We show that STAT5b is important for the in vivo accumulation of CD4+CD25high T cells with regulatory cell function. A patient homozygous for a missense A630P STAT5b mutation displayed immune dysregulation and decreased numbers of CD4+CD25high T cells. STAT5bA630P/A630P CD4+CD25high T cells had low expression of forkhead box P3 and an impaired ability to suppress the proliferation of or to kill CD4+CD25− T cells. Expression of CD25, a component of the high-affinity IL-2R, was also reduced in response to IL-2 or after in vitro propagation. The impact of the STAT5b mutation was selective in that IL-2-mediated up-regulation of the common γ-chain cytokine receptor and perforin, and activation-induced expressions of CD154 and IFN-γ were normal. These results indicate that STAT5b propagates an important IL-2-mediated signal for the in vivo accumulation of functional regulatory T cells.

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.

Healthy young children who acquire CMV have prolonged viral shedding into the urine and saliva, but whether this is attributable to limitations in viral-specific immune responses has not been explored. In this study, we found that otherwise immunocompetent young children after recent primary CMV infection accumulated markedly fewer CMV-specific CD4+ T cells that produced IFN-γ than did adults. These differences in CD4+ T cell function persisted for more than 1 year after viral acquisition, and did not apply to CMV-specific IFN-γ production by CD8+ T cells. The IFN-γ-producing CD4+ T cells of children or adults that were reactive with CMV Ags were mainly the CCR7low cell subset of memory (CD45R0highCD45RAlow) cells. The decreased IFN-γ response to CMV in children was selective, because their CCR7low memory CD4+ T cells and those of adults produced similar levels of this cytokine after stimulation with staphylococcal enterotoxin B superantigen. CD4+ T cells from children also had reduced CMV-specific IL-2 and CD154 (CD40 ligand) expression, suggesting an early blockade in the differentiation of viral-specific CD4+ T cells. Following CMV acquisition, children, but not adults, persistently shed virus in urine, and this was observable for at least 29 mo postinfection. Thus, CD4+ T cell-mediated immunity to CMV in humans is generated in an age-dependent manner, and may have a substantial role in controlling renal viral replication and urinary shedding.

Vγ9Vδ2 T cells are promising candidates for cellular tumor immunotherapy. Due to their HLA-independent mode of action, allogeneic Vγ9Vδ2 T cells can be considered for clinical application. To apply allogeneic Vγ9Vδ2 T cells in adoptive immunotherapy, the methodology used to obtain adequate cell numbers with optimal effector function in vitro needs to be optimized, and clinical safety and efficacy also need to be proven. Therefore, we developed a novel formula to improve the expansion of peripheral γδ T cells from healthy donors. Then, we used a humanized mouse model to validate the therapeutic efficacy of expanded γδ T cells in vivo; furthermore, the expanded γδ T cells were adoptively transferred into late-stage liver and lung cancer patients. We found that the expanded cells possessed significantly improved immune effector functions, including proliferation, differentiation, and cancer cell killing, both in vitro and in the humanized mouse model. Furthermore, a phase I clinical trial in 132 late-stage cancer patients with a total of 414 cell infusions unequivocally validated the clinical safety of allogeneic Vγ9Vδ2 T cells. Among these 132 patients, 8 liver cancer patients and 10 lung cancer patients who received ≥5 cell infusions showed greatly prolonged survival, which preliminarily verified the efficacy of allogeneic Vγ9Vδ2 T-cell therapy. Our clinical studies underscore the safety and efficacy of allogeneic Vγ9Vδ2 T-cell immunotherapy, which will inspire further clinical investigations and eventually benefit cancer patients.

As shown in humanized mice, a population of Vγ9Vδ2 T cells can reduce the severity and mortality of disease caused by infection with human and avian influenza viruses.

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...

Influenza is an acute respiratory viral disease that is transmitted in the first few days of infection. Evasion of host innate immune defenses, including natural killer (NK) cells, is important for the virus's success as a pathogen of humans and other animals. NK cells encounter influenza viruses within the microenvironment of infected cells and are important for host innate immunity during influenza virus infection. It is therefore important to investigate the direct effects of influenza virus on NK cells. In this study, we demonstrated for the first time that influenza virus directly infects and replicates in primary human NK cells. Viral entry into NK cells was mediated by both clathrin-and caveolin-dependent endocytosis rather than through macropinocytosis and was dependent on the sialic acids on cell surfaces. In addition, influenza virus infection induced a marked apoptosis of NK cells. Our findings suggest that influenza virus can directly target and kill NK cells, a potential novel strategy of influenza virus to evade the NK cell innate immune defense that is likely to facilitate viral transmission and may also contribute to virus pathogenesis.