Cell Biology of T Cell Receptor Expression and Regulation.

T cell receptors (TCRs) are protein complexes formed by six different polypeptides. In most T cells, TCRs are composed of αß subunits displaying immunoglobulin-like variable domains that recognize peptide antigens associated with major histocompatibility complex molecules expressed on the surface of antigen-presenting cells. TCRαß subunits are associated with the CD3 complex formed by the γ, δ, ε, and ζ subunits, which are invariable and ensure signal transduction. Here, we review how the expression and function of TCR complexes are orchestrated by several fine-tuned cellular processes that encompass (a) synthesis of the subunits and their correct assembly and expression at the plasma membrane as a single functional complex, (b) TCR membrane localization and dynamics at the plasma membrane and in endosomal compartments, (c) TCR signal transduction leading to T cell activation, and (d) TCR degradation. These processes balance each other to ensure efficient T cell responses to a variety of antigenic stimuli while preventing autoimmunity.

Cholesterol inhibits TCR signaling by directly restricting TCR-CD3 core tunnel motility.

Cholesterol molecules specifically bind to the resting αßTCR to inhibit cytoplasmic CD3ζ ITAM phosphorylation through sequestering the TCR-CD3 complex in an inactive conformation. The mechanisms of cholesterol-mediated inhibition of TCR-CD3 and its activation remain unclear. Here, we present cryoelectron microscopy structures of cholesterol- and cholesterol sulfate (CS)-inhibited TCR-CD3 complexes and an auto-active TCR-CD3 variant. The structures reveal that cholesterol molecules act like a latch to lock CD3ζ into an inactive conformation in the membrane. Mutations impairing binding of cholesterol molecules to the tunnel result in the movement of the proximal C terminus of the CD3ζ transmembrane helix, thereby activating the TCR-CD3 complex in human cells. Together, our data reveal the structural basis of TCR inhibition by cholesterol, illustrate how the cholesterol-binding tunnel is allosterically coupled to TCR triggering, and lay a foundation for the development of immunotherapies through directly targeting the TCR-CD3 complex.

Integration of ζ-deficient CARs into the CD3ζ gene conveys potent cytotoxicity in T and NK cells.

ABSTRACT: Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in nonphysiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR expression and redirection of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T cells exhibited comparable leukemia control to TCRα chain constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.

Alternatively Spliced Variants of Murine CD247 Influence T Cell Development and Activation, Revealing the Importance of the CD3ζ C-Terminal Region.

CD247, also known as CD3ζ, is a crucial signaling molecule that transduces signals delivered by TCR through its three ITAMs. CD3ζ is required for successful thymocyte development. Three additional alternatively spliced variants of murine CD247 have been described, that is, CD3ι, CD3θ, and CD3η, that differ from CD3ζ in the C terminus such that the third ITAM is lost. Previous studies demonstrated defects in T cell development in mice expressing CD3η, but the TCR signaling pathways affected by CD3η and the impacts of the CD3ι and CD3θ on T cell development were not explored. In this study, we used a retrovirus-mediated gene transfer technique to express these three isoforms individually and examined the roles of them on T cell development and activation. Rag1-/- mice reconstituted with CD3θ-expressing bone marrow failed to develop mature T cells. CD3ι-expressing T cells exhibited similar development and activation as cells expressing CD3ζ. In contrast, thymic development was severely impaired in CD3η-reconstituted mice. Single-positive but not double-positive CD3η-expressing thymocytes had reduced TCR expression, and CD5 expression was decreased at the double-positive stage, suggesting a defect in positive selection. Peripheral CD3η-expressing T cells had expanded CD44hi populations and upregulation of exhaustion markers seen by flow cytometry and RNA sequencing analysis. Analysis of early signaling events demonstrated significantly reduced activation of both the PLCγ1 and Akt/mTOR signaling pathways. There was also a reduction in the frequency of activation of CD3η-expressing T cells. These studies reveal the importance of the CD3ζ C-terminal region in T cell development and activation.

Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.

A set point in the selection of the αßTCR T cell repertoire imposed by pre-TCR signaling strength.

Signaling via the T cell receptor (TCR) is critical during the development, maintenance, and activation of T cells. Quantitative aspects of TCR signaling have an important role during positive and negative selection, lineage choice, and ability to respond to small amounts of antigen. By using a mutant mouse line expressing a hypomorphic allele of the CD3ζ chain, we show here that the strength of pre-TCR­mediated signaling during T cell development determines the diversity of the TCRß repertoire available for positive and negative selection, and hence of the final αßTCR repertoire. This finding uncovers an unexpected, pre-TCR signaling­dependent and repertoire­shaping role for ß-selection beyond selection of in-frame rearranged TCRß chains. Our data furthermore support a model of pre-TCR signaling in which the arrangement of this receptor in stable nanoclusters determines its quantitative signaling capacity.

Nonsense CD247 mutations show dominant-negative features in T-cell receptor expression and function.

BACKGROUND: The invariant TCR ζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its 3 immunoreceptor tyrosine-based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects. OBJECTIVE: We sought to analyze and model the potential impact on T-cell receptor (TCR) expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity. METHODS: Jurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with WT CD247 or mutations ablating 1 (Q142X), 2 (Q101X), or 3 (Q70X) ITAMs. RESULTS: Three patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2 and ITAM-3, respectively. Both mutations were associated with low surface CD3 expression and normal intracellular CD247 levels using a transmembrane-specific antibody, but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO cells could not restore normal surface CD3 expression (only 60%, 22%, and 10%, respectively), whereas in WT cells, normal surface CD3 expression was reduced (to 39%, 19%, and 9% of normal levels), and both effects were dependent on ITAM number. All 6 transfectants showed reduced CD69 induction (25% to 50%), indicating that they were unable to signal downstream properly, neither isolated nor associated with WT CD247. CONCLUSIONS: Our results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.

A Mechanical Switch Couples T Cell Receptor Triggering to the Cytoplasmic Juxtamembrane Regions of CD3ζζ.

The eight-subunit T cell receptor (TCR)-CD3 complex is the primary determinant for T cell fate decisions. Yet how it relays ligand-specific information across the cell membrane for conversion to chemical signals remains unresolved. We hypothesized that TCR engagement triggers a change in the spatial relationship between the associated CD3ζζ subunits at the junction where they emerge from the membrane into the cytoplasm. Using three in situ proximity assays based on ID-PRIME, FRET, and EPOR activity, we determined that the cytosolic juxtamembrane regions of the CD3ζζ subunits are spread apart upon assembly into the TCR-CD3 complex. TCR engagement then triggered their apposition. This mechanical switch resides upstream of the CD3ζζ intracellular motifs that initiate chemical signaling, as well as the polybasic stretches that regulate signal potentiation. These findings provide a framework from which to examine triggering events for activating immune receptors and other complex molecular machines.

Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches.

Anticytotoxic T lymphocyte-associated protein 4 (CTLA4) antibodies have shown potent antitumor activity, but systemic immune activation leads to severe immune-related adverse events, limiting clinical usage. We developed novel, conditionally active biologic (CAB) anti-CTLA4 antibodies that are active only in the acidic tumor microenvironment. In healthy tissue, this binding is reversibly inhibited by a novel mechanism using physiological chemicals as protein-associated chemical switches (PaCS). No enzymes or potentially immunogenic covalent modifications to the antibody are required for activation in the tumor. The novel anti-CTLA4 antibodies show similar efficacy in animal models compared to an analog of a marketed anti-CTLA4 biologic, but have markedly reduced toxicity in nonhuman primates (in combination with an anti-PD1 checkpoint inhibitor), indicating a widened therapeutic index (TI). The PaCS encompass mechanisms that are applicable to a wide array of antibody formats (e.g., ADC, bispecifics) and antigens. Examples shown here include antibodies to EpCAM, Her2, Nectin4, CD73, and CD3. Existing antibodies can be engineered readily to be made sensitive to PaCS, and the inhibitory activity can be optimized for each antigen's varying expression level and tissue distribution. PaCS can modulate diverse physiological molecular interactions and are applicable to various pathologic conditions, enabling differential CAB antibody activities in normal versus disease microenvironments.

Expression of unique gene signature distinguishes TCRαß+ /BCR+ dual expressers from CD3+ CD14+ doublets.

Increasing evidence shows pathophysiological significance of rare immune cells, necessitating the need for reliable and proper methods for their detection and analysis. We have recently identified a new lymphocyte that coexpresses lineage markers of T- and B-cells including T cell receptor and B cell receptor (called dual expressers, DEs). Because of the peculiar phenotype of DEs, we used multiple techniques to authenticate their identity (fluorescence-activated cell sorting [FACS], scRNAseq, EBV cell lines, and imaging flow cytometry). In an recent article published in this journal, Burel and colleagues successfully detected DEs using FACS and imaging microscopy. Yet they claimed, based on the profile of what they called naturally occurring CD3+ CD14+ T cell/monocyte complexes that the scRNAseq signature of DEs resembles that of cell-cell complexes. Serious flaws in their analysis, however, invalidate their conclusions. Unlike the CD3+ CD14+ complexes, DEs have a distinct identity due to expression of a unique set of signature genes. Without a clear explanation, Burel and colleagues excluded these genes from their analysis, thereby effectively stripped DEs from their identity. Inclusion of these genes as described in this communication restores the identity of DEs. Moreover, contrary to the claim of Burel and colleagues, B- and T-cell specific genes are similarly expressed in DE cells.

Specific gut commensal flora locally alters T cell tuning to endogenous ligands.

Differences in gut commensal flora can dramatically influence autoimmune responses, but the mechanisms behind this are still unclear. We report, in a Th1-cell-driven murine model of autoimmune arthritis, that specific gut commensals, such as segmented filamentous bacteria, have the ability to modulate the activation threshold of self-reactive T cells. In the local microenvironment of gut-associated lymphoid tissues, inflammatory cytokines elicited by the commensal flora dynamically enhanced the antigen responsiveness of T cells that were otherwise tuned down to a systemic self-antigen. Together with subtle differences in early lineage differentiation, this ultimately led to an enhanced recruitment of pathogenic Th1 cells and the development of a more severe form of autoimmune arthritis. These findings define a key role for the gut commensal flora in sustaining ongoing autoimmune responses through the local fine tuning of T-cell-receptor-proximal activation events in autoreactive T cells.

Persistent antigen and germinal center B cells sustain T follicular helper cell responses and phenotype.

T follicular helper (Tfh) cells provide help to B cells and are crucial for establishment of germinal center (GC) reactions, including production of high-affinity antibodies and generation of memory B cells and long-lived plasma cells. Here we report that the magnitude of the Tfh cell response was dictated by the amount of antigen and directly correlated with the magnitude of the GC B cell response. In addition, maintenance of the Tfh cell phenotype required sustained antigenic stimulation by GC B cells. In lymphopenic conditions, a strong and prolonged Tfh cell response led to bystander B cell activation, hypergammaglobulinemia, and production of poly- and self-reactive antibodies. These data demonstrate that antigen dose determines the size and duration of the Tfh cell response and GC reaction, highlight the transient nature of the Tfh cell phenotype, and suggest a link between overstimulation of Tfh cells and the development of dysregulated humoral immune responses.

CD3Z polymorphisms and promoter hypermethylation in dermatomyositis - the role of cytosine-phosphate-guanine-related single nucleotide polymorphisms.

BACKGROUND: Decreased expression of the T cell receptor (TCR) ζ-chain has been reported in autoimmune diseases. Recent evidence suggests that this deficiency may be due to polymorphisms in the CD3Z (CD247) gene and/or due to promoter hypermethylation. METHODS: Altogether 131 subjects - 36 with dermatomyositis (DM) and 95 healthy controls were genotyped for rs1052230 G > C and rs1052231 T > A polymorphisms using TaqMan assay. The rs840015 G > A polymorphism was analyzed by direct sequencing. The promoter methylation status was analyzed by Sanger sequencing of bisulfite converted DNA. RESULTS: The rs1052230GC genotype and C allele and the rs1052231TA genotype and T allele were found to correlate with photosensitivity as well as the rs1052230C/rs1052231T haplotype. The rs1052231TA genotype was found to be associated with cutaneous disease. The rs840015GG genotype was found increased among patients with DM, leading to increased OR 2.4. On the contrary, the rs840015GA genotype appeared to be protective for the development of DM. From the 11 cytosine-phosphate-guanine (CpG) islands analyzed, only the 8th island showed a difference in its methylation due to the polymorphism rs840015 G > A within this island, as our results suggest. In this way the presence of AA genotype led to no methylation and the presence of the GG genotype was associated with hemimethylation. CONCLUSION: The CD247 rs1052230 G > C and rs1052231 T > A polymorphisms appeared to have a disease-modifying role. The rs840015GA genotype being associated with reduced methylation has a protective role for the development of dermatomyositis and our results suggest that CpG related single nucleotide polymorphisms may play an important role in autoimmunity.

CAR- and TRuC-redirected regulatory T cells differ in capacity to control adaptive immunity to FVIII.

Regulatory T cells (Tregs) control immune responses in autoimmune disease, transplantation, and enable antigen-specific tolerance induction in protein-replacement therapies. Tregs can exert a broad array of suppressive functions through their T cell receptor (TCR) in a tissue-directed and antigen-specific manner. This capacity can now be harnessed for tolerance induction by "redirecting" polyclonal Tregs to overcome low inherent precursor frequencies and simultaneously augment suppressive functions. With the use of hemophilia A as a model, we sought to engineer antigen-specific Tregs to suppress antibody formation against the soluble therapeutic protein factor (F)VIII in a major histocompatibility complex (MHC)-independent fashion. Surprisingly, high-affinity chimeric antigen receptor (CAR)-Treg engagement induced a robust effector phenotype that was distinct from the activation signature observed for endogenous thymic Tregs, which resulted in the loss of suppressive activity. Targeted mutations in the CD3ζ or CD28 signaling motifs or interleukin (IL)-10 overexpression were not sufficient to restore tolerance. In contrast, complexing TCR-based signaling with single-chain variable fragment (scFv) recognition to generate TCR fusion construct (TRuC)-Tregs delivered controlled antigen-specific signaling via engagement of the entire TCR complex, thereby directing functional suppression of the FVIII-specific antibody response. These data suggest that cellular therapies employing engineered receptor Tregs will require regulation of activation thresholds to maintain optimal suppressive function.

Renal Cell Carcinoma-Infiltrating CD3low Vγ9Vδ1 T Cells Represent Potentially Novel Anti-Tumor Immune Players.

Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3low Vγ9Vδ1 T cells with a terminally differentiated effector memory phenotype with up-regulated activation/exhaustion molecules were newly detected as predominant TILs, and the cytotoxic activity of these cells against RCC was confirmed in vitro. In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, "exhausted" T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.

Blood type change identifies late dominance reversal of chimerism after double umbilical cord blood transplantation with review of the literature.

BACKGROUND: A 30-year-old man underwent double umbilical cord blood transplantation (UCBT) for acute myeloid leukemia (AML) with reduced intensity conditioning. The cords had identical HLA types and were each a 5/6 match to the patient. Following transplantation, cord 2 initially dominated all tested cell populations. At day +306, we observed an unusual reversal of dominance chimerism pattern in which cord 1 instead dominated all tested populations. STUDY DESIGN & METHODS: Polymerase chain reaction (PCR)-based short tandem repeat (STR) assays were performed on the peripheral blood and bone marrow samples. The white blood cell (WBC) populations from the peripheral blood were manipulated for testing to create subpopulations enriched for CD3, CD33, and CD56. RESULTS: Chimerism studies on day +77 showed the following: cord 1: 44%-CD3; 0%-CD33; 16%-CD56; cord 2: 56%-CD3; 100%-CD33; 84%-CD56. Cord 2 initially dominated in all tested cell populations. Chimerism studies performed on post-transplantation day +306 uncovered a reversal of dominance chimerism pattern in which cord 1 now dominated in all cell populations (cord 1: 82%-CD3; >95%-CD33; 67%-CD56; cord 2: 18%-CD3; <5%-CD33; 33%-CD56). Between days +127 and +244, the patient's blood type shifted from B Rh-positive to A Rh-negative. CONCLUSION: The change in the patient's blood type identified a late reversal of dominance chimerism pattern. This is a rare occurrence, previously cited only once, which is inconsistent with published data that early high CD3 counts and unseparated bone marrow chimerism predominance at day +100 predict long-term cord dominance in double UCBT in the vast majority of cases.

A novel 4-mRNA signature predicts the overall survival in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive cancer of myeloid cells with high levels of heterogeneity and great variability in prognostic behaviors. Cytogenetic abnormalities and genetic mutations have been widely used in the prognostic stratification of AML to assign patients into different risk categories. Nevertheless, nearly half of AML patients assigned to intermediate risk need more precise prognostic schemes. Here, 336 differentially expressed genes (DEGs) between AML and control samples and 206 genes representing the intratumor heterogeneity of AML were identified. By applying a LASSO Cox regression model, we generated a 4-mRNA prognostic signature comprising KLF9, ENPP4, TUBA4A and CD247. Higher risk scores were significantly associated with shorter overall survival, complex karyotype, and adverse mutations. We then validated the prognostic value of this 4-mRNA signature in two independent cohorts. We also proved that incorporation of the 4-mRNA-based signature in the 2017 European LeukemiaNet (ELN) risk classification could enhance the predictive accuracy of survival in patients with AML. Univariate and multivariate analyses showed that this signature was independent of traditional prognostic factors such as age, WBC count, and unfavorable cytogenetics. Finally, the molecular mechanisms underlying disparate outcomes in high-risk and low-risk AML patients were explored. Therefore, our findings suggest that the 4-mRNA signature refines the risk stratification and prognostic prediction of AML patients.

Combination of CD19 and CD22 CAR-T cell therapy in relapsed B-cell acute lymphoblastic leukemia after allogeneic transplantation.

The prognosis of relapsed acute lymphoblastic leukemia (ALL) after allogeneic transplantation is dismal when treated with conventional approaches. While single-target CD19 or CD22 chimeric antigen receptor (CAR) T-cell therapy has achieved high complete remission (CR) rates in refractory/relapsed B-ALL, it could not maintain a durable remission in most patients. To prolong relapse-free survival, we sequentially combined CD19 and CD22 CAR-T cells to treat post-transplant relapsed B-ALL patients with both CD19/CD22 antigen expression on lymphoblasts. Patient-derived donor cells were collected to produce CAR-T cells that were transfected by lentiviral vectors encoding second generation CARs composed of CD3ζ and 4-1BB. The second T-cell infusion was scheduled at least 1 month, and usually within 6 months after the first CAR-T treatment. Twenty-seven adult and pediatric patients, including 11 (41%) with extramedullary diseases (EMD), received the first CD19 CAR-T and 23 (85%) achieved CR. Subsequently, 21 out of 27 patients received the second CD22 CAR-T and were followed-up for a median of 19.7 (range, 5.6-27.3) months; 14 cases remained in CR, seven relapsed and two of them died from disease progression; Kaplan-Meier survival analysis showed overall survival and event-free survival rates of 88.5% and 67.5%, respectively, at both 12 months and 18 months. CAR-T associated graft-versus-host disease (GVHD) occurred in 23% of patients, with 8% new-onset acute GVHD and 15% persistent or worsened pre-existing cGVHD before CAR-T. This combination strategy of sequential CD19 and CD22 CAR-T therapy significantly improved the long-term survival in B-ALL patients who relapsed after transplantation.

Nanovesicles released by OKT3 hybridoma express fully active antibodies.

Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.