Rapid identification of the copy number of α-globin genes by capillary electrophoresis analysis

ObjectivesThe current study aimed at the rapid identification of the copy number of α-globin genes for the diagnosis of α-thalassemia.Design and methodsTo identify the copy number of α-globin genes in α-thalassemia, we developed a novel method using a multiplex polymerase chain reaction (PCR) in combination with the CE analysis.ResultsThe proposed method provides a rapid detection of the common α-globin gene deletions. Sixty-six patients with α-thalassemia and 46 normal controls were included in the present study. The obtained results showed good correlation with those obtained by gap PCR. Moreover, a low amount of maternal cell contamination in the fetus specimen for the prenatal diagnosis of hemoglobin Barts hydrops fetalis as well as the rare multiplicated α-globin genes can be identified using this method.ConclusionThis method provides a convenient and efficient tool for the rapid identification of the copy number of α-globin genes in α-thalassemia and the individuals with α-globin gene multiplication.     

Shank-interacting protein–like 1 promotes tumorigenesis via PTEN inhibition in human tumor cells

Inactivation of phosphatase and tensin homolog (PTEN) is a critical step during tumorigenesis, and PTEN inactivation by genetic and epigenetic means has been well studied. There is also evidence suggesting that PTEN negative regulators (PTEN-NRs) have a role in PTEN inactivation during tumorigenesis, but their identity has remained elusive. Here we have identified shank-interacting protein–like 1 (SIPL1) as a PTEN-NR in human tumor cell lines and human primary cervical cancer cells. Ectopic SIPL1 expression protected human U87 glioma cells from PTEN-mediated growth inhibition and promoted the formation of HeLa cell–derived xenograft tumors in immunocompromised mice. Conversely, siRNA-mediated knockdown of SIPL1 expression inhibited the growth of both HeLa cells and DU145 human prostate carcinoma cells in vitro and in vivo in a xenograft tumor model. These inhibitions were reversed by concomitant knockdown of PTEN, demonstrating that SIPL1 affects tumorigenesis via inhibition of PTEN function. Mechanistically, SIPL1 was found to interact with PTEN through its ubiquitin-like domain (UBL), inhibiting the phosphatidylinositol 3,4,5-trisphosphate (PIP₃) phosphatase activity of PTEN. Furthermore, SIPL1 expression correlated with loss of PTEN function in PTEN-positive human primary cervical cancer tissue. Taken together, these observations indicate that SIPL1 is a PTEN-NR and that it facilitates tumorigenesis, at least in part, through its PTEN inhibitory function.     

Mouse and human iNKT cell agonist β-mannosylceramide reveals a distinct mechanism of tumor immunity

Type 1 or invariant NKT (iNKT) cell agonists, epitomized by α-galactosylceramide, protect against cancer largely by IFN-γ-dependent mechanisms. Here we describe what we believe to be a novel IFN-γ-independent mechanism induced by β-mannosylceramide, which also defines a potentially new class of iNKT cell agonist, with an unusual β-linked sugar. Like α-galactosylceramide, β-mannosylceramide directly activates iNKT cells from both mice and humans. In contrast to α-galactosylceramide, protection by β-mannosylceramide was completely dependent on NOS and TNF-α, neither of which was required to achieve protection with α-galactosylceramide. Moreover, at doses too low for either alone to protect, β-mannosylceramide synergized with α-galactosylceramide to protect mice against tumors. These results suggest that treatment with β-mannosylceramide provides a distinct mechanism of tumor protection that may allow efficacy where other agonists have failed. Furthermore, the ability of β-mannosylceramide to synergize with α-galactosylceramide suggests treatment with this class of iNKT agonist may provide protection against tumors in humans.     

Genetic modification of T cell clones for therapy of human viral and malignant diseases

Our laboratory has pursued T cell therapy withantigen-specific T cell clones as means to treat humandisease. Our studies in the prevention of CMV disease     

Extrafollicular B cell activation by marginal zone dendritic cells drives T cell–dependent antibody responses

Dendritic cells (DCs) are best known for their ability to activate naive T cells, and emerging evidence suggests that distinct DC subsets induce specialized T cell responses. However, little is known concerning the role of DC subsets in the initiation of B cell responses. We report that antigen (Ag) delivery to DC-inhibitory receptor 2 (DCIR2) found on marginal zone (MZ)–associated CD8α⁻ DCs in mice leads to robust class-switched antibody (Ab) responses to a T cell–dependent (TD) Ag. DCIR2⁺ DCs induced rapid up-regulation of multiple B cell activation markers and changes in chemokine receptor expression, resulting in accumulation of Ag-specific B cells within extrafollicular splenic bridging channels as early as 24 h after immunization. Ag-specific B cells primed by DCIR2⁺ DCs were remarkably efficient at driving naive CD4 T cell proliferation, yet DCIR2-induced responses failed to form germinal centers or undergo affinity maturation of serum Ab unless toll-like receptor (TLR) 7 or TLR9 agonists were included at the time of immunization. These results demonstrate DCIR2⁺ DCs have a unique capacity to initiate extrafollicular B cell responses to TD Ag, and thus define a novel division of labor among splenic DC subsets for B cell activation during humoral immune responses.Upon recognition of T cell–dependent (TD) or T cell–independent (TI) antigen (Ag), B cells differentiate into short-lived antibody (Ab)-forming cells (AFCs), which are critical for providing frontline protection against the spread of blood-borne pathogens such as Salmonella typhimurium and influenza (Gerhard et al., 1997; Cunningham et al., 2007; Rothaeusler and Baumgarth, 2010). Alternatively, cognate interaction of B cells with CD4⁺ T cells results in the formation of germinal centers (GCs) and selection of high-affinity clones for differentiation to memory B cells and long-lived plasma cells (Jacob et al., 1991).  Although GC responses and affinity maturation have been extensively studied, much less is known concerning the early events that govern B cell activation and how they influence the decision to produce extrafollicular AFC responses versus GC B cell differentiation.The context in which B cells encounter Ag is highly influenced by the size, nature, and form of the Ag itself (Roozendaal et al., 2009). Although direct recognition of small, soluble Ag by the BCR can occur in vivo (Pape et al., 2007), acquisition of membrane-associated Ag is also an efficient means to trigger B cell activation (Carrasco and Batista, 2006; Depoil et al., 2008). Multiple APCs can present Ag to B cells including follicular DCs, subcapsular sinus and marginal zone (MZ) macrophages, and DCs (Wykes and MacPherson, 2000; Huang et al., 2005; Qi et al., 2006; Phan et al., 2009; Roozendaal et al., 2009; Suzuki et al., 2009). Among these, DCs in particular have been shown in vitro to influence a range of B cell processes including proliferation, differentiation, and Ig class-switch recombination (CSR; Dubois et al., 1998; Fayette et al., 1998; Litinskiy et al., 2002; Craxton et al., 2003). DC-mediated presentation of Ag to B cells in vivo has been shown to enhance TI Ab responses to immune complexes internalized by FcγRIIb on splenic DCs, as well as TI responses against Streptococcus pneumoniae mediated by blood-derived DCs (Balázs et al., 2002; Bergtold et al., 2005; Dubois and Caux, 2005).In contrast, it is unclear what, if any, role DC–B cell interactions may have during humoral responses to TD Ag. Some evidence has suggested that DCs directly present Ag to B cells during TD immune responses. Qi et al. (2006) showed that adoptively transferred DCs can transfer hen egg lysozyme (HEL) to Ag-specific B cells in the lymph node; however, neither the DC subset responsible for the Ag presentation nor the subsequent B cell response was evaluated. Earlier studies showed that adoptive transfer of Ag-bearing DCs was sufficient to induce TD Ab responses; however, because adoptive transfer strategies were used, the role of Ag uptake in situ by resident DC subsets remained unclear (Wykes et al., 1998; Berney et al., 1999). More recent studies using mAbs to deliver Ag directly to APCs in vivo demonstrated Ab responses after Ag uptake by several C-type lectin receptors (CLRs) including FIRE (F4/80-like receptor), CIRE (C-type lectin immune receptor), Dectin-1, Clec12a, and Clec9a (Corbett et al., 2005; Caminschi et al., 2008; Lahoud et al., 2009). Because the CLRs targeted in these studies are also expressed on macrophages, plasmacytoid DCs (pDCs), and/or B cells, it is again unclear which APC populations were required for the observed Ab induction. In sum, many questions remain concerning the induction of Ab responses by DCs.Here, we describe a novel mechanism underlying DC-mediated induction of Ab responses after Ag uptake by DC-inhibitory receptor 2 (DCIR2), a CLR found exclusively on a subset of MZ-associated CD8α⁻ DCs (Dudziak et al., 2007). Using mAbs to deliver Ag in vivo, we show that Ag uptake by DCIR2, but not DEC205 found on CD8α⁺ DCs, induces robust IgG1-restricted TD Ab responses without the addition of any adjuvant. Although DCIR2⁺ DCs are known to preferentially present peptide–MHCII complexes to CD4 T cells after Ag delivery to DCIR2 (Dudziak et al., 2007), we found that DCIR2⁺ DCs also present Ag to B cells and facilitate their rapid activation in vivo. As early as 24 h after immunization, activated Ag-specific B cells accumulated in extrafollicular splenic bridging channels, coincident with the location of DCIR2⁺ DCs in situ. Importantly, DC-primed Ag-specific B cells were highly efficient APCs for driving naive CD4 T cell proliferation, suggesting that B cell–mediated Ag presentation is a key component to sustaining CD4 T cell responses after Ag delivery to DCs. In the absence of adjuvants, DCIR2 DCs induced extrafollicular Ab responses that did not involve GC formation or affinity maturation of serum Ab. Instead, inclusion of agonists for toll-like receptor (TLR) 7 or TLR9 (but not TLR3, TLR5, or RIG-I) shifted the B cell differentiation program toward GC formation and affinity maturation. Thus, our data show that DCIR2⁺ MZ-associated DCs are uniquely equipped and/or positioned to prime B cells and initiate extrafollicular Ab responses to TD Ag.     

Thromboxane A2 acts as tonic immunoregulator by preferential disruption of low-avidity CD4+ T cell–dendritic cell interactions

Interactions between dendritic cells (DCs) and T cells control the decision between activation and tolerance induction. Thromboxane A2 (TXA2) and its receptor TP have been suggested to regulate adaptive immune responses through control of T cell–DC interactions. Here, we show that this control is achieved by selectively reducing expansion of low-avidity CD4⁺ T cells. During inflammation, weak tetramer-binding TP-deficient CD4⁺ T cells were preferentially expanded compared with TP-proficient CD4⁺ T cells. Using intravital imaging of cellular interactions in reactive peripheral lymph nodes (PLNs), we found that TXA2 led to disruption of low- but not high-avidity interactions between DCs and CD4⁺ T cells. Lack of TP correlated with higher expression of activation markers on stimulated CD4⁺ T cells and with augmented accumulation of follicular helper T cells (T_FH), which correlated with increased low-avidity IgG responses. In sum, our data suggest that tonic suppression of weak CD4⁺ T cell–DC interactions by TXA2–TP signaling improves the overall quality of adaptive immune responses.T cells have evolved to quickly react to potentially dangerous microbes by recognizing pathogen-derived peptide (p)-MHC complexes displayed on antigen-presenting cells, in particular DCs. Because T cells are selected in the thymus for their ability to recognize self-pMHC complexes (Morris and Allen, 2012) and numerous self-reactive T cells are released into the periphery (Su et al., 2013), peripheral tolerance education is critical to avoid activation of autoreactive T cells. Studies using intravital two-photon microscopy (2PM) of reactive PLNs have shed light on the dynamic T cell–DC interactions and their correlation with full versus curtailed T cell activation and tolerance induction. The amount of cognate pMHC complexes on activated DCs is critical in determining the transition of a highly motile scanning-mode T cell to an immotile, stably interacting one (Cahalan and Parker, 2006; Henrickson and von Andrian, 2007; Bajénoff and Germain, 2007). Such stable T cell–DC interactions (>8h) are a prerequisite for full effector T cell differentiation (Rachmilewitz and Lanzavecchia, 2002). Thus, in presence of high amounts of cognate pMHC on activated DCs, T cells decelerate rapidly, whereas T cells show a motile DC sampling behavior when cognate pMHC levels are low. Altered peptide ligands (APLs) with reduced affinity for a given TCR also decrease the length of T cell–DC interactions, limiting T cell activation. Under tolerogenic conditions (i.e., in the absence of co-stimulation), 2PM studies uncovered shortened T cell–DC interactions (Hugues et al., 2004) although this is still controversial (Shakhar et al., 2005). Similarly, the presence of regulatory T (T reg) cells reduces T cell–DC interactions and subsequent T cell activation (Tadokoro et al., 2006; Tang et al., 2006).A perhaps counterintuitive recent finding has revealed a significant increase in CD8⁺ T cell immune response avidity in presence of T reg cells (Pace et al., 2012). This is due to T reg cell–mediated suppression of excessive interactions between DCs and CD8⁺ T cells bearing TCRs with low avidity for pMHC complexes. In the absence of T reg cells, uncontrolled CCR5 ligand secretion by activated DCs induces attraction of bystander TCR clones with low affinity for pMHC complexes, which decreases overall avidity and memory T cell generation of the resulting immune response. Whether a comparable mechanism also exists to selectively support activation of high avidity CD4⁺ T cells by immunoregulatory factors is currently unknown.The short-lived arachidonic acid–derived lipid thromboxane A2 (TXA2) has been suggested to regulate adaptive immune responses (Kabashima et al., 2003). Activated DCs and other cell types produce TXA2, which binds its G-protein coupled receptor TP expressed in thymocytes and naive but not effector/memory CD4⁺ and CD8⁺ T cells. Addition of high amounts of the TP agonist I-BOP induces chemokinesis in naive T cells and decreases in vitro aggregate formation between T cells and DCs, causing reduced T cell activation (Kabashima et al., 2003). Combined with the observation that TXA2 levels rapidly rise in reactive PLN during immune responses (Moore et al., 1989), these data suggest a model where TXA2 may act as a general suppressor of T cell–DC interactions. In line with this hypothesis, aged TP-deficient T cells develop lymphoid hyperplasia and high antibody titers (Kabashima et al., 2003). Yet, it has remained unknown how TXA2 signaling affects dynamic CD4⁺ T cell interactions with DC displaying varying pMHC abundance and affinity in vivo, and how this impacts avidity patterns of responding T cells.Here, we show that during sterile and microbial inflammation, absence of TP resulted in increased expansion of low-avidity CD4⁺ T cells. Using 2PM imaging of cellular interactions in reactive PLNs, we report that paracrine TXA2 signaling preferentially disrupted low-avidity interactions between DCs and OT-II CD4⁺ T cells induced by low cognate pMHC levels or low-affinity peptide. As a consequence, TP^−/− OT-II CD4⁺ T cells show increased expression of early activation markers, as well as augmented accumulation of follicular helper T cells (T_FH) compared with WT OT-II CD4⁺ cells. High numbers of TP^−/− T_FH correlated with increased low-avidity IgG production, thus thwarting the overall quality of the adaptive immune response. In sum, our data uncover a previously unappreciated contribution of a tolerance-inducing mechanism for preferential activation of high avidity CD4⁺ T cells.     

Outcomes of acute leukemia patients transplanted with naive T cell–depleted stem cell grafts

BACKGROUND. Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HCT). In mice, naive T cells (T_N) cause more severe GVHD than memory T cells (T_M). We hypothesized that selective depletion of T_N from human allogeneic peripheral blood stem cell (PBSC) grafts would reduce GVHD and provide sufficient numbers of hematopoietic stem cells and T_M to permit hematopoietic engraftment and the transfer of pathogen-specific T cells from donor to recipient, respectively.METHODS. In a single-arm clinical trial, we transplanted 35 patients with high-risk leukemia with T_N-depleted PBSC grafts following conditioning with total body irradiation, thiotepa, and fludarabine. GVHD prophylactic management was with tacrolimus immunosuppression alone. Subjects received CD34-selected PBSCs and a defined dose of T_M purged of CD45RA⁺ T_N. Primary and secondary objectives included engraftment, acute and chronic GVHD, and immune reconstitution.RESULTS. All recipients of T_N-depleted PBSCs engrafted. The incidence of acute GVHD was not reduced; however, GVHD in these patients was universally corticosteroid responsive. Chronic GVHD was remarkably infrequent (9%; median follow-up 932 days) compared with historical rates of approximately 50% with T cell–replete grafts. T_M in the graft resulted in rapid T cell recovery and transfer of protective virus-specific immunity. Excessive rates of infection or relapse did not occur and overall survival was 78% at 2 years.CONCLUSION. Depletion of T_N from stem cell allografts reduces the incidence of chronic GVHD, while preserving the transfer of functional T cell memory.TRIAL REGISTRATION. ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT 00914940","term_id":"NCT00914940"}}NCT 00914940).FUNDING. NIH, Burroughs Wellcome Fund, Leukemia and Lymphoma Society, Damon Runyon Cancer Research Foundation, and Richard Lumsden Foundation.     

Suppressing T cell motility induced by anti–CTLA-4 monotherapy improves antitumor effects

A promising strategy for cancer immunotherapy is to disrupt key pathways regulating immune tolerance, such as cytotoxic T lymphocyte–associated protein 4 (CTLA-4). However, the determinants of response to anti–CTLA-4 mAb treatment remain incompletely understood. In murine models, anti–CTLA-4 mAbs alone fail to induce effective immune responses to poorly immunogenic tumors but are successful when combined with additional interventions, including local ionizing radiation (IR) therapy. We employed an established model based on control of a mouse carcinoma cell line to study endogenous tumor-infiltrating CD8⁺ T lymphocytes (TILs) following treatment with the anti–CTLA-4 mAb 9H10. Alone, 9H10 monotherapy reversed the arrest of TILs with carcinoma cells in vivo. In contrast, the combination of 9H10 and IR restored MHC class I–dependent arrest. After implantation, the carcinoma cells had reduced expression of retinoic acid early inducible–1 (RAE-1), a ligand for natural killer cell group 2D (NKG2D) receptor. We found that RAE-1 expression was induced by IR in vivo and that anti-NKG2D mAb blocked the TIL arrest induced by IR/9H10 combination therapy. These results demonstrate that anti–CTLA-4 mAb therapy induces motility of TIL and that NKG2D ligation offsets this effect to enhance TILs arrest and antitumor activity.     

Memory T cell–driven differentiation of naive cells impairs adoptive immunotherapy

Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell–T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory–induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell–based immunotherapies.     

MHC class II–restricted antigen presentation by plasmacytoid dendritic cells inhibits T cell–mediated autoimmunity

Although plasmacytoid dendritic cells (pDCs) express major histocompatibility complex class II (MHCII) molecules, and can capture, process, and present antigens (Ags), direct demonstrations that they function as professional Ag-presenting cells (APCs) in vivo during ongoing immune responses remain lacking. We demonstrate that mice exhibiting a selective abrogation of MHCII expression by pDCs develop exacerbated experimental autoimmune encephalomyelitis (EAE) as a consequence of enhanced priming of encephalitogenic CD4⁺ T cell responses in secondary lymphoid tissues. After EAE induction, pDCs are recruited to lymph nodes and establish MHCII-dependent myelin-Ag–specific contacts with CD4⁺ T cells. These interactions promote the selective expansion of myelin-Ag–specific natural regulatory T cells that dampen the autoimmune T cell response. pDCs thus function as APCs during the course of EAE and confer a natural protection against autoimmune disease development that is mediated directly by their ability to present of Ags to CD4⁺ T cells in vivo.Conventional DCs (cDCs) play well established roles in the induction of immunity and tolerance. Both functions require antigen (Ag)-specific interactions between T cells and cDCs in secondary lymphoid tissues. The outcome of these interactions depends on the modulation and integration of three signals: TCR engagement by peptide–MHC complexes, the recruitment of costimulatory and adhesion molecules, and the delivery of soluble mediators (Lebedeva et al., 2005). Under steady-state conditions, cDCs reside in peripheral tissues and lymphoid organs in an immature state characterized by low cell surface expression of MHC class II (MHCII), costimulatory, and adhesion molecules. Immature cDCs continuously capture and present self-Ags, circulate from tissues to lymphoid organs, and maintain tolerance by inducing the deletion of autoreactive T cells or the development of regulatory T cells (T reg cells; Steinman et al., 2003). Signals associated with inflammation, infections, or tissue damage induce cDC maturation, a process involving complex phenotypical changes, including the up-regulation of MHCII, costimulatory, and adhesion molecules, the secretion of inflammatory mediators, and altered migratory properties. Activation of naive T cells by mature cDCs results in clonal expansion and differentiation into effector and memory T cells.Plasmacytoid DCs (pDCs) constitute a unique DC subtype found mainly in the blood and secondary lymphoid organs. The activation of pDCs by infections triggers the secretion of large quantities of type I IFN, suggesting that they have crucial innate functions (Colonna et al., 2004). However, pDCs also express MHCII molecules and undergo a maturation process similar to that of cDCs (Villadangos and Young, 2008). Furthermore, pDCs can internalize, process, and present Ags to CD4⁺ T cells and cross-present Ags to CD8⁺ T cells (Hoeffel et al., 2007; Sapoznikov et al., 2007; Di Pucchio et al., 2008; Young et al., 2008). These findings had suggested that pDCs can function as APCs. However, whether pDCs indeed function as APCs in vivo during ongoing immune responses, and whether this promotes T cell–mediated immunity and/or the maintenance of self-tolerance, remained unsolved issues.pDCs can participate in the maintenance of peripheral tolerance. The induction of T reg cells by pDCs was shown to confer tolerance to cardiac allografts, prevent asthmatic reactions to inhaled Ags, and protect against graft versus host disease (de Heer et al., 2004; Ochando et al., 2006; Hadeiba et al., 2008). pDCs can also induce tolerance by promoting deletion of pathogenic T cells (Goubier et al., 2008) or inhibiting effector CD4⁺ T cell responses in a relapsing model of experimental autoimmune encephalomyelitis (EAE; Bailey-Bucktrout et al., 2008). As these studies relied mainly on antibody-mediated ablation of pDCs, they could not discriminate between innate and adaptive functions of these cells. It therefore remained unknown if pDCs function as tolerogenic APCs in these systems.We have investigated whether MHCII-mediated Ag presentation by pDCs instructs CD4⁺ T cell responses during EAE, a mouse model for multiple sclerosis (MS; Wekerle, 2008). EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG) was found to be severely exacerbated in mice exhibiting a selective abrogation of MHCII expression by pDCs. Conversely, EAE was dampened by the adoptive transfer of WT, but not MHCII-deficient, pDCs. EAE induction triggered the recruitment of pDCs to LNs, where they engaged in MHCII-dependent and MOG-specific interactions with CD4⁺ T cells. This inhibited the development of pathogenic T cells during the priming phase of the disease by promoting the selective expansion of natural T reg cells. Our results demonstrate that Ag-presentation by pDCs can inhibit T cell–mediated autoimmunity and can thus determine the outcome of adaptive immune responses in vivo.     

Myocardial scar identification based on analysis of Look–Locker and 3D late gadolinium enhanced MRI

The aim of this study is to introduce and evaluate an approach for objective and reproducible scar identification from late gadolinium enhanced (LGE) MR by analysis of LGE data with post-contrast T₁ mapping from a routinely acquired T₁ scout Look–Locker (LL) sequence. In 90 post-infarction patients, a LL sequence was acquired prior to a three-dimensional LGE sequence covering the entire left ventricle. In 60/90 patients (training set), the T₁ relaxation rates of remote myocardium and dense myocardial scar were linearly regressed to that of blood. The learned linear relationship was applied to 30/90 patients (validation set) to identify the remote myocardium and dense scar, and to normalize the LGE signal intensity to a range from 0 to 100 %. A 50 % threshold was applied to identify myocardial scar. In the validation set, two observers independently performed manual scar identification, annotated reference regions for the full-width-half-maxima (FWHM) and standard deviation (SD) method, and analyzed the LL sequence for the proposed method. Compared with the manual, FWHM, and SD methods, the proposed method demonstrated the highest inter-class correlation coefficient (0.997) and Dice overlap index (98.7 ± 1.3 %) between the two observers. The proposed method also showed excellent agreement with the gold-standard manual scar identification, with a Dice index of 89.8 ± 7.5 and 90.2 ± 6.6 % for the two observers, respectively. Combined analysis of LL and LGE sequences leads to objective and reproducible myocardial scar identification in post-infarction patients.     

Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell–mediated clearance of HIV-1–infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity–mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4⁺ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8⁺ T cell clearance of CD4⁺ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected–patient resting CD4⁺ T cells. Moreover, DARTs mediated CD8⁺ T cell clearance of HIV from resting CD4⁺ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals.     

The molecular bases of δ/αβ T cell–mediated antigen recognition

αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1⁺ human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity.αβ and γδ T cells, which express highly polymorphic TCRs on their surface, play a vital role in immunity. In humans, the majority of T cells use TCRs derived from the α and β TCR gene loci, whereupon the αβTCR architecture is composed of the variable (Vα), joining (Jα), and constant (Cα) gene segments that form the TCR-α chain, whereas the Vβ, Dβ (diversity), Jβ, and Cβ gene segments constitute the TCR-β chain (Turner et al., 2006). Multiple TCR genes within the α and β loci, coupled with random nucleotide (N) additions at V-(N)-J, V-(N)-D, and D-(N)-J junctional regions, underpin the vast αβTCR repertoire (Turner et al., 2006). This diversity is manifested within the Vα and Vβ domains, each of which contains three complementarity-determining regions (CDRs), collectively forming the antigen (Ag) recognition site of the αβTCR. The αβ T cell diversity provides the capability of αβTCRs to recognize a range of antigenic determinants presented by polymorphic and monomorphic Ag-presenting molecules (Godfrey et al., 2008; Bhati et al., 2014).αβTCRs are typically considered to recognize short peptide (p) fragments bound within the Ag-binding cleft of molecules encoded by the polymorphic MHC. Here, the αβTCR accommodates a wide range of pMHC landscapes with a polarized and approximately conserved docking mode, whereby the Vα and Vβ domains are positioned over the α2 and α1 helices of MHC-I, respectively (Gras et al., 2012). Alternately, some αβ T cells are activated by lipid-based Ags presented by MHC-I–like molecules belonging to the CD1 family (Brigl and Brenner, 2004). The CD1d system, which presents lipid Ags to type I and type II NKT cells, is the best understood in terms of lipid Ag recognition (Girardi and Zajonc, 2012; Rossjohn et al., 2012). Here, a semi-invariant NKT TCR (Vα24-Jα18 in humans), which typifies type I NKT cells, binds a wide range of chemically distinct ligands in a conserved docking mode, whereby the TCR sits in a parallel manner above the F′ pocket of CD1d (Rossjohn et al., 2012). As such, the NKT TCR has been likened to an innate-like pattern recognition receptor (Scott-Browne et al., 2007). In contrast, type II NKT cells can adopt differing docking strategies in binding to CD1d-restricted lipid-based ligands and exhibit features that more closely resemble that of αβTCR recognition in adaptive immunity (Girardi et al., 2012; Patel et al., 2012; Rossjohn et al., 2012). It has also recently been established that mucosal-associated invariant T cells (MAIT cells), which express a semi-invariant αβTCR, recognize vitamin B–based metabolites presented by the monomorphic MHC-I–related protein (MR1; Kjer-Nielsen et al., 2012; Corbett et al., 2014). Here, the MAIT TCR draws upon features typified by innate and adaptive immunity in recognizing these small molecule metabolites (Patel et al., 2013; Eckle et al., 2014). Accordingly, the αβTCR lineage shows remarkable versatility in recognizing three distinct classes of ligands (Bhati et al., 2014).The γδ T cell lineage uses γδTCRs that are derived from the γ and δ TCR gene loci (O’Brien et al., 2007; Vantourout and Hayday, 2013). γδ T cells and αβ T cells develop from common intrathymic precursors but branch into separate lineages at the time when they undergo TCR gene rearrangement and differentiation (Xiong and Raulet, 2007; Ciofani and Zúñiga-Pflücker, 2010). γδ T cells rearrange Vγ and Jγ genes that join to the γ constant (Cγ) gene to form the TCR-γ chain, whereas rearrangement of Vδ, Dδ, and Jδ genes join to the δ constant (Cδ) gene to form the TCR-δ chain. Similar to αβTCRs, γδTCRs possess six CDR loops, three from each chain, which mediate Ag recognition (Bhati et al., 2014). The number of Vγ and Vδ genes in humans is relatively low (8× Vδ and 6× Vγ genes), and further limitation in repertoire diversity comes from restricted pairing of particular Vδ and Vγ genes. However, the potential to use the three Dδ genes, even in multiple copies, combined with N region modifications, dramatically increases TCR-δ diversity (O’Brien et al., 2007; Born et al., 2013). In contrast to αβ T cells, in which Vα and Vβ TCR chains are generally very diverse, some Vγ and Vδ TCR chains show tissue-specific and functional biases. For example, Vδ2⁺ γδ T cells tend to produce inflammatory cytokines such as IFN-γ and TNF, predominate in human blood, and migrate to sites of inflammation, whereas Vδ1⁺ γδ T cells tend to produce regulatory cytokines such as IL-10 and home to noninflamed tissues such as spleen and gut (O’Brien et al., 2007). Compared with αβ T cells, much less is known about what types of Ags are recognized by γδ T cells, although it is generally accepted that γδTCRs confer different specificity and functional characteristics (Vantourout and Hayday, 2013). Some γδTCRs can recognize Ags directly, whereas other studies have demonstrated that γδTCRs can recognize cell surface and soluble protein and peptide Ags and microbial metabolites in the absence of classical Ag-presenting molecules (Born et al., 2013; Vavassori et al., 2013; Sandstrom et al., 2014). Some γδ T cells can respond to Ag-presenting molecules in a ligand-independent manner, such as the MHC-II molecule or the MHC class I–like molecules T10/T22 and endothelial protein C receptor (EPCR), whereas others can recognize lipid-based Ags presented by members of the CD1 family (Born et al., 2013). The molecular bases of γδTCR recognition of CD1d-lipid Ag complexes were recently reported (Luoma et al., 2013; Uldrich et al., 2013).Thus, αβ T cells and γδ T cells act in concert, using distinct TCRs to survey a wide range of Ags to enable protective immunity. Interestingly, the human Vδ gene locus is embedded within the Vα locus, and some human Vδ genes (Vδ4-Vδ8) encoded by TRDV 4, 5, 6, 7, and 8 are also referred to as Vα genes (Vα6, 21, 17, 28, and 14.1) encoded by TRAV 14, 23, 29, 36, and 38-2, respectively (Lefranc and Rabbitts, 1990), because these are capable of rearranging to either Dδ-Jδ-Cδ or Jα-Cα genes. Because these V genes can be used by both γδ and αβ T cell lineages, when paired with Cα, they are termed Vα genes, whereas they are termed Vδ genes when paired with Cδ (Lefranc and Rabbitts, 1990). However, the majority of human γδ T cells use the Vδ1, Vδ2, and Vδ3 variable regions, encoded by TRDV 1, 2, and 3 genes (O’Brien et al., 2007; Mangan et al., 2013). Although these do not have alternate TRAV names, these can also rearrange to Jα-Cα genes, and at least Vδ1 and Vδ3 can be expressed as a functional Vδ-Jα-Cα TCR chain that can pair with a functional TCR-β chain (Miossec et al., 1990; Peyrat et al., 1995). Here we describe a flow cytometry–based method for identifying Vδ1⁺ TCR-β⁺ cells, which we have termed δ/αβ T cells, based on their expression of TCRs comprising a TCR-δ variable gene 1 (Vδ1) joined to a TCR Jα and TCR Cα genes and paired with an array of TCR-β chains. These δ/αβ T cells were readily detectable in most humans and included cells with specificity for both peptide- and lipid-based Ags presented by MHC-I molecules and CD1d, respectively. We have determined the cell surface phenotype, Ag specificity, and functional capacity of a population of these cells. Using x-ray crystallography, we have elucidated the structural architecture of two δ/αβTCRs and show how these TCRs can recognize monomorphic and polymorphic Ag-presenting molecules via distinct mechanisms. Accordingly, we highlight a population of δ/αβ T cells that bind Ag by way of both Vδ and Vβ genes, thus reflecting a greater level of diversity and functional potential within the T cell lineage.     

CD4+ T helper cells use CD154–CD40 interactions to counteract T reg cell–mediated suppression of CD8+ T cell responses to influenza

CD4⁺ T cells promote CD8⁺ T cell priming by licensing dendritic cells (DCs) via CD40–CD154 interactions. However, the initial requirement for CD40 signaling may be replaced by the direct activation of DCs by pathogen-derived signals. Nevertheless, CD40–CD154 interactions are often required for optimal CD8⁺ T cell responses to pathogens for unknown reasons. Here we show that CD40 signaling is required to prevent the premature contraction of the influenza-specific CD8⁺ T cell response. CD40 is required on DCs but not on B cells or T cells, whereas CD154 is required on CD4⁺ T cells but not CD8⁺ T cells, NKT cells, or DCs. Paradoxically, even though CD154-expressing CD4⁺ T cells are required for robust CD8⁺ T cell responses, primary CD8⁺ T cell responses are apparently normal in the absence of CD4⁺ T cells. We resolved this paradox by showing that the interaction of CD40-bearing DCs with CD154-expressing CD4⁺ T cells precludes regulatory T cell (T reg cell)–mediated suppression and prevents premature contraction of the influenza-specific CD8⁺ T cell response. Thus, CD4⁺ T helper cells are not required for robust CD8⁺ T cell responses to influenza when T reg cells are absent.Primary CD8⁺ T cell responses often require help from CD4⁺ T cells, which produce cytokines and provide co-stimulation, including the engagement of CD40 by its ligand CD154 (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998). In one model, CD4⁺ T cells engage CD40 on DCs and license them to become efficient antigen-presenting cells for naive CD8⁺ T cells (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998). However, other models suggest that CD4⁺ T cells provide help to CD8⁺ T cells by activating B cells and promoting CD40-dependent antibody responses (Bachmann et al., 2004) or that they engage CD40 on CD8⁺ T cells (Bourgeois et al., 2002) and directly promote CD8⁺ T cell activation or survival.Interestingly, CD4⁺ T cell help is not required to prime all CD8⁺ T cells responses. Whereas CD8⁺ T cell responses to noninflammatory antigens are impaired in the absence of CD4⁺ T cells or CD40 signaling (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998; Feau et al., 2011), primary responses to some pathogens occur independently of CD4⁺ T cells or CD40 signaling (Whitmire et al., 1996, 1999; Shedlock and Shen, 2003; Shedlock et al., 2003; Sun and Bevan, 2003), possibly because of the direct activation of DCs through pathogen recognition receptors (Hamilton et al., 2001). Curiously, primary CD8⁺ T cell responses to influenza virus require CD40 signaling (Lee et al., 2003a) but not CD4⁺ T cells (Belz et al., 2002), suggesting that other cell types may express CD154 and license CD40-expressing targets in the absence of CD4⁺ T cells. Consistent with this view, activated CD8⁺ T cells (Hernandez et al., 2007; Wong et al., 2008) and natural killer T cells (NKT) express CD154 (Tomura et al., 1999) and may license DCs (Hernandez et al., 2007, 2008; Wong et al., 2008) and help B cells (Chang et al., 2012) in the absence of CD4⁺ T cells. In addition, CD154 is expressed on activated DCs (Johnson et al., 2009) and may directly activate CD40-expressing CD8⁺ T cells. However, the actual role of CD40 signaling and the cellular basis of CD40-mediated help to CD8⁺ T cells help are not fully understood.Whereas helper CD4⁺ T cells promote T and B cell responses, FoxP3-expressing CD4⁺ regulatory T cells (T reg cells) suppress them (Kim et al., 2007; Campbell and Koch, 2011; Chung et al., 2011; Dietze et al., 2011; Linterman et al., 2011). Although the potent suppressive activity of T reg cells is neutralized during infection to allow robust immune responses to pathogens, T reg cells are also involved in the late stages of immune responses to resolve inflammation and curtail immunopathology (Suvas et al., 2003; Fulton et al., 2010; McNally et al., 2011). However, the relationship between CD40-mediated CD4⁺ T cell help and the immunosuppressive activity of T reg cells in CD8⁺ T cell responses to pathogens remains unexplored.Here we determined what cells use CD40–CD154 interactions and how CD40 signaling promotes CD8⁺ T cell responses to influenza. We found that CD4⁺ T cells were the only cells to functionally express CD154 and that DCs were the only cells that required CD40 for optimal CD8⁺ T cell responses to influenza. However, rather than licensing DCs to prime naive CD8⁺ T cells, CD40 signaling was required to prevent the early contraction of the CD8⁺ T cell response. Despite the necessity for CD154 on CD4⁺ T cells, we also observed apparently normal CD8⁺ T cell responses in the absence of CD4⁺ T cells. Finally, we showed that CD8⁺ T cell responses were normal or even enhanced when T reg cells were depleted and that additional CD40 blockade did not change the CD8⁺ T cell response. Thus, our data demonstrate that CD154-expressing CD4⁺ T cells stimulate DCs through CD40 to counteract T reg cell–mediated suppression of the CD8⁺ T cell response during the contraction phase of the immune response.