Dendritic cells drive memory CD8 T-cell homeostasis via IL-15 transpresentation

Interleukin-15 (IL-15) is crucial for the development of naive and memory CD8 T cells and is delivered through a mechanism called transpresentation. Previous studies showed that memory CD8 T cells require IL-15 transpresentation by an as yet unknown cell of hematopoietic origin. We hypothesized that dendritic cells (DCs) transpresent IL-15 to CD8 T cells, and we examined this by developing a transgenic model that limits IL-15 transpresentation to DCs. In this study, IL-15 transpresentation by DCs had little effect on restoring naive CD8 T cells but contributed to the development of memory-phenotype CD8 T cells. The generation of virus-specific, memory CD8 T cells was partially supported by IL-15Ralpha(+) DCs through the preferential enhancement of a subset of KLRG-1(+)CD27(-) CD8 T cells. In contrast, these DCs were largely sufficient in driving normal homeostatic proliferation of established memory CD8 T cells, suggesting that memory CD8 T cells grow more dependent on IL-15 transpresentation by DCs. Overall, our study clearly supports a role for DCs in memory CD8 T-cell homeostasis but also provides evidence that other hematopoietic cells are involved in this function. The identification of DCs fulfilling this role will enable future studies to better focus on mechanisms regulating T-cell homeostasis.     

IL-15/IL-15Ralpha-mediated avidity maturation of memory CD8+ T cells

T cell avidity is critical to viral clearance, but mechanisms of CD8(+) T cell avidity maturation are poorly understood. Here, we find that IL-15 mediates two mechanisms of avidity maturation. (i) By selection at the population level, IL-15 promotes greater survival of high- compared with low-avidity cytotoxic T lymphocytes (CTLs). High-avidity CTLs express higher levels of IL-15Ralpha and persist longer by homeostatic proliferation. (ii) At the individual cell level, IL-15 induces higher levels of surface coreceptor CD8alphabeta, increasing functional avidity. IL-15 during priming selects or induces higher-avidity CTLs. Conversely, high-avidity CTLs are diminished in IL-15Ralpha knockout mice. These results provide an explanation of CD8+ T cell avidity maturation and may contribute to the design of novel vaccines.     

The IL-15/IL-15Ralpha on cell surfaces enables sustained IL-15 activity and contributes to the long survival of CD8 memory T cells

We previously described unique features of the IL-15 receptor (IL-15R)alpha. IL-15Ralpha by itself forms stable complexes with IL-15 on cell surfaces and presents IL-15 in trans to neighboring natural killer/T cells. Moreover, the membrane IL-15/IL-15Ralpha complexes (membIL-15) undergo endosomal internalization but survive lysosomal degradation, allowing the complexes to recycle back to the cell surface. Here, we show that membIL-15+ cells act as a persistent source of IL-15 for the surrounding microenvironment (intercellular reservoir effect). Additionally, membIL-15+ cells give rise to augmented retention of IL-15 in the circulation as well as in tissues. Curiously, IL-15 retention was particularly associated with lungs, rather than with lymph nodes, in normal unstimulated mice, which correlated with the preferential homing of antigen-specific CD8 T cells to lungs during their contraction phase in an IL-15Ralpha-dependent manner. Furthermore, membIL-15, unlike soluble IL-15, caused sustained IL-15 signal transduction in the target cells. Collectively, these characteristics define IL-15 as a unique cytokine with prolonged in vivo survival and sustained biological action on the target cells, which may account for the proposed persistent action of IL-15 that helps the long-term survival of functional CD8 memory T cells in vivo.     

Cytokine-dependent bystander hepatitis due to intrahepatic murine CD8 T-cell activation by bone marrow-derived cells

BACKGROUND & AIMS: Intrahepatic accumulation of CD8+ T cells following antigen-specific activation has been demonstrated in a number of transgenic models and also in extrahepatic viral infections. In some transgenic models, intrahepatic accumulation of cytotoxic T lymphocytes is associated with hepatitis. This observation suggests that hepatocellular damage may occur in some forms of immune-mediated hepatitis on the basis of a "bystander injury," whereby cytotoxic T lymphocytes accumulating in the liver mediate injury to hepatocytes in a nonspecific manner. Mouse transgenic models were therefore developed to investigate whether bystander damage to non-antigen-bearing hepatocytes occurs in vivo. METHODS: T cell receptor transgenic T cells were adoptively transferred into transgenic mice ubiquitously expressing the specific antigen, or into bone marrow radiation chimeras in which hepatocytes did not express the antigen. RESULTS: Selective accumulation of transgenic CD8+ T cells in the liver of intact recipients could be detected within 2 hours of transfer, despite ubiquitous antigenic expression. T cells retained in the liver were activated and induced hepatitis. Similar results were obtained using bone marrow chimeras, suggesting that antigen expression by hepatocytes was not required either for intrahepatic accumulation or for subsequent hepatitis. This "bystander hepatitis" was dependent on tumor necrosis factor alpha and interferon gamma. CONCLUSIONS: Intrahepatic accumulation of activated CD8+ T cells and subsequent hepatitis can result from primary activation of CD8+ T cells by liver resident bone marrow-derived cells, inducing bystander damage to non-antigen-bearing hepatocytes. This mechanism may play a role in some forms of biologically significant hepatitis, including autoimmune hepatitis and hepatitis associated with extrahepatic diseases.     

CD4+CD25+ regulatory T cells control CD8+ T-cell effector differentiation by modulating IL-2 homeostasis

CD4(+)CD25(+) regulatory T cells (Treg) play a crucial role in the regulation of immune responses. Although many mechanisms of Treg suppression in vitro have been described, the mechanisms by which Treg modulate CD8(+) T cell differentiation and effector function in vivo are more poorly defined. It has been proposed, in many instances, that modulation of cytokine homeostasis could be an important mechanism by which Treg regulate adaptive immunity; however, direct experimental evidence is sparse. Here we demonstrate that CD4(+)CD25(+) Treg, by critically regulating IL-2 homeostasis, modulate CD8(+) T-cell effector differentiation. Expansion and effector differentiation of CD8(+) T cells is promoted by autocrine IL-2 but, by competing for IL-2, Treg limit CD8(+) effector differentiation. Furthermore, a regulatory loop exists between Treg and CD8(+) effector T cells, where IL-2 produced during CD8(+) T-cell effector differentiation promotes Treg expansion.     

c-Myc acts downstream of IL-15 in the regulation of memory CD8 T-cell homeostasis

A subset of CD8 T cells in normal mice, expressing high levels of activation markers such as CD44, shares many properties with antigen-specific memory CD8 T cells. Homeostasis of CD44(high) CD8 T cells depends upon cytokines such as interleukin-15 (IL-15); however, the downstream signaling pathways regulating IL-15-dependent homeostatic proliferation are poorly defined. Surprisingly, we show here that haploinsufficiency of the protooncogene c-myc leads to a highly selective decrease in CD44(high) CD8 T cells in mice. Although steady-state proliferation and survival of CD44(high) CD8 T cells appeared not to be dependent on c-Myc, homeostatic proliferation of c-myc(+/-) CD44(high) CD8 T cells in lymphopenic hosts was strongly reduced, and the residual homeostatic proliferation of these cells appeared to occur independently of IL-15. Moreover, c-myc(+/-) CD44(high) CD8 T cells responded very poorly to purified IL-15 in vitro. Backcrossing of c-myc(+/-) mice to IL-15(-/-) mice revealed that the number of CD44(high) CD8 T cells decreased in an additive fashion in mice heterozygous for c-myc and IL-15. Finally homeostatic proliferation of antigen-specific memory CD44(high) CD8 T cells was also impaired in c-myc(+/-) mice. Collectively, our data identify c-Myc as a novel downstream component of the IL-15-dependent pathway controlling homeostatic proliferation of memory CD44(high) CD8 T cells.     

Altered IL-7Ralpha expression with aging and the potential implications of IL-7 therapy on CD8+ T-cell immune responses

We investigated the effects of aging on the IL-7-mediated CD8+ T-cell survival pathway and of IL-7 therapy on T-cell immunity. Cells expressing IL-7 receptor (IL-7R) alphahigh and alphalow were identified in a CD45RA+ effector memory (EM(CD45RA+), CD45RA+CCR7-) CD8+ T-cell subset. Elderly subjects (65 years and older) had an increased frequency of EM(CD45RA+) IL-7Ralphalow) CD8+ T cells, leading to decreased STAT5 phosphorylation and survival responses to IL-7 compared with young subjects (40 years and younger). These EM(CD45RA+) IL-7Ralphalow cells were largely antigen experienced (CD27-CD28-), replicatively senescent (CD57+), and perforinhigh CD8+ T cells that had decreased IL-7Ralpha mRNA, independent of guanine and adenine binding protein alpha (GABPalpha) and growth factor independence-1 (GFI1) expression. In measuring T-cell receptor (TCR) repertoires of EM(CD45RA+) CD8+ T cells, the elderly had a limited repertoire in IL-7Ralphahigh and IL-7Ralphalow cells, whereas the young had a diverse repertoire in IL-7Ralphahigh but not in IL-7Ralphalow cells. These findings suggest that aging affects IL-7Ralpha expression by EM(CD45RA+) CD8+ T cells, leading to impaired signaling and survival responses to IL-7, and that IL-7 therapy may improve the survival of EM(CD45RA+) CD8+ T cells with a diverse TCR repertoire in the young but not in the elderly.     

IL-15R alpha expression on CD8+ T cells is dispensable for T cell memory

The generation and maintenance of immunological memory requires the activation, expansion, and persistent proliferation of antigen-specific T cells. Recent work suggests that IL-15 may be important for this process. Surprisingly, we now find that expression of the high-affinity receptor for IL-15, IL-15R alpha, on T cells is dispensable for the generation or maintenance of memory CD8(+) T cells. By contrast, IL-15R alpha expression on cells other than T cells is absolutely critical for this function. These findings may be related to IL-15R alpha's ability to present IL-15 in trans to low-affinity IL-15R beta gamma(c) receptors on memory CD8(+) T cells. These unexpected results provide insights into how IL-15R alpha supports memory CD8(+) T cells.     

IL-7 and IL-15 differentially regulate CD8+ T-cell subsets during contraction of the immune response

Although it is known that interleukin-7 (IL-7) and IL-15 influence the survival and turnover of CD8+ T cells, less is known about how these cytokines affect different subsets during the course of the immune response. We find that IL-7 and IL-15 differentially regulate CD8+ T-cell subsets defined by KLRG1 and CD127 expression during the contraction phase of the immune response. The provision of IL-15, or the related cytokine IL-2, during contraction led to the preferential accumulation of KLRG1(hi)CD127(lo) CD8+ T cells, whereas provision of IL-7 instead favored the accumulation of KLRG1(lo)CD127(hi) cells. While IL-7 and IL-15 both induced proliferation of KLRG1(lo) cells, KLRG1(hi) cells exhibited an extraordinarily high level of resistance to cytokine-driven proliferation in vivo despite their dramatic accumulation upon IL-15 administration. These results suggest that IL-15 and IL-2 greatly improve the survival of KLRG1(hi) CD8+ T cells, which are usually destined to perish during contraction, without inducing proliferation. As the availability of IL-15 and IL-2 is enhanced during periods of extended inflammation, our results suggest a mechanism in which a population of cytokine-dependent KLRG1(hi) CD8+ T cells is temporarily retained for improved immunity. Consideration of these findings may aid in the development of immunotherapeutic strategies against infectious disease and cancer.     

Steady-state dendritic cells expressing cognate antigen terminate memory CD8+ T-cell responses

Antigen stimulation of naive T cells in conjunction with strong costimulatory signals elicits the generation of effector and memory populations. Such terminal differentiation transforms naive T cells capable of differentiating along several terminal pathways in response to pertinent environmental cues into cells that have lost developmental plasticity and exhibit heightened responsiveness. Because these cells exhibit little or no need for the strong costimulatory signals required for full activation of naive T cells, it is generally considered memory and effector T cells are released from the capacity to be inactivated. Here, we show that steady-state dendritic cells constitutively presenting an endogenously expressed antigen inactivate fully differentiated memory and effector CD8(+) T cells in vivo through deletion and inactivation. These findings indicate that fully differentiated effector and memory T cells exhibit a previously unappreciated level of plasticity and provide insight into how memory and effector T-cell populations may be regulated.     

IL-15 enhances survival and function of HIV-specific CD8+ T cells

HIV-specific CD8(+) T cells are prone to undergo apoptosis, and this may affect their ability to control HIV infection. Because CD8-mediated immune responses play a key role in controlling HIV infection, enhancing the survival and effector function of HIV-specific CD8(+) T cells may augment their ability to control HIV virus. We show here that interleukin 15 (IL-15) potently inhibits spontaneous and CD95/Fas-induced apoptosis of HIV-specific CD8(+) T cells. IL-15 inhibits apoptosis in both CD45RA(-)CD62L(-) and CD45RA(+)CD62L(-) effector memory subpopulations of these cells. Furthermore, IL-15 greatly enhances the survival of HIV-specific CD8(+) T cells in long-term cultures. Finally, IL-15 directly enhances activation, interferon gamma (IFNgamma) production, and direct ex vivo cytotoxicity of HIV-specific CD8(+) T cells. Thus, IL-15 potently enhances the survival and effector function of HIV-specific CD8(+) T cells and, therefore, may prove useful in augmenting the antiviral function of these cells.     

ITK and IL-15 support two distinct subsets of CD8+ T cells

CD8(+) T cells are commonly divided into na?ve CD44(lo)CD122(lo) and "memory phenotype" CD44(hi)CD122(hi) cells. Here we show data suggesting that these two cell populations represent independent CD8(+) T cell subsets. Whereas IL-15(-/-) mice lack CD44(hi)CD122(hi) CD8(+) T cells, mice deficient in the kinase ITK lack CD44(lo)CD122(lo) cells among CD8(+) T cells. The same defects were observed during thymus development. CD44(hi)CD122(hi) cells were found among double-positive thymocytes and increased in frequency during CD8 development in wild-type mice. At the mature stage, IL-15(-/-) mice harbored virtually no CD44(hi)CD122(hi) CD8(+) thymocytes. In contrast, ITK(-/-) mice lacked CD44(lo)CD122(lo) CD8(+) cells at this stage. We generated mice with genetic deletions in both IL-15 and ITK and observed a severe reduction of all CD8(+) T cells. The two CD44(lo)CD122(lo) and CD44(hi)CD122(hi) CD8(+) T cell subsets differed in the periphery in that natural killer (NK) receptor expression was found only on CD44(hi)CD122(hi) CD8(+) T cells. This expression was paralleled by their ability to respond to both T cell receptor and NK receptor engagements. In contrast, CD44(lo)CD122(lo) CD8(+) T cells mounted stronger responses to T cell receptor stimulation but failed to recognize NK receptor ligands. Thus, whereas ITK-dependent CD44(lo)CD122(lo) CD8(+) T cells appear to represent conventional CD8(+) T cells, IL-15-dependent CD44(hi)CD122(hi) CD8(+) T cells may have functions in both adaptive and innate immunity.     

Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+ CD28+ T lymphocyte responses

ObjectiveTo develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8⁺ CD28⁺ cytotoxic T lymphocyte (CTL) responses.MethodsCell-sized Dynabeads® M-450 Epoxy beads coated with H-2K^b: Ig-TRP2_180-188and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8⁺CD28⁺ CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs.ResultsDimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8⁺CD28⁺CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- γ under the stimulation of H-2K^b: Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells.ConclusionsThe new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8⁺ CD28⁺ CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.     

IL-15 mimics T cell receptor crosslinking in the induction of cellular proliferation, gene expression, and cytotoxicity in CD8+ memory T cells

Generation of CD8(+) memory T cells requires antigenic stimulation through T cell receptor (TCR); however, maintenance of CD8(+) memory T cells seems to be mediated by cytokines, such as IL-15, in a TCR-independent manner. Compared with the TCR-induced activation, less is known about the mechanisms of IL-15 action. We report here a comparative and kinetic analysis of the responses of memory phenotype CD8(+) T cells to IL-15 or TCR (anti-CD3) stimulation in vitro. These two stimuli induce highly similar responses in memory phenotype CD8(+) T cells as measured by cellular proliferation, gene expression changes, synthesis of effector molecules (IFNgamma, tumor necrosis factor beta, granzyme B, and perforin), and induction of cytotoxicity. From 189 genes/expressed sequence tags (ESTs) whose expression changed in CD8(+) memory T cells after IL-15 and anti-CD3 stimulation identified by cDNA microarray analysis, 77% of the genes/ESTs exhibit a highly similar pattern of expression between IL-15 and anti-CD3-treated cells, and only 16% and 7% of the genes/ESTs are differentially expressed in response to IL-15 and anti-CD3 treatments, respectively. These results show that IL-15 and anti-CD3 stimulation induced remarkably similar gene expression and effector function. Thus, IL-15 acts not only as a crucial growth factor but also as an antigen-independent activator of effector functions for CD8(+) memory T cells.     

IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T cells

IL-15 and IL-2 possess similar properties, including the ability to induce T cell proliferation. However, whereas IL-2 can promote apoptosis and limit CD8(+) memory T cell survival and proliferation, IL-15 helps maintain a memory CD8(+) T cell population and can inhibit apoptosis. We sought to determine whether IL-15 could enhance the in vivo function of tumor/self-reactive CD8(+) T cells by using a T cell receptor transgenic mouse (pmel-1) whose CD8(+) T cells recognize an epitope derived from the self/melanoma antigen gp100. By removing endogenous IL-15 by using tumor-bearing IL-15 knockout hosts or supplementing IL-15 by means of exogenous administration, as a component of culture media or as a transgene expressed by adoptively transferred T cells, we demonstrate that IL-15 can improve the in vivo antitumor activity of adoptively transferred CD8(+) T cells. These results provide several avenues for improving adoptive immunotherapy of cancer in patients.     

TLR3 ligand stimulates fully functional memory CD8+ T cells in the absence of CD4+ T-cell help

We investigated whether Toll-like receptor ligands (TLR-Ls) can bypass the requirement for CD4(+) T-cell help in the induction of fully efficient memory CD8(+) T cells (cytotoxic T lymphocytes [CTLs]). "Helpless" CTLs were induced by a synthetic CD8(+) T-cell epitope administered with TLR3-L and TLR9-L, but not with TLR2/6-L, TLR4-L, or TLR7-L. The up-regulation of MHC-I and costimulatory molecules by dendritic cells following TLR stimulation was not sufficient for the priming of "helpless" CTLs, which depended essentially on the induction of a strong IFN-alpha/beta response. The "helpless" CTLs induced by TLR-Ls differentiated into fully functional memory CTLs able to proliferate as well as their "helped" counterparts upon challenge, in the absence of CD4(+) T-cell help.     

白细胞介素-15对骨髓增生异常综合征骨髓造血干/祖细胞增殖的影响

目的：观察白细胞介素(IL-15)对体外培养的骨髓增生异常综合征(MDS)患者CD34^ 细胞增殖作用。方法：应用单克隆抗体免疫磁珠分离系统提取MDS患者CD34^ 细胞，以加IL-15组为实验组，不加IL-15组为对照组，进行液体和甲基纤维素半固体集落培养，计算培养后细胞数和CFU—E、BFU—E、CFU—GM、CFU—GEMM等集落数，并用MTT比色法检测IL-15对MDS患者CD34^ 细胞增殖的抑制作用，流式细胞术检测上述培养细胞周期的变异情况。结果：11例MDS对象平均CD34^ 细胞比例、回收率、纯度和富集倍数均达要求，MTT比色法检测IL-15对CD34^ 细胞的增殖作用呈最佳浓度效应，最佳浓度为20μg／L，细胞增殖抑制最低峰值时间为8d。用0μg／L IL-15(对照组)和20μg／L IL-15(实验组)作用MDS CD34^ 细胞，计数显示培养细胞最大增殖倍数和集落形成比率实验组均较对照组明显增加，IL-15作用后各细胞周期G1、S、G2期比例有明显改变，与对照组比较，差异有统计学意义。结论：IL-15对MDS CD34^ 细胞有促增殖效应，与其它造血生长因子具有协同作用，对MDS治疗可能有一定的应用前景。     

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor-negative cells that facilitate bone marrow repopulation with hematopoietic stem cells

Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8(+) cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8(+) cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8alpha(+)CD3epsilon(+) T-cell receptor- negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34(+) cells. In vitro, cFCs cocultured with CD34(+) cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8(+)CD3(+)TCR(-) cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.     

CD25+CD4+ T cells contribute to the control of memory CD8+ T cells

Previously we demonstrated that IL-15 and IL-2 control the number of memory CD8+ T cells in mice. IL-15 induces, and IL-2 suppresses the division of these cells. Here we show that CD25+CD4+ regulatory T cells play an important role in the IL-2-mediated control of memory phenotype CD8+ T cell number. In animals, the numbers of CD25+CD4+ T cells were inversely correlated with the numbers of memory phenotype CD8+ T cells with age. Treatment with anti-IL-2 caused CD25+CD4+ T cells to disappear and, concurrently, increased the numbers of memory phenotype CD8+ T cells. This increase in the numbers of CD8+ memory phenotype T cells was not manifest in animals lacking CD4+ cells. Importantly, adoptive transfer of CD25+CD4+ T cells significantly reduced division of memory phenotype CD8+ T cells. Thus, we conclude that CD25+CD4+ T cells are involved in the IL-2-mediated inhibition of memory CD8+ T cell division and that IL-2 controls memory phenotype CD8+ T cell numbers at least in part through maintenance of the CD25+CD4+ T cell population.