Innate Immune Molecule NLRC5 Protects Mice From Helicobacter-induced Formation of Gastric Lymphoid Tissue.

BACKGROUND & AIMS: Helicobacter pylori induces strong inflammatory responses that are directed at clearing the infection, but if not controlled, these responses can be harmful to the host. We investigated the immune-regulatory effects of the innate immune molecule, nucleotide-binding oligomerization domain-like receptors (NLR) family CARD domain-containing 5 (NLRC5), in patients and mice with Helicobacter infection. METHODS: We obtained gastric biopsies from 30 patients in Australia. We performed studies with mice that lack NLRC5 in the myeloid linage (Nlrc5møKO) and mice without Nlrc5 gene disruption (controls). Some mice were gavaged with H pylori SS1 or Helicobacter felis; 3 months later, stomachs, spleens, and sera were collected, along with macrophages derived from bone marrow. Human and mouse gastric tissues and mouse macrophages were analyzed by histology, immunohistochemistry, immunoblots, and quantitative polymerase chain reaction. THP-1 cells (human macrophages, controls) and NLRC5-/- THP-1 cells (generated by CRISPR-Cas9 gene editing) were incubated with Helicobacter and gene expression and production of cytokines were analyzed. RESULTS: Levels of NLRC5 messenger RNA were significantly increased in gastric tissues from patients with H pylori infection, compared with patients without infection (P < .01), and correlated with gastritis severity (P < .05). H pylori bacteria induced significantly higher levels of chemokine and cytokine production by NLRC5-/- THP-1 macrophages than by control THP-1 cells (P < .05). After 3 months of infection with H felis, Nlrc5mø-KO mice developed gastric hyperplasia (P < .0001), splenomegaly (P < .0001), and increased serum antibody titers (P < .01), whereas control mice did not. Nlrc5mø-KO mice with chronic H felis infection had increased numbers of gastric B-cell follicles expressing CD19 (P < .0001); these follicles had features of mucosa-associated lymphoid tissue lymphoma. We identified B-cell-activating factor as a protein that promoted B-cell hyperproliferation in Nlrc5mø-KO mice. CONCLUSIONS: NLRC5 is a negative regulator of gastric inflammation and mucosal lymphoid formation in response to Helicobacter infection. Aberrant NLRC5 signaling in macrophages can promote B-cell lymphomagenesis during chronic Helicobacter infection.

The lipoprotein HP1454 of Helicobacter pylori regulates T-cell response by shaping T-cell receptor signalling.

Helicobacter pylori (HP) is a Gram-negative bacterium that chronically infects the stomach of more than 50% of human population and represents a major cause of gastric cancer, gastric lymphoma, gastric autoimmunity, and peptic ulcer. It still remains to be elucidated, which HP virulence factors are important in the development of gastric disorders. Here, we analysed the role of the HP protein HP1454 in the host-pathogen interaction. We found that a significant proportion of T cells isolated from HP patients with chronic gastritis and gastric adenocarcinoma proliferated in response to HP1454. Moreover, we demonstrated in vivo that HP1454 protein drives Th1/Th17 inflammatory responses. We further analysed the in vitro response of human T cells exposed either to an HP wild-type strain or to a strain with a deletion of the hp1454 gene, and we revealed that HP1454 triggers the T-cell antigen receptor-dependent signalling and lymphocyte proliferation, as well as the CXCL12-dependent cell adhesion and migration. Our study findings prove that HP1454 is a crucial bacterial factor that exerts its proinflammatory activity by directly modulating the T-cell response. The relevance of these results can be appreciated by considering that compelling evidence suggest that chronic gastric inflammation, a condition that paves the way to HP-associated diseases, is dependent on T cells.

CD25 deficiency: A new conformational mutation prevents the receptor expression on cell surface.

CD25 deficiency is a very rare autosomal recessive disorder that shows a clinical phenotype highly overlapping IPEX syndrome with an increased susceptibility to viral, bacterial, and fungal infections. It is due to mutations in the IL2Rα gene that codes for the α subunit of the IL2 receptor complex. Here we report the characterization of a novel IL2Rα gene mutation leading to a severe protein conformational alteration that abrogates its cell surface expression in a child presenting with early-onset IPEX-like disorder. Cytofluorimetric analysis revealed the total absence of CD25 cell surface expression and addressed IL2Rα molecular investigation. The early clinical and molecular diagnosis of CD25 deficiency in this patient promptly led to hematopoietic stem cell transplantation (HSCT), allowing complete resolution of the symptoms and definitive cure of the disease.

p66Shc deficiency in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia enhances leukemogenesis by altering the chemokine receptor landscape.

The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eµ-TCL1/p66Shc-/- mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eµ-TCL1 mice. Eµ-TCL1/p66Shc-/- mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells' level of p66Shc expression. p66Shc expression declined with disease progression in Eµ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.

Placental bed research: II. Functional and immunological investigations of the placental bed.

Research on the placenta as the interface between the mother and the fetus has been undertaken for some 150 years, and in 2 subsequent reviews, we attempted to summarize the situation. In the first part, we described the discovery of unique physiological modifications of the uteroplacental spiral arteries, enabling them to cope with a major increase in blood flow necessary to ensure proper growth of the fetus. These consist of an invasion of the arterial walls by trophoblast and a progressive disappearance of its normal structure. Researchers then turned to the pathophysiology of the placental bed and in particular to its maternal vascular tree. This yielded vital information for a better understanding of the so-called great obstetrical syndromes (preeclampsia, fetal growth restriction, premature labor and delivery, placenta accreta). Systematic morphological investigations of the uteroplacental vasculature showed that preeclampsia is associated with decreased or failed transformation of spiral arteries and the persistence of endothelial and smooth muscle cells in segments of their myometrial portion. Here we report on recent functional investigations of the placental bed, including in situ biophysical studies of uteroplacental blood flow and vascular resistance, and manipulation of uteroplacental perfusion. These new methodologies have provided a novel way of identifying pregnancies in which remodeling is impaired. In animals it is now possible to manipulate uteroplacental blood flow, leading to an enhancement of fetal growth; this opens the way to trials in abnormal human pregnancies. In this second part, we explored a new, extremely important area of research that deals with the role of specific subsets of leukocytes and macrophages in the placental bed. The human first-trimester decidua is rich in leukocytes called uterine natural killer cells. Both macrophages and uterine natural killer cells increase in number from the secretory endometrium to early pregnancy and play a critical role in mediating the process of spiral artery transformation by inducing initial structural changes. It seems therefore that vascular remodeling of spiral arteries is initiated independently of trophoblast invasion. Dysregulation of the immune system may lead to reproductive failure or pregnancy complications, and in this respect, recent studies have advanced our understanding of the mechanisms regulating immunological tolerance during pregnancy, with several mechanisms being proposed for the development of tolerance to the semiallogeneic fetus. In particular, these include several strategies by which the trophoblast avoids maternal recognition. Finally, an important new dimension is being explored: the likelihood that pregnancy syndromes and impaired uteroplacental vascular remodeling may be linked to future maternal and even the child's cardiovascular disease risk. The functional evidence underlying these observations will be discussed.

Role of Mycobacterium avium lysate INF-γ, IL-17, and IL-2 ELISPOT assays in diagnosing nontuberculous mycobacteria lymphadenitis in children.

Nontuberculous mycobacteria are the most frequent cause of chronic cervical lymphadenitis in childhood. The aim of the study was to evaluate the performance of IL-2, IL-17, and INF-γ in-house enzyme-linked immunospot assays using a Mycobacterium avium lysate, in order to identify a noninvasive diagnostic method of nontuberculous mycobacteria infection. Children with subacute and chronic lymphadenopathies or with a previous diagnosis of nontuberculous mycobacteria lymphadenitis were prospectively enrolled in the study. Sixty children with lymphadenitis were included in our study: 16 with confirmed infection (group 1), 30 probable infected (group 2) and 14 uninfected (group 3). Significantly higher median cytokine values were found in group 1 vs group 2, in group 1 vs group 3, and in group 2 vs group 3 considering IL-2-based enzyme-linked immunospot assay (p = 0.015, p < 0.001, p = 0.004, respectively). INF-γ-based enzyme-linked immunospot assay results were significantly higher in group 2 vs group 3 (p = 0.010). Differences between infected and uninfected children were not significant considering IL-17 assays (p = 0.431). Mycobacterium avium lysate IL-2 and INF-γ-based enzyme-linked immunospot assays seem to be promising noninvasive diagnostic techniques for discriminating children with nontuberculous mycobacteria lymphadenitis and noninfected subjects.

ß2 Glycoprotein I Recognition Drives Th1 Inflammation in Atherosclerotic Plaques of Patients with Primary Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is characterized by recurrent arterial/venous thrombosis and miscarriages in the persistent presence of autoantibodies against phospholipid-binding proteins (aPLs), such as ß2 glycoprotein I (ß2GPI). In addition to the aPL thrombophilic effect, arterial thrombosis was related to accelerated atherosclerosis in animal models; however, contrasting findings were reported in primary APS patients with regard to the increased number of plaques or abnormal arterial wall thickness. We investigated the cytokine production induced by ß2GPI in activated T cells that infiltrate in vivo atherosclerotic lesions of primary APS patients with atherothrombosis. We also examined the helper function of ß2GPI-specific T cells for monocyte matrix metalloproteinase-9 and tissue factor production, as well as their cytolytic potential and their helper function for Ab production. APS patients with atherothrombosis harbor in vivo-activated CD4+ T cells that recognize ß2GPI in atherothrombotic lesions. ß2GPI induces T cell proliferation and IFN-γ expression in plaque-derived T cell clones. ß2GPI-specific T cells display helper function for monocyte matrix metalloproteinase-9 and tissue factor production and promote Ig production in autologous B cells. Moreover, plaque-derived ß2GPI-specific CD4+ T lymphocytes express perforin-mediated and Fas/Fas ligand-mediated cytotoxicity. ß2GPI, and especially the DI domain, drive a local Th1 inflammatory response, with subsequent plaque instability that eventually favors atherothrombosis. This finding may explain the association between aPLs and arterial thrombosis, despite the lack of evidence of surrogate markers for atherosclerosis in primary APS.

Cytokine BAFF released by Helicobacter pylori-infected macrophages triggers the Th17 response in human chronic gastritis.

BAFF is a crucial cytokine that affects the activity of both innate and adaptive immune cells. It promotes the expansion of Th17 cells in autoimmune disorders. With this study, we investigated the BAFF/Th17 responses in Helicobacter pylori-induced gastritis in humans. Our results show that the mucosa from Helicobacter(+) patients with chronic gastritis is enriched in IL-17 and BAFF, whereas the two cytokines are weakly expressed in Helicobacter(-) patients with chronic gastritis; moreover, the expression of both BAFF and IL-17 decreases after bacteria eradication. We demonstrate that BAFF accumulates in macrophages in vivo and that it is produced by monocyte-derived macrophages in vitro, after Helicobacter stimulation. Application of BAFF on monocytes triggers the accumulation of reactive oxygen species that are crucial for the release of pro-Th17 cytokines, such as IL-23, IL-1ß, and TGF-ß. Moreover, BAFF directly promotes the differentiation of Th17 cells. In conclusion, our results support the notion that an axis BAFF/Th17 exists in chronic gastritis of Helicobacter(+) patients and that its presence strictly depends on the bacterium. Moreover, we demonstrated that BAFF is able to drive Th17 responses both indirectly, by creating a pro-Th17 cytokine milieu through the involvement of innate immune cells, and directly, via the differentiation of T cells toward the specific profile. The results obtained in this study are of great interest for Helicobacter-related diseases and the development of novel therapeutic strategies based on the inhibition of the BAFF/IL-17 response.

Chlamydophila pneumoniae phospholipase D (CpPLD) drives Th17 inflammation in human atherosclerosis.

Phospholipases are produced from bacterial pathogens causing very different diseases. One of the most intriguing aspects of phospholipases is their potential to interfere with cellular signaling cascades and to modulate the host-immune response. Here, we investigated the role of the innate and acquired immune responses elicited by Chlamydophila pneumoniae phospholipase D (CpPLD) in the pathogenesis of atherosclerosis. We evaluated the cytokine and chemokine production induced by CpPLD in healthy donors' monocytes and in vivo activated T cells specific for CpPLD that infiltrate atherosclerotic lesions of patients with C. pneumoniae antibodies. We also examined the helper function of CpPLD-specific T cells for monocyte matrix metalloproteinase (MMP)-9 and tissue factor (TF) production as well as the CpPLD-induced chemokine expression by human venular endothelial cells (HUVECs). We report here that CpPLD is a TLR4 agonist able to induce the expression of IL-23, IL-6, IL-1ß, TGF-ß, and CCL-20 in monocytes, as well as CXCL-9, CCL-20, CCL-4, CCL-2, ICAM-1, and VCAM-1 in HUVECs. Plaque-derived T cells produce IL-17 in response to CpPLD. Moreover, CpPLD-specific CD4(+) T lymphocytes display helper function for monocyte MMP-9 and TF production. CpPLD promotes Th17 cell migration through the induction of chemokine secretion and adhesion molecule expression on endothelial cells. These findings indicate that CpPLD is able to drive the expression of IL-23, IL-6, IL-1ß, TGF-ß, and CCL-20 by monocytes and to elicit a Th17 immune response that plays a key role in the genesis of atherosclerosis.

Immunity, inflammation, and vaccines for Helicobacter pylori.

Helicobacter pylori colonizes mucosa, activates Toll-like and Nod-like receptors, and usually elicits a gastric T-helper 1/17 (Th1/Th17) type of immune response. Among several bacterial factors, the secreted peptidyl prolyl cis, trans-isomerase of H. pylori represents a key factor driving Th17 inflammation. A complex and fascinating balance between H. pylori and host factors takes part in the gastric niche and is responsible for the chronicity of the infection. Novel insights into the innate and adaptive responses against H. pylori, dealing with gastric epithelial cells, cytokines, and immune evasion have been elucidated over the past year and are discussed for the development of an effective vaccine.

Orchestration of inflammation and adaptive immunity in Borrelia burgdorferi-induced arthritis by neutrophil-activating protein A.

OBJECTIVE: Lyme arthritis (LA) is characterized by infiltration of inflammatory cells, mainly neutrophils (polymorphonuclear cells [PMNs]) and T cells, into the joints. This study was undertaken to evaluate the role of the neutrophil-activating protein A (NapA) of Borrelia burgdorferi in eliciting inflammation and in driving the adaptive immune response. METHODS: Levels of NapA, interferon-γ (IFNγ), interleukin-17 (IL-17), and T cell-attracting chemokines were assessed by enzyme-linked immunosorbent assay in synovial fluid from patients with LA. The profile of T cells recruited into the synovia of patients with LA was defined by fluorescence-activated cell sorting analysis. NapA was intraarticularly injected into rat knees, and the cells recruited in synovia were characterized. The role of NapA in recruiting immune cells was confirmed by chemotaxis assays using a Transwell system. RESULTS: NapA, IFNγ, IL-17, CCL2, CCL20, and CXCL10 accumulated in synovial fluid from patients with LA. Accordingly, T cells obtained from these patients produced IFNγ or IL-17, but notably, some produced both cytokines. NapA promoted neutrophil and T lymphocyte recruitment both in vitro and in vivo. Interestingly, the infiltration of T cells not only resulted from the chemotactic activity of NapA but also relied on the chemokines produced by PMNs exposed to NapA. CONCLUSION: We provide evidence that NapA functions as one of the main bacterial products involved in the pathogenesis of LA. Accordingly, we show that, at very early stages of LA, NapA accumulates and, in turn, orchestrates the recruitment of inflammatory cells into the joint cavity. Thereafter, with the contribution of recruited cells, NapA promotes the infiltration of T cells producing IL-17 and/or IFNγ.

Tumor-associated macrophages as major source of APRIL in gastric MALT lymphoma.

Lymphoid hyperplasia of gastric mucosa associated with Helicobacter pylori (HP) infection represents a preneoplastic condition of the mucosa associated lymphoid tissue (MALT), which may evolve to a B-cell lymphoma. While it is well established that the initial neoplastic proliferation of B cells is antigen-driven and dependent on the helper activity of HP-specific T cells, it needs to be elucidated which cytokine or soluble factor(s) promote B-cell activation and lymphomagenesis. Herein, we originally report that gastric MALT lymphoma express high levels of a proliferation inducing ligand (APRIL), a novel cytokine crucial in sustaining B-cell proliferation. By immunohistochemistry, we demonstrate that APRIL is produced almost exclusively by gastric lymphoma-infiltrating macrophages located in close proximity to neoplastic B cells. We also show that macrophages produce APRIL on direct stimulation with both HP and HP-specific T cells. Collectively, our results represent the first evidence for an involvement of APRIL in gastric MALT lymphoma development in HP-infected patients.

Helicobacter pylori attachment-blocking antibodies protect against duodenal ulcer disease.

The majority of the world population carry the gastric pathogen Helicobacter pylori. Fortunately, most individuals experience only low-grade or no symptoms, but in many cases the chronic inflammatory infection develops into severe gastric disease, including duodenal ulcer disease and gastric cancer. Here we report on a protective mechanism where H. pylori attachment and accompanying chronic mucosal inflammation can be reduced by antibodies that are present in a vast majority of H. pylori carriers. These antibodies block binding of the H. pylori attachment protein BabA by mimicking BabA's binding to the ABO blood group glycans in the gastric mucosa. However, many individuals demonstrate low titers of BabA blocking antibodies, which is associated with an increased risk for duodenal ulceration, suggesting a role for these antibodies in preventing gastric disease.

T cells in gastric cancer: friends or foes.

Gastric cancer is the second cause of cancer-related deaths worldwide. Helicobacter pylori is the major risk factor for gastric cancer. As for any type of cancer, T cells are crucial for recognition and elimination of gastric tumor cells. Unfortunately T cells, instead of protecting from the onset of cancer, can contribute to oncogenesis. Herein we review the different types, "friend or foe", of T-cell response in gastric cancer.

The adenylate cyclase toxins of Bacillus anthracis and Bordetella pertussis promote Th2 cell development by shaping T cell antigen receptor signaling.

The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2-driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4(+) T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2-polarizing concentrations of ET and CyaA selectively inhibit TCR-dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR-signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3zeta phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4(+) T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins.

Helicobacter pylori-derived neutrophil-activating protein increases the lifespan of monocytes and neutrophils.

An invariable feature of Helicobacter pylori-infected gastric mucosa is the persistent infiltration of inflammatory cells. The neutrophil-activating protein (HP-NAP) has a pivotal role in triggering and orchestrating the phlogistic process associated with H. pylori infection. Aim of this study was to address whether HP-NAP might further contribute to the inflammation by increasing the lifespan of inflammatory cells. We report that HP-NAP is able to prolong the lifespan of monocytes, in parallel with the induction of the anti-apoptotic proteins A1, Mcl-1, Bcl-2 and Bcl-X(L). This effect does not result from a direct action on the apoptotic machinery, but rather it requires the release of endogenous pro-survival factors, such as interleukin-1beta, which probably acts in synergy with other unidentified mediators. We also report that HP-NAP promotes the survival of Ficoll-purified neutrophils in a monocyte-dependent fashion: indeed, mononuclear cell depletion of Ficoll-purified neutrophils completely abolished the pro-survival effect by HP-NAP. In conclusion, our data reinforce the notion that HP-NAP has a pivotal role in sustaining a prolonged activation of myeloid cells.

Novel immunotherapeutic strategies of gastric cancer treatment.

Gastric cancer (GC) is the fourth most common cancer and the second most frequent cause of cancer-related deaths, accounting for 10.4% of cancer deaths worldwide. Despite the improvements, estimated cure rates for patients with advanced stages remain poor, and in the metastatic setting, chemotherapy is the mainstay of palliative therapy and results in objective response rates (ORRs) of only 20-40% and median overall survivals (OS) of 8-10 months. Therefore, many investigators believe that the potential for making significant progress lies in understanding and exploiting the molecular biology of these tumors to investigate new therapeutic strategies to combat GC, such as specific immunotherapy. In this paper, we analyze the different approaches used for immune-based (especially dendritic and T cells) therapies to gastric cancer treatment and discuss the results obtained in preclinical models as in clinical trials.

T cells and adoptive immunotherapy: recent developments and future prospects in gastrointestinal oncology.

Gastrointestinal oncology is one of the foremost causes of death: the gastric cancer accounts for 10.4% of cancer deaths worldwide, the pancreatic cancer for 6%, and finally, the colorectal cancer for 9% of all cancer-related deaths. For all these gastrointestinal cancers, surgical tumor resection remains the primary curative treatment, but the overall 5-year survival rate remains poor, ranging between 20-25%; the addition of combined modality strategies (pre- or postoperative chemoradiotherapy or perioperative chemotherapy) results in 5-year survival rates of only 30-35%. Therefore, many investigators believe that the potential for making significant progress lies on understanding and exploiting the molecular biology of gastrointestinal tumors to investigate new therapeutic strategies such as specific immunotherapy. In this paper we will focus on recent knowledge concerning the role of T cells and the use of T adoptive immunotherapy in the treatment of gastrointestinal cancers.

ADP-heptose enables Helicobacter pylori to exploit macrophages as a survival niche by suppressing antigen-presenting HLA-II expression.

The persistence of Helicobacter pylori in the human gastric mucosa implies that the immune response fails to clear the infection. We found that H. pylori compromises the antigen presentation ability of macrophages, because of the decline of the presenting molecules HLA-II. Here, we reveal that the main bacterial factor responsible for this effect is ADP-heptose, an intermediate metabolite in the biosynthetic pathway of lipopolysaccharide (LPS) that elicits a pro-inflammatory response in gastric epithelial cells. In macrophages, it upregulates the expression of miR146b which, in turn, would downmodulate CIITA, the master regulator for HLA-II genes. Hence, H. pylori, utilizing ADP-heptose, exploits a specific arm of macrophage response to establish its survival niche in the face of the immune defense elicited in the gastric mucosa.

Increasing LFA-1 Expression Enhances Immune Synapse Architecture and T Cell Receptor Signaling in Jurkat E6.1 Cells.

The Jurkat E6.1 clone has been extensively used as a powerful tool for the genetic and biochemical dissection of the TCR signaling pathway. More recently, these cells have been exploited in imaging studies to identify key players in immunological synapse (IS) assembly in superantigen-specific conjugates and to track the dynamics of signaling molecules on glass surfaces coated with activating anti-CD3 antibodies. By comparison, Jurkat cells have been used only scantily for imaging on supported lipid bilayers (SLBs) incorporating laterally mobile TCR and integrin ligands, which allow to study synaptic rearrangements of surface molecules and the fine architecture of the mature IS, likely due to limitations in the assembly of immune synapses with well-defined architecture. Here we have explored whether upregulating the low levels of endogenous LFA-1 expression on Jurkat E6.1 cells through transduction with CD11a- and CD18-encoding lentiviruses can improve IS architecture. We show that, while forced LFA-1 expression did not affect TCR recruitment to the IS, E6.1 LFA-1 high cells assembled better structured synapses, with a tighter distribution of signaling-competent TCRs at the center of the IS. LFA-1 upregulation enhanced protein phosphotyrosine signaling on SLBs but not at the IS formed in conjugates with SEE-pulsed APCs, and led to the constitutive formation of an intracellular phosphotyrosine pool co-localizing with endosomal CD3ζ. This was paralleled by an increase in the levels of p-ZAP-70 and p-Erk both under basal conditions and following activation, and in enhanced Ca2+ mobilization from intracellular stores. The enhancement in early signaling E6.1 LFA-1 high cells did not affect expression of the early activation marker CD69 but led to an increase in IL-2 expression. Our results highlight a new role for LFA-1 in the core architecture of the IS that can be exploited to study the spatiotemporal redistribution of surface receptors on SLBs, thereby extending the potential of E6.1 cells and their derivatives for fine-scale imaging studies.