IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation.

Cytokines that are induced by infection may contribute to the initiation of immune responses through their ability to stimulate dendritic cells (DCs). In this paper, we have addressed the role of IL-15 in DC activation, investigating its expression by DCs in response to three different signals of infection and examining its ability to stimulate DCs. We report that the expression of both IL-15 and the IL-15 receptor alpha-chain are increased in splenic DCs from mice inoculated with dsRNA (poly(I:C)), LPS, or IFN-alphabeta, and in purified murine splenic DCs treated with IFN-alphabeta in vitro. Furthermore, IL-15 itself was able to activate DCs, as in vivo or in vitro exposure of splenic DCs to IL-15 resulted in an up-regulation of costimulatory molecules, markedly increased production of IFN-gamma by DC and an enhanced ability of DCs to stimulate Ag-specific CD8(+) T cell proliferation. The magnitude of all of the IL-15-induced changes in DCs was reduced in mice deficient for the IFN-alphabeta receptor, suggesting a role for IFN-alphabeta in the stimulation of DCs by IL-15. These results identify IL-15 as a stimulatory cytokine for DCs with the potential for autocrine activity and link its effects to expression of IFN-alphabeta.

T cell death and memory.

In typical immune responses, contact with antigen causes naive T cells to proliferate and differentiate into effector cells. After the pathogen is destroyed, most effector T cells are eliminated-thereby preserving the primary T cell repertoire-but some cells survive and form long-lived memory cells. During each stage of this process, the life or death fate of T cells is strictly regulated.

An IFN-gamma-dependent pathway controls stimulation of memory phenotype CD8+ T cell turnover in vivo by IL-12, IL-18, and IFN-gamma.

Unlike naive T cells, memory phenotype (CD44(high)) T cells exhibit a high background rate of turnover in vivo. Previous studies showed that the turnover of memory phenotype CD8(+) (but not CD4(+)) cells in vivo can be considerably enhanced by products of infectious agents such as LPS. Such stimulation is TCR independent and hinges on the release of type I IFNs (IFN-I) which leads to the production of an effector cytokine, probably IL-15. In this study, we describe a second pathway of CD44(high) CD8(+) stimulation in vivo. This pathway is IFN-gamma rather than IFN-I dependent and is mediated by at least three cytokines, IL-12, IL-18, and IFN-gamma. As for IFN-I, these three cytokines are nonstimulatory for purified T cells and under in vivo conditions probably act via production of IL-15.

Type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo.

Type I interferons (IFN-I) are rapidly induced following infection and play a key role in nonspecific inhibition of virus replication. Here we have investigated the effects of IFN-I on the generation of antigen-specific antibody responses. The data show that IFN-I potently enhance the primary antibody response to a soluble protein, stimulating the production of all subclasses of IgG, and induce long-lived antibody production and immunological memory. In addition, endogenous production of IFN-I was shown to be essential for the adjuvant activity of CFA. Finally, IFN-I enhanced the antibody response and induced isotype switching when dendritic cells were the only cell type responding to IFN-I. The data reveal the potent adjuvant activity of IFN-I and their important role in linking innate and adaptive immunity.

The development, maturation, and turnover rate of mouse spleen dendritic cell populations.

Three distinct subtypes of dendritic cells (DC) are present in mouse spleen, separable as CD4(-)8alpha(-), CD4(+)8alpha(-), and CD4(-)8alpha(+) DC. We have tested whether these represent stages of development or activation within one DC lineage, or whether they represent separate DC lineages. All three DC subtypes appear relatively mature by many criteria, but all retain a capacity to phagocytose particulate material in vivo. Although further maturation or activation could be induced by bacterially derived stimuli, phagocytic capacity was retained, and no DC subtype was converted to the other. Continuous elimination of CD4(+)8(-) DC by Ab depletion had no effect on the levels of the other DC subtypes. Bromodeoxyuridine labeling experiments indicated that all three DC subtypes have a rapid turnover (half-life, 1.5-2.9 days) in the spleen, with none being the precursor of another. The three DC subtypes showed different kinetics of development from bone marrow precursors. The CD8alpha(+) spleen DC, apparently the most mature, displayed an extremely rapid turnover based on bromodeoxyuridine uptake and the fastest generation from bone marrow precursors. In conclusion, the three splenic DC subtypes behave as rapidly turning over products of three independent developmental streams.

Stimulation of memory T cells by cytokines.

Mature T cells can be classified on the basis of cell surface markers into naïve- and memory-phenotype cells. These phenotypically-defined subsets exhibit distinct kinetic behaviour in vivo. Thus, naïve-phenotype T cells persist long-term in a non-dividing state, while memory-phenotype T cells include cycling cells and have a more rapid rate of turnover. We have investigated the possibility that the different kinetic behaviour of naïve- and memory-phenotype T cells reflects a differential responsiveness to cytokines. It was discovered that memory-, but not naïve-, phenotype T cells were stimulated to proliferate by a variety of infection-induced cytokines. These results suggest that cytokines contribute to the high background rate of turnover exhibited by memory T cells.

Stimulation of naïve and memory T cells by cytokines.

On the basis of cell surface markers, mature T cells are considered to have either a naïve or a memory phenotype. These cells exhibit distinct types of kinetic behaviour in vivo. While naïve-phenotype cells persist long term in a non-dividing state, memory-phenotype T cells include cycling cells and exhibit a more rapid rate of turnover; this has also been shown to be true for cells that can be definitively identified as naïve or memory T cells respectively. The number of memory-phenotype (CD44hi) CD8+ T cells entering cell cycle is greatly increased after in vivo exposure to viruses, bacteria or components of bacteria. Accelerated turnover of memory T cells also occurs after the injection of a variety cytokines that are induced by infectious agents, including type I interferon (IFN-I). Although naïve-phenotype T cells do not divide in response to these cytokines, they do exhibit signs of activation, including upregulation of CD69 after exposure to IFN-I. These findings suggest that the dissimilar in vivo kinetics of naïve- and memory-phenotype T cells might reflect their divergent responses to cytokines. Furthermore, the ability of infection-induced cytokines to stimulate non-specific proliferation of memory-phenotype T cells and partial activation of naïve-phenotype T cells implies that they play a complex role during primary immune responses to infectious agents.

Type I interferon-mediated stimulation of T cells by CpG DNA.

Immunostimulatory DNA and oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNA) are strongly stimulatory for B cells and antigen-presenting cells (APCs). We report here that, as manifested by CD69 and B7-2 upregulation, CpG DNA also induces partial activation of T cells, including naive-phenotype T cells, both in vivo and in vitro. Under in vitro conditions, CpG DNA caused activation of T cells in spleen cell suspensions but failed to stimulate highly purified T cells unless these cells were supplemented with APCs. Three lines of evidence suggested that APC-dependent stimulation of T cells by CpG DNA was mediated by type I interferons (IFN-I). First, T cell activation by CpG DNA was undetectable in IFN-IR-/- mice. Second, in contrast to normal T cells, the failure of purified IFN-IR-/- T cells to respond to CpG DNA could not be overcome by adding normal IFN-IR+ APCs. Third, IFN-I (but not IFN-gamma) caused the same pattern of partial T cell activation as CpG DNA. Significantly, T cell activation by IFN-I was APC independent. Thus, CpG DNA appeared to stimulate T cells by inducing APCs to synthesize IFN-I, which then acted directly on T cells via IFN-IR. Functional studies suggested that activation of T cells by IFN-I was inhibitory. Thus, exposing normal (but not IFN-IR-/-) T cells to CpG DNA in vivo led to reduced T proliferative responses after TCR ligation in vitro.

The induction of in vivo proliferation of long-lived CD44hi CD8+ T cells after the injection of tumor cells expressing IFN-alpha1 into syngeneic mice.

The tumorigenicity of transplantable tumor cells in mice is reduced by transduction with cytokine genes, including IFN-alpha and interleukin (IL) 12. Although T cells are considered important in tumor rejection, the mechanism by which genetically modified tumor cells stimulate the immune system has not been examined. In this study, the in vivo proliferation of T-cell subsets in mice transplanted with cytokine-producing syngeneic tumor cells was assessed by administering the DNA precursor bromodeoxyuridine. The injection of viable cells producing IFN-alpha or IL-12 caused a marked proliferation of CD8+ T lymphocytes in both the spleen and lymph nodes. Proliferation was most prominent among memory-phenotype CD44hi CD8+ T cells. In contrast, proliferation of CD8+ T cells did not occur in mice injected with control cells or with cells expressing IL-4, granulocyte colony-stimulating factor, or IFN-gamma. Pulse-chase studies in mice injected with IFN-alpha-producing cells showed that a proportion of proliferating CD8+ T cells survived for at least 70 days, suggesting that long-lived memory cells are induced using such an approach. In summary, these results, together with previous studies on the host immune reactivity triggered by the injection of tumor cells expressing IFN-alpha, represent a strong rationale for considering IFN-alpha as a powerful T-cell adjuvant for the generation of more effective cancer vaccines.

CD40 ligand-mediated interactions are involved in the generation of memory CD8(+) cytotoxic T lymphocytes (CTL) but are not required for the maintenance of CTL memory following virus infection.

CD8(+) cytotoxic T lymphocytes (CTL) play a key role in the control of many virus infections, and the need for vaccines to elicit strong CD8(+) T-cell responses in order to provide optimal protection in such infections is increasingly apparent. However, the mechanisms involved in the induction and maintenance of CD8(+) CTL memory are currently poorly understood. In this study, we investigated the involvement of CD40 ligand (CD40L)-mediated interactions in these processes by analyzing the memory CTL response of CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV). The maintenance of memory CD8(+) CTL precursors (CTLp) at stable frequencies over time was not impaired in CD40L-deficient mice. By contrast, the initial generation of memory CTLp was affected. CD40L-deficient mice produced lower levels of CD8(+) CTLp during the primary immune response to LCMV than did wild-type controls, despite the fact that the LCMV-specific effector CTL response of CD40L-deficient mice was indistinguishable from that of control animals. The differentiation of naïve CD8(+) T cells into effector and memory CTL thus involves pathways that can be discriminated from each other by their requirement for CD40L-mediated interactions. Expression of CD40L by CTLp themselves was not an essential step during their expansion and differentiation from naïve CD8(+) cells into memory CTLp; instead, the reduction in memory CTLp generation in CD40L-deficient mice was likely a consequence of defects in the CD4(+) T-cell response mounted by these animals. These results thus suggest a previously unappreciated role for CD40L in the generation of CD8(+) memory CTLp, the probable nature of which is discussed.

Bystander stimulation of T cells in vivo by cytokines.

Immune responses to infectious agents, especially viruses, are often associated with extensive proliferation of T cells and transient enlargement of the lymphoid tissues. Since the precursor frequency of T cells for specific antigen is low, the bulk of the T cells proliferating in the primary response are presumably stimulated via non-antigen-specific mechanisms, e.g. via cytokines elicited by the infectious agent concerned. Such 'bystander' stimulation of T cells occurs in mice injected with agents that elicit production of type I interferon (IFN I). Induction of IFN I in vivo causes marked stimulation of the CD44hi subset of CD8+ T cells and is prominent after injection of live viruses or products of bacteria such as lipopolysaccharide. Cytokines elicited by infectious agents may act as adjuvants during the primary response and could serve to boost the survival of long-lived memory cells.

Anti-viral immunity: spotting virus-specific T cells.

Historically, quantitation of virus-specific CD8+ T cells has been accomplished by limiting dilution analysis of cytotoxic precursor cells. Recent studies have shown that this technique greatly underestimates the actual number of antigen-specific cells and have provided new insight into anti-viral immune responses.

Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15.

Proliferation of memory-phenotype (CD44hi) CD8+ cells induced by infectious agents can be mimicked by injection of type I interferon (IFN I) and by IFN I-inducing agents such as lipopolysaccharide and Poly I:C; such proliferation does not affect naive T cells and appears to be TCR independent. Since IFN I inhibits proliferation in vitro, IFN I-induced proliferation of CD8+ cells in vivo presumably occurs indirectly through production of secondary cytokines, e.g., interleukin-2 (IL-2) or IL-15. We show here that, unlike IL-2, IL-15 closely mimics the effects of IFN I in causing strong and selective stimulation of memory-phenotype CD44hi CD8+ (but not CD4+) cells in vivo; similar specificity applies to purified T cells in vitro and correlates with much higher expression of IL-2Rbeta on CD8+ cells than on CD4+ cells.

Lifespan of gamma/delta T cells.

Information on the turnover and lifespan of murine gamma/delta cells was obtained by administering the DNA precursor, bromodeoxyuridine (BrdU), in the drinking water and staining lymphoid cells for BrdU incorporation. For TCR-gamma/delta (Vgamma2) transgenic mice, nearly all gamma/delta thymocytes became BrdU+ within 2 d and were released rapidly into the peripheral lymphoid tissues. These recent thymic emigrants (RTEs) underwent phenotypic maturation in the periphery for several days, but most of these cells died within 4 wk. In adult thymectomized (ATx) transgenic mice, only a small proportion of gamma/delta cells survived as long-lived cells; most of these cells had a slow turnover and retained a naive phenotype. As in transgenic mice, the majority of RTEs generated in normal mice (C57BL/6) appeared to have a restricted lifespan as naive cells. However, in marked contrast to TCR transgenic mice, most of the gamma/delta cells surviving in ATx normal mice had a rapid turnover and displayed an activated/memory phenotype, implying a chronic response to environmental antigens. Hence, in normal mice many gamma/delta RTEs did not die but switched to memory cells.

T cell stimulation in vivo by lipopolysaccharide (LPS).

Lipopolysaccharide (LPS) from gram-negative bacteria causes polyclonal activation of B cells and stimulation of macrophages and other APC. We show here that, under in vivo conditions, LPS also induces strong stimulation of T cells. As manifested by CD69 upregulation, LPS injection stimulates both CD4 and CD8(+) T cells, and, at high doses, stimulates naive (CD44(lo)) cells as well as memory (CD44(hi)) cells. However, in terms of cell division, the response of T cells after LPS injection is limited to the CD44(hi) subset of CD8(+) cells. In contrast with B cells, proliferative responses of CD44(hi) CD8(+) cells require only very low doses of LPS (10 ng). Based on studies with LPS-nonresponder and gene-knockout mice, LPS-induced proliferation of CD44(hi) CD8(+) cells appears to operate via an indirect pathway involving LPS stimulation of APC and release of type I (alpha, beta) interferon (IFN-I). Similar selective stimulation of CD44(hi) CD8(+) cells occurs in viral infections and after injection of IFN-I, implying a common mechanism. Hence, intermittent exposure to pathogens (gram-negative bacteria and viruses) could contribute to the high background proliferation of memory-phenotype CD8(+) cells found in normal animals.

Factors controlling the turnover of T memory cells.

Most of the T cells participating in the primary immune response are rapidly eliminated, but small numbers of these cells survive and differentiate into long-lived memory cells. Information on the life history of memory cells can be obtained by studying the component of memory-phenotype T cells found in normal animals; these cells are presumed to represent memory cells specific for various environmental antigens. For CD8+ cells, in vivo exposure to viruses and certain other infectious agents causes a large proportion of memory-phenotype (CD44hi) cells to enter the cell cycle. In this situation, stimulation of CD44hi CD8+ cells does not seem to require T-cell receptor ligation and appears to reflect release of various cytokines, especially type I interferon. The capacity of infectious agents to induce non-antigen-specific stimulation of T cells may play a role in boosting the survival of memory cells and perhaps also in providing an adjuvant function during the primary response.

Induction of bystander T cell proliferation by viruses and type I interferon in vivo.

T cell proliferation in vivo is presumed to reflect a T cell receptor (TCR)-mediated polyclonal response directed to various environmental antigens. However, the massive proliferation of T cells seen in viral infections is suggestive of a bystander reaction driven by cytokines instead of the TCR. In mice, T cell proliferation in viral infections preferentially affected the CD44hi subset of CD8+ cells and was mimicked by injection of polyinosinic-polycytidylic acid [poly(I:C)], an inducer of type I interferon (IFN I), and also by purified IFN I; such proliferation was not associated with up-regulation of CD69 or CD25 expression, which implies that TCR signaling was not involved. IFN I [poly(I:C)]-stimulated CD8+ cells survived for prolonged periods in vivo and displayed the same phenotype as did long-lived antigen-specific CD8+ cells. IFN I also potentiated the clonal expansion and survival of CD8+ cells responding to specific antigen. Production of IFN I may thus play an important role in the generation and maintenance of specific memory.

In vivo response of mature T cells to Mlsa antigens. Long-term progeny of dividing cells include cells with a naive phenotype.

To characterize T cells surviving elimination following strong in vivo responses, bromodeoxyuridine (BrdU) was used to mark the V beta 6+ CD4+ T cells proliferating after exposure to MLsa superantigens. Though a portion of the surviving V beta 6+ cells failed to label with BrdU, many were BrdU+, indicating that some proliferating cells escape elimination. Both the portion of V beta 6+ cells responding to MLs Ag in vivo and the number of responding cells that escaped elimination were influenced by the MHC haplotype; weaker responses correlated with enhanced survival of BrdU+ V beta 6+ cells. As expected, some of these cells expressed cell surface markers generally associated with an activated/memory phenotype. Significantly, however, a large proportion of BrdU+ V beta 6+ cells were phenotypically indistinguishable from naive cells.