High expression of CD26 accurately identifies human bacteria‐reactive MR1‐restricted MAIT cells

Mucosa‐associated invariant T (MAIT) cells express the semi‐invariant T‐cell receptor TRAV1–2 and detect a range of bacteria and fungi through the MHC‐like molecule MR1. However, knowledge of the function and phenotype of bacteria‐reactive MR1‐restricted TRAV1–2⁺ MAIT cells from human blood is limited. We broadly characterized the function of MR1‐restricted MAIT cells in response to bacteria‐infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacteria‐reactive MR1‐restricted T cells shared effector functions of cytolytic effector CD8⁺ T cells. By analysing an extensive panel of phenotypic markers, we determined that CD26 and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8⁺ subsets we demonstrated that high expression of CD26 on CD8⁺ TRAV1–2⁺ cells identified with high specificity and sensitivity, bacteria‐reactive MR1‐restricted T cells from human blood. CD161^hi was also specific for but lacked sensitivity in identifying all bacteria‐reactive MR1‐restricted T cells, some of which were CD161^dim. Using cell surface expression of CD8, TRAV1–2, and CD26^hi in the absence of stimulation we confirm that bacteria‐reactive T cells are lacking in the blood of individuals with active tuberculosis and are restored in the blood of individuals undergoing treatment for tuberculosis.     

Processing‐dependent and ‐independent pathways for recognition of iodinated contrast media by specific human T cells

Background One to three percent of patients exposed to intravenously injected iodinated contrast media (CM) develop delayed hypersensitivity reactions. Positive patch test reactions, immunohistological findings, and CM‐specific proliferation of T cells in vitro suggest a pathogenetic role for T cells. We have previously demonstrated that CM‐specific T cell clones (TCCs) show a broad range of cross‐reactivity to different CM. However, the mechanism of specific CM recognition by T cell receptors (TCRs) has not been analysed so far. Objective To determine how T cells specifically recognize CM. Methods CM‐specific TCCs were generated from human blood of three CM‐allergic patients and a specific TCR was transfected into a mouse T cell hybridoma. Functional analysis such as proliferation assays, IL‐2 secretion assays, and calcium influx experiments were performed using irradiated, glutaraldehyde‐fixed, CM‐pre‐incubated, human leucocyte antigen (HLA)‐DR‐matched or ‐mismatched antigen‐presenting cells (APCs), and HLA‐blocking antibodies. Results We identified two mechanisms of T cell stimulation: some TCCs and the transfectant reacted to CM independent of uptake by APCs because proliferation/IL‐2 secretion occurred in the presence of glutaraldehyde‐fixed APCs, and intracellular calcium increased within seconds after drug addition. Other TCCs required functional APCs, compatible with uptake and presentation of CM on MHC‐class II molecules, as implied by three findings: (1) glutaraldehyde fixation of APCs abrogated presentation; (2) CM could not be washed away from CM‐pre‐incubated APCs; and (3) the optimal pulsing time was 10–20 h. Because allogeneic, MHC‐matched, CM‐pulsed APCs could induce proliferative responses as well, the ability of CM uptake and presentation is not unique to APCs from patients with CM‐induced delayed hypersensitivity. Conclusion Our data suggest that CM may be stimulatory for T cells either by direct binding to the MHC–TCR complex or by binding after uptake and processing by APCs. This questions the assumed inert nature of CM. Cite this as: M. Keller, M. Lerch, M. Britschgi, V. Tâche, B. O. Gerber, M. Lüthiuthi, P. Lochmatter, G. Kanny, A. J. Bircher, C. Christiansen and W. J. Pichler, Clinical & Experimental Allergy, 2010 (40) 257–268.     

Identification of a dengue virus‐specific HLA‐A*0201‐restricted CD8+ T cell epitope

In this study, a combination of epitope‐prediction programs and in vitro assays was used to identify dengue virus (DENV)‐specific CD8⁺ T cell epitopes. Peripheral blood mononuclear cells (PBMCs) isolated from patients who recovered from dengue fever were stimulated with candidate epitope peptides derived from DENV, which were predicted by using SYFPEITHI and RANKpep epitope‐prediction programs. The IFN‐γ ELISpot results and the results of intracellular staining of IFN‐γ showed that peptides NS4b_40 (TLYAVATTI), E_256 (QEGAMHTAL), NS3_205 (LPAIVREAI), NS5_210 (SRNSTHEMY), and NS3_207 (AIVREAIKR) could induce the recall response of CD8⁺ T cells. Furthermore, the results of the MHC–peptide complex stabilization assay revealed that peptide NS4b_40 (TLYAVATTI) has a high affinity for HLA‐A*0201 molecules. The IFN‐γ ELISpot results and staining of intracellular IFN‐γ confirmed that this peptide could induce high‐level CD8⁺ T cell response in HLA‐A*0201 positive PBMCs. Peptide NS4b_40 (TLYAVATTI) was identified as a novel DENV‐specific HLA‐A*0201‐restricted CD8⁺ T cell epitope. J. Med. Virol. 82:642–648, 2010. © 2010 Wiley‐Liss, Inc.     

Ultra‐high‐field RF coil development for evaluating upper extremity imaging applications

The purpose of this study is to develop and evaluate a custom‐designed 7  T MRI coil and explore its use for upper extremity applications. An RF system composed of a transverse electromagnetic transmit coil and an eight‐channel receive‐only array was developed for 7  T upper extremity applications. The RF system was characterized and evaluated using scattering parameters and B₁⁺ mapping. Finite difference time domain simulations were performed to evaluate the B₁⁺ field distribution and specific absorption rate for the forearm region of the upper extremity. High‐resolution 7  T images were acquired and compared with those at 3 T. The simulation and experimental results show very good B₁⁺ field homogeneity across the forearm. High‐resolution images of musculotendinous, osseocartilaginous, and neurovascular structures in the upper extremity are presented with T₁ volumetric interpolated breath‐hold examination, T₂ double‐echo steady state, T₂* susceptibility weighted imaging (SWI), diffusion tensor imaging, and time‐of‐flight sequences. Comparison between 3  T and 7  T is shown. Intricate contextual anatomy can be delineated in synovial, fibrocartilaginous, interosseous, and intraosseous trabecular structures of the forearm, as well as palmar and digital vascular anatomy (including microvascular detail in SWI). Ultra‐high‐field 7  T imaging holds great potential in improving the sensitivity and specificity of upper extremity imaging, especially in wrist and hand pathology secondary to bone, ligament, nerve, vascular, and other soft or hard tissue etiology.     

The flexibility of the TCR allows recognition of a large set of naturally occurring epitope variants by HIV-specific cytotoxic T lymphocytes.

Pathogens attempt to evade immune recognition by expressing mutated antigens. The present study shows that two mechanisms happen in vivo during the course of HIV infection to limit the escape of antigenic variants from cytotoxic T lymphocyte (CTL) recognition: recognition of several epitope variants by the same TCR and generation of several CTL populations specific for a single epitope but recognizing different variant sequences. We have studied two CTL populations directed towards the HIV-p24gag amino acids 176--184 QASQEVKNW epitope, presented by HLA-B5301. Both CTL populations were derived from a long-term asymptomatic HIV-infected child and they express different TCR. Each of the two CTL recognizes five of the 10 naturally occurring variants. These variants are distinct for both CTL and thus a total of eight variants are recognized. Thus, polyclonality of CTL specific for the same epitope but differing in variant sequences recognized may improve the control of variant viruses' replication in vivo. In addition to cross-recognition of several variant epitopes, promiscuous recognition of exogenous peptides complexed to allogeneic HLA-B molecules occurs, showing that the TCR can tolerate amino acid changes on both the peptide and the MHC molecule. This flexibility of the TCR is probably of great importance for control of viruses with high genetic variability, such as HIV.     

Proline substitution independently enhances H‐2Db complex stabilization and TCR recognition of melanoma‐associated peptides

The immunogenicity of H‐2D^b (D^b) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T‐cell cross‐reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D^b in complex with an optimized version of the melanoma‐associated epitope gp100_25–33. Furthermore, the p3P modification directly increased the affinity of the D^b/gp100_25–33‐specific T‐cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D^b/gp100_25–33 complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition.     

Development of an epitope panel for consistent identification of antigen‐specific T‐cells in humans

We aimed to establish a panel of MHC–peptide multimers suitable as a positive control in the detection of HLA A*0201 restricted antigen specific T cells (ASTC) by flow cytometry. MHC Dextramers were loaded with HLA A*0201 binding peptides from viral antigens and melanoma targets identified from a literature search and in silico prediction. Peripheral blood mononuclear cells (PBMC) from healthy donors were analysed with the MHC Dextramers using flow cytometry. The best performing epitopes were tested on PBMC from patients undergoing testing for Mycobacterium tuberculosis infection to assess the coverage of this epitope panel. Of 21 candidate epitopes, ASTC could be detected against 12 (57·1%) in at least one of 18 healthy blood donors. Reactivity to two or more epitopes was seen in 17 of the 18 donors (94·4%). We selected the six best‐performing epitopes and demonstrated a positive response in 42 (97·7%) of 43 patient samples (healthy, latent and active M. tuberculosis infection). The selected panel of six antigenic epitopes sufficed as a positive control in the detection of ASTC in HLA A*0201. Performance was robust in different stages of latent and active M. tuberculosis infection, indicating reliability also during infection.     

Diversity and clonotypic composition of influenza‐specific CD8+ TCR repertoires remain unaltered in the absence of Aire

TCR repertoire diversity is important for the protective efficacy of CD8⁺ T cells, limiting viral escape and cross‐reactivity between unrelated epitopes. The exact mechanism for selection of restricted versus diverse TCR repertoires is far from clear, although one thought is that the epitopes resembling self‐peptides might select a limited array of TCR due to the deletion of autoreactive TCR. The molecule Aire promotes the expression of tissue‐specific Ag on thymic medullary epithelial cells and the deletion of autoreactive cells, and in the absence of Aire autoreactive cells persist. However, the contribution of Aire‐dependent peptides to the selection of the Ag‐specific TCR repertoire remains unknown. In this study, we dissect restricted (D^bNP₃₆₆%⁺CD8⁺) and diverse (D^bPA₂₂₄%⁺CD8⁺, K^dNP₁₄₇%⁺CD8⁺) TCR repertoires responding to three influenza‐derived peptides in Aire‐deficient mice on both B6 and BALB/c backgrounds. Our study shows that the number, qualitative characteristics and TCR repertoires of all influenza‐specific, D^bNP₃₆₆%⁺CD8⁺, D^bPA₂₂₄%⁺CD8⁺ and K^dNP₁₄₇%⁺CD8⁺ T cells are not significantly altered in the absence of Aire. This provides the first demonstration that the selection of an Ag‐specific T‐cell repertoire is not significantly perturbed in the absence of Aire.     

Butyrophilin 3A (BTN3A,CD277)‐specific antibody 20.1 differentially activates Vγ9Vδ2 TCR clonotypes and interferes with phosphoantigen activation

Phosphoantigens (PAgs)‐like HMBPP ((E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate) and butyrophilin 3 (BTN3A, CD277)‐specific monoclonal antibody 20.1 induce TCR‐mediated activation of Vγ9Vδ2 T cells. Here, we compared murine reporter cells transduced with Vγ9Vδ2 TCRs G115, D1C55, and MOP for the activation in culture with human RAJI cells and PAgs or mAb 20.1 and its single‐chain (sc) derivative. All transductants responded readily to PAg but only TCR MOP γ‐chain‐expressing cells responded to mAb/sc 20.1. Furthermore, both antagonist and agonist mAb and sc of the agonist mAb inhibited the PAg response of TCR‐transduced murine reporter cells. These findings suggest that, in contrast to stimulation by physiological stimulators (PAg), the responsiveness to mAb 20.1 depends strongly on CDR3 sequences of the TCR, and that mAb 20.1 can interfere with the PAg‐response. Mouse or human origin of reporter cells might affect the mAb 20.1 response since all three TCR‐mediated mAb 20.1‐induced activation of TCR‐transduced Jurkat cells. The pronounced differences between PAg and mAb 20.1‐induced activation observed here help to understand the often contradictory published data. This study provides novel perspectives on the physiological mechanism of Vγ9Vδ2 T‐cell activation, and highlights the complex mode of action of BTN3A‐specific antibodies as agents in cancer immunotherapy.     

Next generation sequencing for TCR repertoire profiling: Platform‐specific features and correction algorithms

The TCR repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform‐specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of next generation sequencing applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.     

Organization of the resting TCR in nanoscale oligomers

Nucleotide oligomerization and binding domain (NOD)-like receptors (NLRs) are a major constituent of the cytosolic innate immune-sensing machinery and participate in a wide array of pathways including nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK), inflammasome, and type I interferon (IFN) signaling. NLRs have known roles in autoimmune, autoinflammatory, and infectious diseases. With respect to virus infection, NLRP3 is the most extensively studied NLR, including mechanisms of activation and inhibition. Furthermore, the importance of NLRP3 in both innate and adaptive immunity has been demonstrated. In comparison to NLRP3, the roles of other NLRs during virus infection are only just emerging. NLRC2 is an important activator of innate antiviral signaling and was recently found to mitigate inflammation during virus infection through autophagy. Finally, functions for NLRX1 in immune modulation and reactive oxygen species production require further examination and the importance of NLRC5 as a transactivator of major histocompatibility complex (MHC) class I and antigen presentation is currently developing. In this review, we discuss current knowledge pertaining to viruses and NLRs as well as areas of potential research, which will help advance the study of NLR biology during virus infection.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

Memory/effector T cells in TCR transgenic mice develop via recognition of enteric antigens by a second, endogenous TCR.

The majority of clonotypic CD4(+) T cells in the intestinal lamina propria of DO11.10 TCR transgenic mice have an activated/memory phenotype and produce effector cytokines despite the absence of prior exposure to ovalbumin (OVA), the transgene-specific antigen. A small number of splenic T cells have a similar phenotype. Clonotypic T cells from Peyer's patch are intermediate in both phenotype and effector cytokine production. Flow cytometric analysis of cells isolated from thymectomized, OVA-naive DO11.10 mice treated with continuous administration of BrdU indicated that a significant fraction of clonotype-positive T cells in the lamina propria and Peyer's patch were in the cell cycle, with significantly fewer cycling cells in the spleen. Most of the cycling cells from each anatomic site expressed low levels of CD45RB. Effector cytokine expression was enriched in the CD45RB(low) populations. These memory/effector cell populations were eliminated in DO11.10/SCID and DO11.10/RAG-2(-/-) mice, suggesting that recognition of non-OVA antigens through a second, non-clonotypic TCR was driving differentiation of memory/effector cells in naive BALB/c DO11.10 mice. Clonotypic CD4(+) T cells isolated from DO11.10, but not from DO11.10/SCID or DO11.10/RAG-2(-/-) mice, were stimulated to enter the cell cycle by antigen-presenting cells pulsed with an intestinal bacterial antigen extract. These data provide direct evidence that enteric bacterial antigens can activate transgenic T cells through a second, non-clonotypic TCR, and support the notion that the development and turnover of memory/effector cells in vivo is driven by the intestinal flora.     

2D TCR–pMHC–CD8 kinetics determines T‐cell responses in a self‐antigen‐specific TCR system

Two‐dimensional (2D) kinetic analysis directly measures molecular interactions at cell–cell junctions, thereby incorporating inherent cellular effects. By comparison, three‐dimensional (3D) analysis probes the intrinsic physical chemistry of interacting molecules isolated from the cell. To understand how T‐cell tumor reactivity relates to 2D and 3D binding parameters and to directly compare them, we performed kinetic analyses of a panel of human T‐cell receptors (TCRs) interacting with a melanoma self‐antigen peptide (gp100₂₀₉–₂₁₇) bound to peptide‐major histocompatibility complex in the absence and presence of co‐receptor CD8. We found that while 3D parameters are inadequate to predict T‐cell function, 2D parameters (that do not correlate with their 3D counterparts) show a far broader dynamic range and significantly improved correlation with T‐cell function. Thus, our data support the general notion that 2D parameters of TCR–peptide‐major histocompatibility complex–CD8 interactions determine T‐cell responsiveness and suggest a potential 2D‐based strategy to screen TCRs for tumor immunotherapy.     

On certain two‐signal perspectives of lymphocyte activation and inactivation,thymic G‐quadruplexes,and the role of aggregation in self/not‐self discrimination

Distinctive “two signal” paths in immunology, taken by researchers with different academic backgrounds, seem to have both contained facets of the truth. Having been influenced by education at a medical school where Almroth Wright’s early contributions were not forgotten, the author’s “path less followed” led to views that began to gain recognition late in the twentieth century when the intimate relationship between innate and acquired immunity became more apparent.     

Clonotype‐specific avidity influences the dynamics and hierarchy of virus‐specific regulatory and effector CD4+ T‐cell responses

A key component of immunity against viruses, CD4⁺ T cells expand and differentiate into functional subsets upon primary infection, where effector (Teff) cells facilitate infection control and regulatory (Treg) cells mitigate immunopathology. After secondary infection, Teff cells mount a robust response from the memory pool. Here, we show that Treg‐cell responses are diminished upon secondary infection, and Treg‐cell response dynamics are associated more with T‐cell receptors (TCRs) repertoire and avidity than with epitope specificity. In the murine model, the IA^bM₂₀₉ epitope of respiratory syncytial virus is recognized by both CD4⁺ Treg and Teff cells, while the IA^bM2₂₆ epitope is recognized almost exclusively by CD4⁺ Teff cells expressing high avidity TCR Vβ8.1/8.2 and dominating the CD4⁺ T‐cell response during primary and secondary infections. IA^bM₂₀₉‐Teff cells express relatively low avidity TCRs during early primary infection, but high avidity TCR Vβ7‐expressing IA^bM₂₀₉‐Teff cells emerge during the late phase, and become dominant after secondary infection. The emerging high avidity IA^bM₂₀₉‐Teff cells outcompete IA^bM₂₀₉‐Treg cells that share the same epitope, but have low avidity and are restricted to TCR Vβ2 and Vβ6 subpopulations. These data indicate that MHC‐peptide‐TCR interactions can produce different kinetic and functional profiles in CD4⁺ T‐cell populations even when responding to the same epitope.     

How chronic viral infections impact on antigen‐specific T‐cell responses

Persistent viral infections are, by definition, associated with ineffective antiviral immunity, in particular those infections caused by viruses that are highly productive and replicative (including HIV, HBV and HCV). The reasons for ineffective antiviral immunity in these types of infections are complex and manifold, and only recently a more comprehensive picture of the parameters responsible for attenuation of immune function is emerging. One reason for poor viral control in these types of infections is the functional deterioration of antiviral T‐cell responses and understanding the underlying mechanisms is of key importance. This review summarizes our current knowledge of cell‐intrinsic and cell‐extrinsic parameters that contribute to T‐cell exhaustion during chronic viral infections and discusses related implications for host survival, immunopathology, and control of infection.     

Central tolerance spares the private high‐avidity CD4+ T‐cell repertoire specific for an islet antigen in NOD mice

Although central tolerance induces the deletion of most autoreactive T cells, some autoreactive T cells escape thymic censorship. Whether potentially harmful autoreactive T cells present distinct TCRαβ features remains unclear. Here, we analyzed the TCRαβ repertoire of CD4⁺ T cells specific for the S100β protein, an islet antigen associated with type 1 diabetes. We found that diabetes‐resistant NOD mice deficient for thymus specific serine protease (TSSP), a protease that impairs class II antigen presentation by thymic stromal cells, were hyporesponsive to the immunodominant S100β_1‐15 epitope, as compared to wild‐type NOD mice, due to intrathymic negative selection. In both TSSP‐deficient and wild‐type NOD mice, the TCRαβ repertoire of S100β‐specific CD4⁺ T cells though diverse showed a specific bias for dominant TCRα rearrangements with limited CDR3α diversity. These dominant TCRα chains were public since they were found in all mice. They were of intermediate‐ to low‐avidity. In contrast, high‐avidity T cells expressed unique TCRs specific to each individual (private TCRs) and were only found in wild‐type NOD mice. Hence, in NOD mice, the autoreactive CD4⁺ T‐cell compartment has two major components, a dominant and public low‐avidity TCRα repertoire and a private high‐avidity CD4⁺ T‐cell repertoire; the latter is deleted by re‐enforced negative selection.