Our findings suggest a potentially novel therapeutic approach to seasonal, zoonotic avian, and pandemic influenza-the use of phosphoantigens to activate gammadelta T cells against influenza virus infections.

While few children and young adults have cross-protective antibodies to the pandemic H1N1 2009 (pdmH1N1) virus, the illness remains mild. The biological reasons for these epidemiological observations are unclear. In this study, we demonstrate that the bulk memory cytotoxic T lymphocytes (CTLs) established by seasonal influenza viruses from healthy individuals who have not been exposed to pdmH1N1 can directly lyse pdmH1N1-infected target cells and produce gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣). Using influenza A virus matrix protein 1 (M1 58-66 ) epitope-specific CTLs isolated from healthy HLA-A2 ؉ individuals, we further found that M1 58-66 epitope-specific CTLs efficiently killed both M1 58-66 peptide-pulsed and pdmH1N1-infected target cells ex vivo. These M1 58-66 -specific CTLs showed an effector memory phenotype and expressed CXCR3 and CCR5 chemokine receptors. Of 94 influenza A virus CD8 T-cell epitopes obtained from the Immune Epitope Database (IEDB), 17 epitopes are conserved in pdmH1N1, and more than half of these conserved epitopes are derived from M1 protein. In addition, 65% (11/17) of these epitopes were 100% conserved in seasonal influenza vaccine H1N1 strains during the last 20 years. Importantly, seasonal influenza vaccination could expand the functional M1 58-66 epitope-specific CTLs in 20% (4/20) of HLA-A2 ؉ individuals. Our results indicated that memory CTLs established by seasonal influenza A viruses or vaccines had cross-reactivity against pdmH1N1. These might explain, at least in part, the unexpected mild pdmH1N1 illness in the community and also might provide some valuable insights for the future design of broadly protective vaccines to prevent influenza, especially pandemic influenza.

IntroductionCD4 ϩ CD25 ϩ Foxp3 ϩ regulatory T cells (Treg) are negative regulators of immune responses to self-and foreign antigens and play a critical role in maintaining immune tolerance by suppressing pathologic immune responses in autoimmune diseases, transplant allograft rejection, and graft-versus-host disease (GVHD). [1][2][3] On adoptive transfer in rodents, Treg were found to control experimental autoimmune diseases, 4 inhibit GVHD, 5,6 and prevent transplant allograft rejection, 7,8 indicating that Treg-based therapy has a great therapeutic potential for these diseases in humans.An important obstacle to Treg-based therapy has been the limited numbers of these cells that are available, as only approximately 1% to 2% of circulating human CD4 ϩ T cells are Treg. Several groups have developed protocols to expand a large number of polyclonal CD4 ϩ CD25 ϩ Treg in vitro with repeated stimulation by either CD3 and CD28 mAbs or artificial antigenpresenting cells (APCs) for activation through CD3 and CD28, together with exogenous high-dose interleukin-2 (IL-2). [9][10][11] However, polyclonal Treg may cause global immune suppression. 4,7 In addition, because there are only few antigen-specific Treg in the population of the polyclonal Treg, very large numbers of nonspecifically expanded Treg are required to inhibit bone-marrow allograft rejection in animal models. 12 All of these characteristics of polyclonal Treg hamper their clinical applications.In contrast, adoptive transfer of antigen-specific Treg has been shown to prevent and treat T cell-mediated inflammatory diseases with high efficiency. In animal models, small numbers of antigen-specific Treg can suppress experimental autoimmune diseases 13 and prevent GVHD and allograft rejection in bone marrow and solid organ transplantation. 14,15 Importantly, the transfer of antigen-specific Treg prevented target antigenmediated T-cell responses, such as GVHD and allograft rejection, but did not compromise host general immunity, including the graft-versus-tumor activity and antiviral immunity. 5,[15][16][17] Based on these studies, antigen-specific Treg has substantial promise for human immunotherapy.The reliable induction and expansion of rare antigen-specific Treg are technically challenging. Currently, several protocols for murine antigen-specific Treg induction and expansion have been reported in which either purified CD4 ϩ CD25 Ϫ or CD4 ϩ CD25 ϩ cells were cocultured with autologous dendritic cells (DCs) pulsed with alloantigen in the presence of high-dose IL-2 or directly cocultured with allogeneic DCs. 14,18-20 Similar protocol has also been reported for generation of human antigen-specific Treg recently. 21 In this protocol, antigen-specific CD4 ϩ CD25 ϩ Treg can be generated using the coculture of CD4 ϩ CD25 Ϫ T cells with allogeneic monocyte-derived DCs. However, the large-scale in vitro expansion of alloantigen-specific Treg is difficult because of certain features of DCs. For example, DCs are relatively rare in peripheral blood and are usually derived f...