Erika Assarsson

CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo.

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRαβ+ cells have been described as being either CD4+CD8− or CD4−CD8−. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Vα14-Jα281 TCR chain in conjunction with either a Vβ2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRαβ+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1−/−, and Jα281−/− mice that lack classical NKT cells. Unlike classical NKT cells, 50–60% of these NK1.1+TCRαβ+ cells express CD8 and have a diverse TCR Vβ repertoire. Purified NK1.1−CD8α+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rβ+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.

Memory CD8+ T Cells Provide an Early Source of IFN-γ

During the non-Ag-specific early phase of infection, IFN-γ is believed to be primarily provided by NK and NKT cells in response to pathogen-derived inflammatory mediators. To test whether other cell types were involved in early IFN-γ release, IFN-γ-producing cells were visualized in spleens and lymph nodes of LPS-injected mice. In addition to NK and NKT cells, IFN-γ was also detected in a significant fraction of CD8+ T cells. CD8+ T cells represented the second major population of IFN-γ-producing cells in the spleen (∼30%) and the majority of IFN-γ+ cells in the lymph nodes (∼70%). LPS-induced IFN-γ production by CD8+ T cells was MHC class I independent and was restricted to CD44high (memory phenotype) cells. Experiments performed with C3H/HeJ (LPS-nonresponder) mice suggested that CD8+ T cells responded to LPS indirectly through macrophage/dendritic cell-derived IFN-α/β, IL-12, and IL-18. IFN-γ was also detected in memory CD8+ T cells from mice injected with type I IFN or with poly(I:C), a synthetic dsRNA that mimics early activation by RNA viruses. Taken together, these results suggest that in response to bacterial and viral products, memory T cells may contribute to innate immunity by providing an early non-Ag-specific source of IFN-γ.

Emergence of CD8 + T Cells Expressing NK Cell Receptors in Influenza A Virus-Infected Mice

Both innate and adaptive immune responses play an important role in the recovery of the host from viral infections. In the present report, a subset of cells coexpressing CD8 and NKR-P1C (NK1.1) was found in the lungs of mice infected with influenza A virus. These cells were detected at low numbers in the lungs of uninfected mice, but represented up to 10% of the total CD8+ T cell population at day 10 postinfection. Almost all of the CD8+NK1.1+ cells were CD8αβ+CD3+TCRαβ+ and a proportion of these cells also expressed the NK cell-associated Ly49 receptors. Interestingly, up to 30% of these cells were virus-specific T cells as determined by MHC class I tetramer staining and by intracellular staining of IFN-γ after viral peptide stimulation. Moreover, these cells were distinct from conventional NKT cells as they were also found at increased numbers in influenza-infected CD1−/− mice. These results demonstrate that a significant proportion of CD8+ T cells acquire NK1.1 and other NK cell-associated molecules, and suggests that these receptors may possibly regulate CD8+ T cell effector functions during viral infection.

NK Cells Stimulate Proliferation of T and NK Cells through 2B4/CD48 Interactions

Few studies have addressed the consequences of physical interactions between NK and T cells, as well as physical interactions among NK cells themselves. We show in this study that NK cells can enhance T cell activation and proliferation in response to CD3 cross-linking and specific Ag through interactions between 2B4 (CD244) on NK cells and CD48 on T cells. Furthermore, 2B4/CD48 interactions between NK cells also enhanced proliferation of NK cells in response to IL-2. Overall, these results suggest that NK cells augment the proliferation of neighboring T and NK cells through direct cell-cell contact. These results provide new insights into NK cell-mediated control of innate and adaptive immunity and demonstrate that receptor/ligand-specific cross talk between lymphocytes may occur in settings other than T-B cell or T-T cell interactions.

Cutting Edge: Regulation of CD8+ T Cell Proliferation by 2B4/CD48 Interactions

The biological function of 2B4, a CD48-binding molecule expressed on T cells with an activation/memory phenotype, is not clear. In this report, we demonstrate that proliferation of CD8+ T cells is regulated by 2B4. Proliferative responses of CD8+ T cells were significantly reduced by anti-2B4 Ab. The effects were not potentiated by anti-CD48 Ab, suggesting that the observed responses were driven by 2B4/CD48 interactions. Surprisingly, the 2B4/CD48-dependent proliferative responses were also observed in the absence of APCs. This suggests that 2B4/CD48 interactions can occur directly between T cells. Furthermore, when activated 2B4+CD8+ T cells were mixed with 2B4−CD8+ TCR-transgenic T cells and specific peptide-loaded APC, the proliferation of the latter T cells was inhibited by anti-2B4 Ab. Taken together, this suggests that 2B4 on activated/memory T cells serves as a ligand for CD48, and by its ability to interact with CD48 provides costimulatory-like function for neighboring T cells.

2B4/CD48-Mediated Regulation of Lymphocyte Activation and Function

2B4 (CD244) is a member of the CD2 subset of the Ig superfamily. This molecule is expressed on innate immune cells, including NK cells, and on subsets of T cells. The 2B4 molecule interacts with CD48, which is widely expressed on hemopoietic cells. Although earlier reports demonstrated a role for 2B4 as an activating receptor in both mice and humans, recent studies of 2B4-deficient mice have suggested that 2B4 functions predominantly as an inhibitory receptor in mice. In addition, 2B4 may also act as a costimulatory ligand for cells expressing CD48. Thus, the 2B4 molecule is more multifunctional than previously understood. In this study, we delineate the current view of 2B4-CD48 interactions among lymphocytes and other cells.

Expression of the DX5 antigen on CD8+ T cells is associated with activation and subsequent cell death or memory during influenza virus infection

The antigen recognized by the DX5 antibody (DX5 antigen) is expressed on all murine NK cells. In the present study we found that a proportion of CD8+ T cells (∼5%) also express the DX5 antigen in uninfected mice, and that numbers of CD8+ T cells expressing DX5 are significantly higher in the lungs of influenza virus‐infected mice representing up to 50% of all CD8+ T cells on day 10 post infection. The expression of the DX5 antigen on CD8+ T cells was associated with a memory phenotype in uninfected C57BL/6 mice and with an activation phenotype during influenza virus infection. Interestingly, when lymphocytes were isolated from lungs of influenza virus‐infected mice on day 10 post infection and adoptively transferred into recombination activating gene‐1 (RAG1)‐deficient mice, CD8+DX5+ cells could not be recovered from the recipient mice 2 days later. Moreover, CD8+DX5+ cells were not detected when lung cells were removed from day 10 influenza virus‐infected mice and cultured in vitro for 2 days. However, CD8+DX5+ cells could be detected when apoptosis inhibitors were added to these cultures, suggesting that the CD8+DX5+ cells underwent apoptosis during cell culture. Furthermore, almost all DX5 expressing CD8+ cells from lungs of mice on day 10 post influenza virus infection stained positively with Annexin‐V. Taken together, the data suggest that CD8+ T cells expressing DX5 are associated with an activation/memory phenotype and are biased towards apoptosis.

Cytokine-induced killer T cells kill immature dendritic cells by TCR-independent and perforin-dependent mechanisms

Cytokine‐induced killer (CIK) cells are ex vivo, expanded T cells with proven anticancer activity in vitro and in vivo. However, their functional properties with the exception of their cancer cell‐killing activity are largely unclear. Here, we show that CIK T cells recognize dendritic cells (DC), and although mature DC (mDC) induce CIK T cells to produce IFN‐γ, immature DC (iDC) are killed selectively by them. Moreover, CIK T cell activation by mDC and their destruction of iDC are independent of the TCR. The cytotoxicity of CIK T cells to iDC is perforin‐dependent. Our data have revealed an important regulatory role of CIK cells.

IL-2 down-regulates the expression of TCR and TCR-associated surface molecules on CD8(+) T cells.

CD8+ T cells are known to down‐regulate the TCR complex upon ligation with its cognate MHC class I‐peptide complex. In the present report, we demonstrate that stimulation of CD8+ T cells with cytokines also leads to down‐regulation of the TCR complex and TCR‐associated surface molecules. A significant reduction of TCRα β, CD3, CD8α and CD8β surface expression was observed when CD8+ T cells were cultured in IL‐2 and to a lesser extent in IL‐4 or IL‐15. The down‐regulation was apparent after 2 days of culture and was observed at IL‐2 concentrations as low as 10 U/ml. Using TCR transgenic mice, we found that the down‐regulation was associated with a decreased affinity of CD8+ T cells to MHC class I‐peptide complexes, as determined by MHC class I tetramer staining. Furthermore, the antigen‐specific proliferation of IL‐2‐pre‐activated CD8+ T cells was significantly reduced compared to naive CD8+ T cells orto CD8+ T cells previously stimulated with peptide‐pulsed dendritic cells. Moreover, only CD8αhigh but not CD8αlow cells sorted from IL‐2‐activated CD8+ T cells proliferated in response to specific antigen, although both subsets proliferated equally well to IL‐2. Taken together, these data suggest that the down‐regulation of TCR components and a subsequent decrease in affinity towards MHC class I‐peptide complexes may be a mechanism by which TCR‐dependent proliferation of non‐specifically activated CD8+ T cells is avoided.

Severe Defect in Thymic Development in an Insertional Mutant Mouse Model

Transgenic mice were generated expressing NK1.1, an NK cell-associated receptor, under control of the human CD2 promoter. Unexpectedly, one of the founder lines, Tg66, showed a marked defect in thymic development characterized by disorganized architecture and small size. Mapping of the transgene insertion by fluorescence in situ hybridization revealed integration in chromosome 2, band G. Already from postnatal day 3, the thymic architecture was disturbed with a preferential loss of cortical thymic epithelial cells, a feature that became more pronounced over time. Compared with wild-type mice, total thymic cell numbers decreased dramatically between 10 and 20 days of age. Thymocytes isolated from adult Tg66 mice were predominantly immature double-negative cells, indicating a block in thymic development at an early stage of differentiation. Consequently, Tg66 mice had reduced numbers of peripheral CD4+ and CD8+ T cells. Bone marrow from Tg66 mice readily reconstituted thymi of irradiated wild-type as well as RAG-deficient mice. This indicates that the primary defect in Tg66 mice resided in nonhemopoietic stromal cells of the thymus. The phenotype is observed in mice heterozygous for the insertion and does not resemble any known mutations affecting thymic development. Preliminary studies in mice homozygous for transgene insertion reveal a more accelerated and pronounced phenotype suggesting a semidominant effect. The Tg66 mice may serve as a useful model to identify genes regulating thymic epithelial cell differentiation, thymic development, and function.