Although CD161(++)IL-18Rα(+) CD8(+) T cells express the ABC-B1 transporter, they have shorter telomeres and less telomerase activity and do not express aldehyde dehydrogenase. Finally, CD161(++)IL-18Rα(+) CD8(+) T cells show similarities to terminally differentiated T cells, expressing IFNγ, KLRG1 and the transcription factor Blimp-1. In conclusion, CD161(++)IL-18Rα(+) CD8(+) T cells lack many features of typical 'stem' cells, but appear rather to be a subset of effector-type cells.

Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy.

Transplant rejection involves a coordinated attack of the innate and the adaptive immune systems of the host. To investigate this dynamic process and the contributions of both donor and host cells, we developed an ear skin graft model suitable for intravital imaging. We found that donor dermal dendritic cells (DCs) migrated rapidly from the graft and were replaced by host CD11b(+) mononuclear cells. The infiltrating host cells captured donor antigen, reached the draining lymph node and cross-primed graft-reactive CD8(+) T cells. Furthermore, we defined the mechanisms by which host T cells target graft cells. We found that primed T cells entered the graft from the surrounding tissue and localized selectively at the dermis-epidermis junction. Later, CD8(+) T cells disseminated throughout the graft and many became arrested. These results provide insights into the antigen presentation pathway and the stepwise progression of CD8(+) T cell activity, thereby offering a framework for evaluating how immunotherapy might abrogate the key steps in allograft rejection.

Blocking type I interferon (IFN) signaling impairs antigen responsiveness of circulating lymphocytes and alters their homing to lymphoid organs: protective role of type I IFN.

BACKGROUND: We recently demonstrated that type I Interferon (IFN) rescues in vitro, human B-lymphocytes from apoptosis via PI3Kδ/Akt, Rho-A, NFκB and Bcl-2/BclXL. In the present study we extended our work to clarify, in vivo, the role of type I IFN signalling on the circulating and lymphoid organs homing lymphocytes. METHODOLOGY: Two groups of mice 13 in each were set: type I IFN signalling blocked mice injected with anti-IFNAR1 antagonist antibody (10 mg/kg body weight) once/day for up to 20 days, and control group were injected with vehicle alone. RESULTS: Flow cytometry analysis to monitor the blood lymphocyte phenotype and proliferation have shown a significant decrease in CD45R/B220(+) [corrected] B cells, CD4(+) and CD8(+) T cells in treated animals. Furthermore, the proliferative capacities of these lymphocyte subsets were significantly decreased in treated animals compared to those of control mice. Marked reduction in the plasma levels of IL-2 and IL-7 (cytokines important for the development of T and B cells) but not of IL-6 or IL-10 was observed in treated mice and this may a cause for emergence decrease in B and T cell numbers. Immunohistochemical studies have further shown a marked reduction in the numbers of CD20(+) B cells in spleen and Peyer's patches and CD3(+) T cells in thymus of treated animals. Moreover, electron microscopy examinations have revealed a loss of lymphocytes with characteristic features of apoptosis. CONCLUSION: Our data confirmed that the in vivo inhibition of type I IFN signaling induce decrease in the numbers and defective functions of circulating and lymphoid organs homing lymphocytes providing a strong evidence for the protective effects of type 1 IFNs (IFN-α/ß) on B and T lymphocytes.