T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual1–4. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.

Human B cells are the primary targets of Epstein Barr virus (EBV) infection. In most cases EBV infection is asymptomatic because of a highly effective host immune response but some individuals develop self-limiting infectious mononucleosis, while others develop EBV-associated lymphoid or epithelial malignancies. The viral and immune factors that determine the outcome of infection are not understood. The EBV life cycle includes a lytic phase, culminating in the production of new viral particles, and a latent phase, during which the virus remains largely silent for the lifetime of the host in memory B cells. Thus, in healthy individuals there is a tightly orchestrated interplay between EBV and the host that allows the virus to persist. To promote viral persistence EBV has evolved a variety of strategies to modulate the host immune response including inhibition of immune cell function, blunting of apoptotic pathways, and interfering with antigen processing and presentation pathways. In this article we focus on mechanisms by which dysregulation of the host B cell, and immune modulation, by the virus can contribute to development of EBV+ B cell lymphomas.

Epstein–Barr virus (EBV) is a human γ-herpesvirus that establishes latency and lifelong infection in host B cells while achieving a balance with the host immune response. When the immune system is perturbed through immunosuppression or immunodeficiency, however, these latently infected B cells can give rise to aggressive B cell lymphomas. Natural killer (NK) cells are regarded as critical in the early immune response to viral infection, but their role in controlling expansion of infected B cells is not understood. Here, we report that NK cells from healthy human donors display increased killing of autologous B lymphoblastoid cell lines (LCLs) harboring latent EBV compared to primary B cells. Coculture of NK cells with autologous EBV+ LCL identifies an NK cell population that produces IFNγ and mobilizes the cytotoxic granule protein CD107a. Multi-parameter flow cytometry and Boolean analysis reveal that these functional cells are enriched for expression of the NK cell receptor NKG2A. Further, NKG2A+ NK cells more efficiently lyse autologous LCL than do NKG2A− NK cells. More specifically, NKG2A+2B4+CD16−CD57−NKG2C−NKG2D+ cells constitute the predominant NK cell population that responds to latently infected autologous EBV+ B cells. Thus, a subset of NK cells is enhanced for the ability to recognize and eliminate autologous, EBV-infected transformed cells, laying the groundwork for harnessing this subset for therapeutic use in EBV+ malignancies.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a proven oncogene that is essential for transformation LMP14 is an EBV-encoded oncoprotein that is essential for transformation of human B lymphocytes (1-3). In B cells LMP1 mimics the CD40 receptor, although unlike CD40, LMP1 functions in a ligand-independent manner and is constitutively active (4). LMP1 activates several cellular signaling pathways culminating in expression of downstream genes involved in cell transformation, survival, and proliferation. Thus, LMP1 plays a central role in EBV-associated tumorigenesis.LMP1 is composed of a short cytoplasmic N-terminal tail required for insertion into the membrane, six membrane-spanning domains that facilitate oligomerization of the protein, and a C-terminal cytoplasmic tail. Within the C terminus of LMP1 are two major signaling domains, C-terminal activation region 1 (CTAR1) and CTAR2. The CTAR interact with tumor necrosis factor receptor-associated factors (TRAFs) and TRAF-associated death domain protein (TRADD) molecules and potentially other adapter molecules to activate NF-B, Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (Erk), and phosphoinositide 3-kinase (PI3K). Several key sites within the C terminus of LMP1 are necessary for proper signaling including the PXQXT motif in the CTAR1 region which mediates binding of TRAFs 1, 2, 3, and 5, and tyrosines 384 and 385 of CTAR2 that are essential for TRADD interaction, the recruitment of TRAF2, and association of receptor-interacting protein (5-11).Activation of NF-B by LMP1 is critical to the survival of EBV-infected B cells. NF-B activation is highly regulated through association with the IB complex that prevents nuclear localization of NF-B. In EBV-infected B cells the activation of NF-B is initiated through interaction of LMP1 with TRAF2 at CTAR1 and TRAF6 at CTAR2, allowing for activation of the IB kinase (12, 13). IB kinase phosphorylates IB␣, thereby targeting it for proteasomal degradation. NF-B is then free to translocate to the nucleus and to activate transcription of downstream genes that promotes cell survival (14,15). Similar to the NF-B pathway, interaction of the adapter protein TRAF2 with both CTAR1 and CTAR2 (indirectly through TRADD) is required for p38 MAPK activation by LMP1 (16). We (17) and others (18) have shown that activation of p38 by LMP1 contributes to the induction of IL-10, an important autocrine growth factor for EBV-infected B cells.Whereas CTAR1 and CTAR2 both contribute to signaling of NF-B and p38, the activation of JNK, PI3K/Akt, and Erk rely on a single CTAR. Specifically, the CTAR2 domain of LMP1 participates in activation of the JNK pathway through interaction with TRAF6, TRAF2, and TRADD (19 -21). JNK kinase