Marc Bonneville

The molecular basis for modulation of human Vγ9Vδ2 T cell responses by CD277/Butyrophilin-3 (BTN3A)-specific antibodies

Background: Phosphoisoprenoid stimulation of Vγ9Vδ2 T cells can be modulated by anti-BTNA3 antibodies. Results: Agonist and antagonist antibodies associate differently with BTN3A structurally and biophysically. Conclusion: Differential binding of antibodies to BTN3A modulates its oligomerization on the cell surface. Significance: Defining how γδ T cells recognize antigen is critical for understanding their functions in the immune response. Human Vγ9Vδ2 T cells are well known for their rapid and potent response to infection and tumorigenesis when in the presence of endogenous or exogenous phosphoisoprenoids. However, the molecular mechanisms behind the activation of this γδ T cell population remains unclear. Evidence pointing to a role for the CD277/butyrophilin-3 (BTN3A) molecules in this response led us to investigate the structures of these molecules and their modifications upon binding to an agonist antibody (20.1) that mimics phosphoisoprenoid-mediated Vγ9Vδ2 activation and an antagonist antibody (103.2) that inhibits this reactivity. We find that the three BTN3A isoforms: BTN3A1, BTN3A2, and BTN3A3, have high structural homology to the B7 superfamily of proteins and exist as V-shaped homodimers in solution, associating through the membrane proximal C-type Ig domain. The 20.1 and 103.2 antibodies bind to separate epitopes on the BTN3A Ig-V domain with high affinity but likely with different valencies based on their binding orientation. These structures directly complement functional studies of this system that demonstrate that BTN3A1 is necessary for Vγ9Vδ2 activation and begin to unravel the extracellular events that occur during stimulation through the Vγ9Vδ2 T cell receptor.

Cross-Reactivity of Herpesvirus-Specific CD8 T Cell Lines Toward Allogeneic Class I MHC Molecules

Although association between persistent viral infection and allograft rejection is well characterized, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. We appraised in this study the alloreactivity of CD8 T cell lines specific for immunodominant epitopes from human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). CD8 T cell lines were generated after sorting with immunomagnetic beads coated with either pp65495–503/A*0201, BMLF1259–267/A*0201, or BZLF154–64/B*3501 multimeric complexes. Alloreactivity of the CD8 T cell lines against allogeneic class I MHC alleles was assessed by screening of (i) TNF-α production against COS-7 cells transfected with as many as 39 individual HLA class I-encoding cDNA, and (ii) cytotoxicity activity toward a large panel of HLA-typed EBV-transformed B lymphoblastoid cell lines. We identified several cross-reactive pp65/A*0201-specific T cell lines toward allogeneic HLA-A*3001, A*3101, or A*3201. Moreover, we described here cross-recognition of HLA-Cw*0602 by BZLF1/B*3501-specific T cells. It is noteworthy that these alloreactive CD8 T cell lines showed efficient recognition of endothelial cells expressing the relevant HLA class I allele, with high level TNF-α production and cytotoxicity activity. Taken together, our data support the notion that herpes virus-specific T cells recognizing allo-HLA alleles may promote solid organ rejection.

Assessment of CD8 involvement in T cell clone avidity by direct measurement of HLA-A2/Mage3 complex density using a high-affinity TCR like monoclonal antibody

Peptide affinity for MHC molecules determines the number of MHC/peptide complexes stabilized at the cell surface in in vitro tests or in vaccination protocols. We isolated a high affinity monoclonal antibody specific for the HLA‐A2/Mage3 complex that enables an equilibrium binding assay to be performed on T2 cell line loaded with a range of Mage3 peptides. Binding of Mage3 to the HLA‐A2 molecule can be modeled by a standard receptor‐ligand interaction characterized by an affinity constant. This model enables the measurement of the affinity of other immunogenic peptides for HLA‐A2 by a competition test and the calculation of the density of complexes stabilized at the T2 cell surface for all peptide concentrations. Quantification of the HLA‐A2/Mage3 complexes at target cell surfaces was used to estimate the number of complexes required to reach cytotoxicity ED50 of human T cell clones sorted from an unprimed repertoire. We confirm with this antibody the direct relationship between clone avidity and TCR affinity, and the moderate contribution of the CD8 co‐receptor in the reinforcement of TCR‐MHC/peptide contact. Nevertheless, CD8 plays a critical role in the amplification of the specific signal to establish an efficient T cell response at low specific complex densities found in physiological situations.

Up-regulation of cytolytic functions of human Vδ2− γδ T lymphocytes through engagement of ILT2 expressed by tumor target cells

In humans, the majority of peripheral blood γδ T cells expresses Vγ9Vδ2 T-cell receptors (TCR) and recognize nonpeptidic phosphorylated antigens. In contrast, most tissue-derived γδ T cells, which are located mainly in spleen and epithelia, preferentially use Vδ1 or Vδ3 chains paired with diverse Vγ chains to form their TCR. Our knowledge about the antigenic specificity and costimulation requirements of human Vδ2(-) γδ T cells remains limited. In an attempt to address this important issue, we characterized the specificity of a monoclonal antibody (mAb 256), screened for its ability to specifically inhibit cytolytic responses of several human Vδ2(-) γδ T-cell clones against transformed B cells. We show that mAb 256 does not target a TCR ligand but blocks key interactions between non-TCR molecules on effector γδ T cells and ILT2 molecule, expressed by tumor targets. In line with the previously reported specificity of this NK receptor for classic and nonclassic major histocompatibility complex (MHC) class I molecules, blockade of MHC class I/ILT2 interactions using MHC class I- or ILT2-specific mAbs and ILT2-Fc molecules inhibited tumor-induced activation of Vγ8Vδ3 T-cell clones. Therefore, this study describes a new cytotoxic T lymphocyte activation pathway involving MHC class I engagement on γδ T cells.

Sensing of cell stress by human γδ TCR-dependent recognition of annexin A2

Significance Human γδ T lymphocytes have innate-like and adaptive-like functions and can circulate in blood or reside in tissues. They are activated by specific antigens recognized by their T-cell receptor and recognize infected and transformed cells, suggesting that cellular stress is involved in specific antigen expression. However, molecular characterization of stress-induced antigens remains elusive, hampering our understanding of the role of γδ T cells in cancer and infections. In the present study we identify annexin A2 as such stress-induced antigen known as a phospholipid-binding protein involved in tumorigenesis, redox potential regulation, and wound healing. Stress-mediated membrane exposure of annexin A2 could thus constitute a danger signal for γδ T cells to recognize various cell dysregulations and protect the host against cancer and infections. Human γδ T cells comprise a first line of defense through T-cell receptor (TCR) recognition of stressed cells. However, the molecular determinants and stress pathways involved in this recognition are largely unknown. Here we show that exposure of tumor cells to various stress situations led to tumor cell recognition by a Vγ8Vδ3 TCR. Using a strategy that we previously developed to identify antigenic ligands of γδ TCRs, annexin A2 was identified as the direct ligand of Vγ8Vδ3 TCR, and was found to be expressed on tumor cells upon the stress situations tested in a reactive oxygen species-dependent manner. Moreover, purified annexin A2 was able to stimulate the proliferation of a Vδ2neg γδ T-cell subset within peripheral blood mononuclear cells and other annexin A2-specific Vδ2neg γδ T-cell clones could be derived from peripheral blood mononuclear cells. We thus propose membrane exposure of annexin A2 as an oxidative stress signal for some Vδ2neg γδ T cells that could be involved in an adaptive stress surveillance.

The Juxtamembrane Domain of Butyrophilin BTN3A1 Controls Phosphoantigen-Mediated Activation of Human Vγ9Vδ2 T Cells.

Vγ9Vδ2 T lymphocytes are the major human peripheral γδ T cell subset, with broad reactivity against stressed human cells, including tumor cells. Vγ9Vδ2 T cells are specifically activated by small phosphorylated metabolites called phosphoantigens (PAg). Stress-induced changes in target cell PAg levels are specifically detected by butyrophilin (BTN)3A1, using its intracellular B30.2 domain. This leads to the activation of Vγ9Vδ2 T cells. In this study, we show that changes in the juxtamembrane domain of BTN3A1, but not its transmembrane domain, induce a markedly enhanced or reduced γδ T cell reactivity. There is thus a specific requirement for BTN3A1’s juxtamembrane domain for correct γδ T cell–related function. This work identified, as being of particular importance, a juxtamembrane domain region of BTN3A molecules identified as a possible dimerization interface and that is located close to the start of the B30.2 domain.

Full restoration of Brucella-infected dendritic cell functionality through Vγ9Vδ2 T helper type 1 crosstalk.

Vγ9Vδ2 T cells play an important role in the immune response to infectious agents but the mechanisms contributing to this immune process remain to be better characterized. Following their activation, Vγ9Vδ2 T cells develop cytotoxic activity against infected cells, secrete large amounts of cytokines and influence the function of other effectors of immunity, notably cells playing a key role in the initiation of the adaptive immune response such as dendritic cells. Brucella infection dramatically impairs dendritic cell maturation and their capacity to present antigens to T cells. Herein, we investigated whether V T cells have the ability to restore the full functional capacities of Brucella-infected dendritic cells. Using an in vitro multicellular infection model, we showed that: 1/Brucella-infected dendritic cells activate Vγ9Vδ2 T cells through contact-dependent mechanisms, 2/activated Vγ9Vδ2 T cells induce full differentiation into IL-12 producing cells of Brucella-infected dendritic cells with functional antigen presentation activity. Furthermore, phosphoantigen-activated Vγ9Vδ2 T cells also play a role in triggering the maturation process of dendritic cells already infected for 24 h. This suggests that activated Vγ9Vδ2 T cells could be used to modulate the outcome of infectious diseases by promoting an adjuvant effect in dendritic cell-based cellular therapies.

Chicago 2014 – 30 years of γδ T cells

The international γδ T cell conference takes place every 2 years. After being held in Denver (USA) in 2004, La Jolla (USA) in 2006, Marseille (France) in 2008, Kiel (Germany) in 2010 and Freiburg (Germany) in 2012, the γδ T cell community gathered this time in Chicago (USA). This conference was organized by Zheng Chen from 16 to 18 May 2014 at his home institution, the University of Illinois College of Medicine, and boasted 180 attendants from all over the world and almost 100 submitted abstracts.

Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells

The major function of CD8 T cells is to kill specifically target cells. Moreover in certain incurable diseases, antigen-specific human CD8 T cells are impaired, and assessment of their cytolytic activity could bring insights into their physiopathological role and ways to restore immune dysfunctions for immunotherapeutic purposes. Despite this, T cell cytolytic function has been seldom analyzed thoroughly in humans, due to the lack of approaches well suited for ex vivo assessment of T cell cytotoxicity. Current techniques require prior in vitro expansion of antigen-specific CD8 T cell populations and the use of immortalized cells as targets to measure the cell-mediated killing. Furthermore, bulk cytotoxic activity is frequently measured using percentage of specific lysis calculations that do not quantify actual target cell death and effector numbers at the single cell level. Here we established a new flow cytometry-based assay that allows accurate single-cell analysis of cytotoxic capacity of primary antigen-specific CD8 T cells generated in vivo in humans after antigenic exposure without in vitro amplification that can be used for specificities restricted by different HLAs as target cells are autologous cells. We show that this assay is robust, highly sensitive irrespective of the frequency of antigen-specific CD8 T cells, and allows accurate calculation of the index of cytotoxic capacity in lytic units. This new assay provides a sensitive method to measure the intrinsic cytotoxic activity of antigen-specific CD8 T cells directly ex vivo on human primary cells.

Stereotaxic administrations of allogeneic human Vγ9Vδ2 T cells efficiently control the development of human glioblastoma brain tumors

ABSTRACT Glioblastoma multiforme (GBM) represents the most frequent and deadliest primary brain tumor. Aggressive treatment still fails to eliminate deep brain infiltrative and highly resistant tumor cells. Human Vγ9Vδ2 T cells, the major peripheral blood γδ T cell subset, react against a wide array of tumor cells and represent attractive immune effector T cells for the design of antitumor therapies. This study aims at providing a preclinical rationale for immunotherapies in GBM based on stereotaxic administration of allogeneic human Vγ9Vδ2 T cells. The feasibility and the antitumor efficacy of stereotaxic Vγ9Vδ2 T cell injections have been investigated in orthotopic GBM mice model using selected heterogeneous and invasive primary human GBM cells. Allogeneic human Vγ9Vδ2 T cells survive and patrol for several days within the brain parenchyma following adoptive transfer and can successfully eliminate infiltrative GBM primary cells. These striking observations pave the way for optimized stereotaxic antitumor immunotherapies targeting human allogeneic Vγ9Vδ2 T cells in GBM patients.