Cells were passaged on reaching confluence and supplemented with fresh medium every other day for a period of 10 days.
Interleukin 2 was obtained from Maurice Gately (Hoffmann-La Roche Inc) through the AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIAID, NIH). Cells were cultured in RPMI 1640 containing heat-inactivated FBS (5%), heat-inactivated human AB serum (5%) (Sigma Aldrich), l-glutamine (2 mM), streptomycin/penicillin (100 IU/100 µg/mL), and recombinant human interleukin 2 (200 IU/mL). 13,14įive million PBMCs were cultured with the EBV + Akata cells, at an effector-to-target cell ratio of 10:1 in a 24-well plate. Thus, the expansion and dominance of Vδ2 + T cells in the blood of adult humans is likely due to selective pressures mediated by pathogen-derived pAgs. At birth, Vδ2 + T cells comprise a small proportion of T cells in the thymus as well as of peripheral blood T cells. 12 Despite this bias, there is no apparent restriction to the pairing of Vγ9JγPCγ1 with other δ chains. 11 A majority of γδ T cells in peripheral blood have TCRs with γ chains encoded by the Vγ9, JγP, and Cγ1 gene segments and Vδ chains encoded by Vδ2. 9,10 The complementarity determining region 3 (CDR3) loops of the δ chain have the highest potential for diversity and are longer than the γ-chain CDR3 loops, which are short and constrained. The δ-chain locus also contains 3 D segments (Dδ1-3), 4 J segments (Jδ1-4), and 1 C region gene. The other 5 segments are Vα segments that are rarely used in δ-chain rearrangements. It comprises 8 V segments (Vδ1-8) of which only Vδ1, Vδ2, and Vδ3 are bona fide Vδ segments. 7,8 The human δ-chain locus lies within the α-chain locus on chromosome 14. The human γ-chain locus is located on chromosome 7 and comprises 2 constant region genes (Cγ1 and Cγ2), 5 joining segments (Jγ1, Jγ2, JγP, JγP1, and JγP2) and 14 variable gene segments, of which only 6 (Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9) are functional. This qualitative difference in response does not correlate with sex, HLA type, or previous exposure to either EBV or cytomegalovirus (CMV). Indeed, after EBV stimulation in vitro, the proliferation of γδ T cells from group 1 donors is ∼10-fold greater than that of the γδ T cells from group 2 donors.
4 For group 1 donors, their natural killer (NK) cells and γδ T cells both make a strong response to EBV, whereas group 2 donors make a strong NK-cell response but a weak γδ T-cell response. With this in vitro model, we previously showed that innate immunity to EBV divides healthy humans into 2 groups. 3 In this investigation, we used an EBV + Burkitt lymphoma cell line to study the innate immune response to EBV infection. This form of EBV latency, termed “latency I,” resembles that present in Burkitt lymphoma, an EBV-associated malignancy. Consequently, only some latently EBV-infected B cells survive to become memory B cells. In vivo, EBV infection is usually controlled by the human host’s immune system. In conclusion, we find that differences in the TCRγ-chain repertoire underlie the differential response of group 1 and group 2 to EBV.ĮBV infects epithelial cells and B cells.
In group 2 donors, EBV activates a small subpopulation of γδ T cells, most expressing Vγ9JγP. The TCRs using Vγ9JγP are closely related to the TCRs of γδ T cells that respond to phosphoantigens. In group 1 donors, EBV activates many γδ T cells expressing Vγ9JγP, inducing proliferation that produces a large population of activated effector cells. In the absence of EBV, group 1 TCRγ chains are enriched for complementarity determining region 3 (CDR3s) containing JγP, whereas group 2 TCRγ chains are enriched for CDR3s containing Jγ2. To investigate the underlying basis for this difference in γδ T-cell immunity to EBV, we used next-generation sequencing to compare the γδ T-cell receptor (TCR) repertoires of groups 1 and 2. Group 1 makes a strong natural killer (NK)–cell and γδ T-cell response, whereas group 2 makes a strong NK-cell response, but a weak γδ T-cell response. Humans form 2 groups based on their innate immunity to Epstein-Barr virus (EBV).
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