T-cell Clusters Research Articles

Spatiotemporally Distinct Interactions with Dendritic Cell Subsets Facilitates CD4+ and CD8+ T Cell Activation to Localized Viral Infection

The dynamics of when and where CD4(+) Tcells provide help for CD8(+) Tcell priming and which dendritic cells (DCs) activate CD4(+) Tcells invivo after localized infection are poorly understood. By using a cutaneous herpes simplex virus infection model combined with intravital 2-photon imaging of the draining lymph node (LN) to concurrently visualize pathogen-specific CD4(+) and CD8(+) Tcells, we found that early priming of CD4(+) Tcells involved clustering with migratory skin DCs. CD8(+) Tcells did not interact with migratory DCs and their activation was delayed, requiring later clustering interactions with LN-resident XCR1(+) DCs. CD4(+) Tcells interacted with these late CD8(+) Tcell clusters on resident XCR1(+) DCs. Together, these data reveal asynchronous Tcell activation by distinct DC subsets and highlight the key role of XCR1(+) DCs as the central platform for cytotoxic T lymphocyte activation and the delivery of CD4(+) Tcell help.

Flow cytometric identification of immunophenotypically aberrant T-cell clusters on skin shave biopsy specimens from patients with mycosis fungoides.

To assess the ability of flow cytometry (FC) to detect putative neoplastic T-cell subsets on skin shave biopsy (SSB) specimens from patients with mycosis fungoides (MF) and to study the immunophenotype of skin-infiltrating tumor cells in MF. SSB specimens from patients with suspected MF were bisected and submitted for both FC and routine histopathology. Six-dimensional gating strategies were applied to identify putative neoplastic cells, independently from their expected immunophenotype. Aberrant T cells were detected by FC in 18 of 33 SBB specimens, of which all had clinicomorphologic features of MF. Of the remaining 15 SSB specimens, six had clinicomorphologic features of MF and nine were diagnosed with benign inflammatory dermatoses. Unexpectedly, CD26 was aberrantly overexpressed in 11 (73%) and lost in three (20%) of 15 SSB specimens from patients with MF where this antigen was evaluated. Other detected aberrancies included CD3 dim- (13/18 [72%]), CD7 dim- (15/18 [83%]), and CD4-/CD8- (3/18 [17%]). FC is capable of identifying putative neoplastic cells on SSB specimens from patients with MF. Bright homogeneous CD26 expression is a common and previously undescribed immunophenotypic aberrancy on MF skin infiltrates.

Local CD4 and CD8 T-Cell Reactivity to HSV-1 Antigens Documents Broad Viral Protein Expression and Immune Competence in Latently Infected Human Trigeminal Ganglia

Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1–infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.

T-cell infiltration correlates with CXCL10 expression in ganglia of cynomolgus macaques with reactivated simian varicella virus.

Ganglia of monkeys with reactivated simian varicella virus (SVV) contained more CD8 than CD4 T cells around neurons. The abundance of CD8 T cells was greater less than 2 months after reactivation than that at later times and correlated with that of CXCL10 RNA but not with those of SVV protein or open reading frame 61 (ORF61) antisense RNA. CXCL10 RNA colocalized with T-cell clusters. After SVV reactivation, transient T-cell infiltration, possibly mediated by CXCL10, parallels varicella zoster virus (VZV) reactivation in humans.

Positive HIV ELISA test, autoimmune hemolytic anemia, and generalized lymphadenopathy: A unifying diagnosis

Positive HIV ELISA test, autoimmune hemolytic anemia, and generalized lymphadenopathy: A unifying diagnosis

Immature T-cell clustering and efficient differentiation require the polarity protein Scribble.

T-cell polarization is required for cell migration and cell-cell interactions, cellular behaviors crucial for lymphocyte differentiation. Despite expression of the epithelial polarity network in T cells, neither its contribution to thymocyte polarity nor its requirement during development is known. We report here that depletion of the polarity protein Scribble in hematopoietic progenitor cells results in inefficient T-cell development characterized by a partial developmental block during the early double-negative (DN) stage of differentiation. Scribble-depleted hematopoietic progenitor cells exhibit a delayed transition into late CD44(lo/-)CD25(+) DN3 cells, evidenced by the accumulation of early CD44(int)CD25(+) DN3 cells. As a consequence, a limited cellular expansion and a reduced frequency of intracellular T-cell receptor β-positive DN3 cells are observed among Scribble-deficient differentiating T cells. Moreover, whereas purified Scribble-depleted DN2 and DN3 cells do not exhibit compromised spontaneous motility, T-cell clustering and prolonged homotypic interactions among such cells are reduced. This deficiency correlates with a lack of polarization of the integrin LFA-1 during T-cell migration or on the initiation of T-cell-T-cell interactions. Scribble is therefore a critical contributor to the clustering of immature T cells, an event shown here to be necessary for efficient developmental progression.

Memory T cells persisting within the brain after local infection show functional adaptations to their tissue of residence

The brain is not routinely surveyed by lymphocytes and is defined as an immuno-privileged site. However, viral infection of the brain results in the infiltration and long-term persistence of pathogen-specific CD8+T cells. These cells survive without replenishment from the circulation and are referred to as resident memory T cells (Trm). Brain Trm selectively express the integrin CD103, the expression of which is dependent on antigen recognition within the tissue. After clearance of virus, CD8+T cells persist in tight clusters, presumably at prior infection hot spots. Antigen persistence is not a prerequisite for T-cell retention, as suggested by the failure to detect viral genomes in the T-cell clusters. Furthermore, we show that an intracranial dendritic cell immunization regimen, which allows the transient introduction of antigen, also results in the generation of memory T cells that persist long term in the brain. Brain Trm die rapidly on isolation from the tissue and fail to undergo recall expansion after adoptive transfer into the bloodstream of antigen-challenged recipients. These ex vivo defects imply a dependency on the local milieu for function and survival. Cumulatively, this work shows that Trm are a specialized population of memory T cells that can be deposited in tissues previously thought to be beyond routine immune surveillance.

Adaptive Evolution of the Chlamydia trachomatis Dominant Antigen Reveals Distinct Evolutionary Scenarios for B- and T-cell Epitopes: Worldwide Survey

Background Chlamydia trachomatis is one of the most disseminated human pathogens, for which no vaccine is available yet. Understanding the impact of the host pressure on pathogen antigens is crucial, but so far it was only assessed for highly-restricted geographic areas. We aimed to evaluate the evolutionary picture of the chlamydial key antigen (MOMP), which is one of the leading multi-subunit vaccine candidates, in a worldwide basis.Methodology/Principal FindingsUsing genetics, molecular evolution methods and mathematical modelling, we analyzed all MOMP sequences reported worldwide, composed by 5026 strains from 33 geographic regions of five continents. Overall, 35.9% of variants were detected. The evolutionary pattern of MOMP amino acid gains/losses was found to differ from the remaining chromosome, reflecting the demanding constraints of this porin, adhesin and dominant antigen. Amino acid changes were 4.3-fold more frequent in host-interacting domains (P<10−12), specifically within B-cell epitopes (P<10−5), where 25% of them are at fixation (P<10−5). According to the typical pathogen-host arms race, this rampant B-cell antigenic variation likely represents neutralization escape mutants, as some mutations were previously shown to abrogate neutralization of chlamydial infectivity in vitro. In contrast, T-cell clusters of diverse HLA specificities are under purifying selection, suggesting a strategy that may lead to immune subversion. Moreover, several silent mutations are at fixation, generating preferential codons that may influence expression, and may also reflect recombination-derived ‘hitchhiking-effect’ from favourable nonsilent changes. Interestingly, the most prevalent C. trachomatis genotypes, E and F, showed a mutation rate 22.3-fold lower than that of the remainder (P<10−20), suggesting more fitted antigenic profiles.Conclusions/SignificanceGlobally, the adaptive evolution of the C. trachomatis dominant antigen is likely driven by its complex pathogenesis-related function and reflects distinct evolutionary antigenic scenarios that may benefit the pathogen, and thus should be taking into account in the development of a MOMP-based vaccine.

ICAM-1 expression on CD8+ T cells negatively regulates IFN-γ production. (50.28)

Abstract The factors that regulate the production of effector cytokines following antigen stimulation of naïve CD8+ T-cells are incompletely characterized. We utilized ICAM-1-/- mice to determine if the expression of ICAM-1 on naïve CD8+ T-cells modulates CD8+ T-cell activation and differentiation. Stimulation of purified wild-type OT-I CD8+ T-cells with purified MHC/Antigen, B7-1, and the inflammatory cytokine IL-12 results in robust proliferation and differentiation into CD8+ effector T-cells producing IFN-γ. This activation is associated with prominent T-cell:T-cell clusters and increased expression of ICAM-1. The presence of IL-12 sustains elevated levels of ICAM-1 on activated CD8+ T-cells and enhances T-cell:T-cell clusters. Activation of ICAM-1-/- OT-I T-cells results in comparable levels of proliferation as wild-type OT-I T-cells. However, ICAM-1-/- CD8+ T-cells fail to form significant homotypic clusters upon activation in this system. In addition, when compared to wild-type OT-I T-cells, antigen-specific activation of ICAM-1-/- OT-I T-cells results in a higher percentage of effector T-cells producing IFN-γ. These results suggest that inflammatory cytokines such as IL-12 may attenuate the generation of IFN-γ+ CD8+ effector T-cells via modulation of ICAM-1 expression following antigen stimulation of naïve CD8+ T-cells.

Expression Pattern of G Protein-Coupled Receptor 30 in LacZ Reporter Mice

Multiple reports implicated the function of G protein-coupled receptor (GPR)-30 with nongenomic effects of estrogen, suggesting that GPR30 might be a G-protein coupled estrogen receptor. However, the findings are controversial and the expression pattern of GPR30 on a cell type level as well as its function in vivo remains unclear. Therefore, the objective of this study was to identify cell types that express Gpr30 in vivo by analyzing a mutant mouse model that harbors a lacZ reporter (Gpr30-lacZ) in the Gpr30 locus leading to a partial deletion of the Gpr30 coding sequence. Using this strategy, we identified the following cell types expressing Gpr30: 1) an endothelial cell subpopulation in small arterial vessels of multiple tissues, 2) smooth muscle cells and pericytes in the brain, 3) gastric chief cells in the stomach, 4) neuronal subpopulations in the cortex as well as the polymorph layer of the dentate gyrus, 5) cell populations in the intermediate and anterior lobe of the pituitary gland, and 6) in the medulla of the adrenal gland. In further experiments, we aimed to decipher the function of Gpr30 by analyzing the phenotype of Gpr30-lacZ mice. The body weight as well as fat mass was unchanged in Gpr30-lacZ mice, even if fed with a high-fat diet. Flow cytometric analysis revealed lower frequencies of T cells in both sexes of Gpr30-lacZ mice. Within the T-cell cluster, the amount of CD62L-expressing cells was clearly reduced, suggesting an impaired production of T cells in the thymus of Gpr30-lacZ mice.

Dendritic cell microvilli: a novel membrane structure associated with the multifocal synapse and T-cell clustering

AbstractPolarizing effects of productive dendritic cell (DC)–T-cell interactions on DC cytoskeleton have been known in some detail, but the effects on DC membrane have been studied to a lesser extent. We found that T-cell incubation led to DC elongation and segregation of characteristic DC veils to the broader pole of the cell. On the opposite DC pole, we observed a novel membrane feature in the form of bundled microvilli. Each villus was approximately 100 nm in diameter and 600 to 1200 nm long. Microvilli exhibited high density of antigen-presenting molecules and costimulatory molecules and provided the physical basis for the multifocal immune synapse we observed during human DC and T-cell interactions. T cells preferentially bound to this site in clusters often contained both CD4+ and CD8+ T cells.

Ectopic Lymph Nodes Within Human Solid Tumors

Ectopic Lymph Nodes Within Human Solid Tumors

Long-Term Survival for Patients With Non–Small-Cell Lung Cancer With Intratumoral Lymphoid Structures

It has been established that the immune system plays an important role in tumor rejection. There is also compelling evidence that immune responses can develop independently of secondary lymphoid organs in tertiary lymphoid structures. We studied the presence and the correlation of tertiary lymphoid structures with clinical outcome in non-small-cell lung cancer (NSCLC), as the prognostic value of these structures in patients with cancer had not yet been established. This retrospective study was performed by immunohistochemistry on paraffin-embedded tissue specimens from 74 patients with early-stage NSCLC. Tertiary lymphoid structures were detected in some tumors but not in nontumoral lungs. Thus we called these structures tumor-induced bronchus-associated lymphoid tissue (Ti-BALT). As in lymph nodes, Ti-BALTs were composed of mature dendritic cell (DC)/T-cell clusters adjacent to B-cell follicles and had features of an ongoing immune response. Because the quantitative counting of Ti-BALT was difficult to achieve, we used mature DCs that homed exclusively in Ti-BALT as a specific marker of these structures. Univariate analysis showed that the density of mature DCs was highly associated with a favorable clinical outcome (overall, disease-specific, and disease-free survival), suggesting that Ti-BALT may participate in antitumoral immunity. The density of tumor-infiltrating lymphocytes, in particular, CD4(+) and T-bet(+) Th1 T cells, was profoundly decreased in tumors weakly infiltrated by mature DCs. The density of mature DCs was found to be a better predictor of clinical outcome than the other parameters tested. The number of tumor-infiltrating mature DCs may identify patients with early-stage NSCLC who have a high risk of relapse.

Establishment of the Porcine Epitheliochorial Placenta Is Associated with Endometrial T‐Cell Recruitment

We assessed implantation-associated quantitative changes in peripheral blood and endometrial T lymphocytes throughout epitheliochorial placenta formation. T-cell subsets were investigated in 10-, 15-, 20-, 30-, and 40-day pregnant and non-pregnant sows by flow cytometry and immunohistochemistry. Endometrial total T, T cytotoxic (Tc), and T helper (Th) cells were in peak numbers at the attachment phase of implantation and Tc cells persisted in high proportions up to placental establishment. The number of gammadelta T lymphocytes was relatively small and implantation-independent. In situ, T cells increased in number with the advancement of implantation and formed T-cell clusters with implantation phase-dependent location. Percentages of peripheral blood T cells were not significantly changed throughout the implantation. Superficial and adeciduate implantation of pigs has a profound effect on the number of total T, Tc, and Th cells and pattern of distribution of endometrial T cells in situ.

Persistent Antigen Presentation after Acute Vesicular Stomatitis Virus Infection

Long-term antigen expression is believed to play an important role in modulation of T-cell responses to chronic virus infections. However, recent studies suggest that immune responses may occur late after apparently acute infections. We have now analyzed the CD8 T-cell response to vesicular stomatitis virus (VSV), which is thought to cause to an infection characterized by rapid virus clearance by innate and adaptive immune system components. Unexpectedly, virus-encoded antigen was detectable more than 6 weeks after intranasal VSV infection in both draining and nondraining lymph nodes by adoptively transferred CD8 T cells. Infection with Listeria monocytogenes expressing the same antigen did not result in prolonged antigen presentation. Weeks after VSV infection, discrete T-cell clustering with dendritic cells within the lymph node was observed after transfer of antigen-specific CD8 T cells. Moreover, memory CD8 T cells as defined by phenotype and function were generated from naïve CD8 T cells entering the response late after infection. These findings suggested that protracted antigen presentation after an apparently acute virus infection may contribute to an ongoing antiviral immune response.

Generation of a synthetic lymphoid tissue–like organoid in mice

Stromal cells play an important role in the formation of the normal organized microarchitecture of secondary lymphoid organs. Here we demonstrate that a tissue-engineered, lymphoid tissue-like organoid, which was constructed by transplantation of stromal cells embedded in biocompatible scaffolds into the renal subcapsular space in mice, had an organized tissue structure similar to secondary lymphoid organs. This organoid contained compartmentalized B-cell and T-cell clusters, high endothelial venule-like vessels, germinal centers and follicular dendritic cell networks. Furthermore, the organoid was transplantable to naive normal or severe combined immunodeficiency (SCID) mice, and antigen-specific, IgG-isotype antibody formation could be induced soon after intravenous administration of the antigen. This simplified system of lymphoid tissue-like organoid construction will facilitate analyses of cell-cell interactions required for development of secondary lymphoid organs and efficient induction of adaptive immune responses, and may have possible applications in the treatment of immune deficiency.

Flow cytometric enumeration of antigen-specific T lymphocytes.

Until the second half of the 1990s, quantification of MHC-restricted, peptide-specific T lymphocytes required cell culture– based techniques of mononuclear cell suspensions that were often laborious, cumbersome, and prone to bias towards cellular subsets with growth advantage. Initially, limiting dilution assays were used. These assays include multiple days of culturing T lymphocytes in the presence of antigen and antigen-presenting cells, and the functional read-outs produced were T-cell proliferation (1) or cytolytic activity (2). However, such assays may not accurately reflect the function and frequency of T cells in vivo, due to selective proliferation and apoptosis that occur over time in culture. Alternatively, assays for T-cell activation have been developed based on the upregulation of the early activation marker CD69 (3) and/or the production of cytokines upon specific stimulation. The cytokines thus being secreted by the T cells can be measured

In situ analysis of the evolution of the primary immune response in murine Chlamydia trachomatis genital tract infection.

Adaptive immune responses contribute to the resolution of Chlamydia trachomatis genital tract infection and protect against reinfection, but our understanding of the mechanisms of those protective responses is incomplete. In this study, we analyzed by in situ immunohistochemistry the progression of the inflammatory and cytokine responses in the genital tracts of mice vaginally infected with C. trachomatis strain mouse pneumonitis. The cellular inflammatory response was characterized by an initial elevation in myeloid cells in the vagina (day 3) and uterine horns (day 7), followed by a marked rise in the number of T cells, predominantly CD4(+) cells. CD8(+) T cells and CD45R(+) B cells were also detected but were much less numerous. Perivascular clusters of CD4(+) T cells, which resembled clusters of T cells seen in delayed-type hypersensitivity responses, were evident by 2 weeks postinfection. Following the resolution of infection, few CD8(+) T cells and CD45R(+) B cells remained, whereas numerous CD4(+) T cells and perivascular clusters of CD4(+) T cells persisted in genital tract tissues. Interleukin-12 (IL-12)- and tumor necrosis factor alpha (TNF-alpha)-producing cells were observed in vaginal tissue by day 3 of infection and in uterine tissues by day 7. Cells producing IL-4 or IL-10 were absent from vaginal tissues at day 3 of infection but were present in uterine tissues by day 7 and were consistently more numerous than IL-12- and TNF-alpha-producing cells. Thus, the evolution of the local inflammatory response was characterized by the accumulation of CD4(+) T cells into perivascular clusters and the presence of cells secreting both Th1- and Th2-type cytokines. The persistence of CD4(+)-T-cell clusters long after infection had resolved (day 70) may provide for a readily mobilizable T-cell response by which previously infected animals can quickly respond to and control a secondary infectious challenge.

Human T-cell responses to Mycoplasma arthritidis-derived superantigen.

When injected into mice, Mycoplasma arthritidis causes a chronic arthritis that resembles rheumatoid arthritis, histologically. The organism produces a superantigen termed Mycoplasma arthritidis mitogen or MAM, that in humans preferentially expands T cells whose antigen receptors express V beta 17. T cells with this phenotype appear to be increased in rheumatoid synovial effusions. We describe a novel approach to isolating and characterizing human MAM-reactive T-cell lines and determining their T-cell receptor (TCR) V beta usage. Lines were prepared from T cells that clustered with dendritic cells during a 2-day exposure to MAM. Cluster and noncluster fractions of T cells were then expanded by using feeder cells and a polyclonal mitogen. Most of the MAM reactivity was found in dendritic T-cell clusters, as were most of the T cells expressing TCR V beta 17. After expansion, 76% of the cluster-derived T-cell lines were MAM reactive, while no reactivity was seen in cell lines derived from the noncluster fraction. Of the MAM-reactive lines, 49% expressed V beta 17 on some or all of the cells. Cell lines from both cluster and noncluster fractions were analyzed for TCR V beta mRNA expression by PCR amplification. Other V beta genes (5.1, 7, 8, 12, and 20) were found to be expressed by lines that were MAM reactive, although these were not a major component of the cluster-derived T cells. Some non-cluster-derived lines expressed V beta s 17, 12, and 7, but these proved to be nonreactive to MAM. Therefore, dendritic cells can be used to immunoselect and characterize T cells that express superantigen-reactive TCRs.

Plasmacytoid T-cell clusters in non-specific lymphadenitis.

The occurrence and morphology of Lymphoblastennester (clusters of lymphoblasts) were analyzed in 500 unselected cases of nonspecific lymphadenitis. Sixty-eight cases showed such clusters, which consist of uniform-looking medium-sized cells. Based on the results of recent immunologic investigations, these cells may be interpreted as T-cells with plasmacytoid features ("plasmacytoid T-cells', PTC). PTC were usually located near venules in the pulp, but not in the generally hyperplastic T-nodules. There was usually no relation to hyperplasia of B-regions (follicles). Although occasional mitotic figures and basophilic blast cells were found at the edges of PTC clusters, it is possible that PTC develop through transformation of T-lymphocytes. PTC often showed pyknotic nuclei and a tendency to perish, suggesting that they are end cells. Necrotic cells were phagocytosed by macrophages, which occasionally caused a starry sky pattern like that seen in germinal centers. Sometimes there were also a few interdigitating reticulum cells in the clusters. The function of PTC is still obscure; they might secrete lymphokines.