Population dynamics of naive and memory CD8 T cell responses after antigen stimulations in vivo

The extent to which the progeny of one primary memory CD8 T cell differs from the progeny of one naive CD8 T cell of the same specificity remains an unresolved question. To explore cell-autonomous functional differences between naive and memory CD8 T cells that are not influenced by differences in the priming environment, an experimental model has been developed in which physiological numbers of both populations of cells were cotransferred into naive hosts before Ag stimulation. Interestingly, naive CD8 T cells undergo greater expansion in numbers than do primary memory CD8 T cells after various infections or immunizations. The intrinsic ability of one naive CD8 T cell to give rise to more effector CD8 T cells than one memory CD8 T cell is independent of the number and quality of primary memory CD8 T cells present in vivo. The sustained proliferation of newly activated naive CD8 T cells contributed to their greater magnitude of expansion. Additionally, longitudinal analyses of primary and secondary CD8 T cell responses revealed that on a per-cell basis naive CD8 T cells generate higher numbers of long-lived memory cells than do primary memory CD8 T cells. This enhanced "memory generation potential" of responding naive CD8 T cells occurred despite the delayed contraction of secondary CD8 T cell responses. Taken together, the data in this study revealed previously unappreciated differences between naive and memory CD8 T cells and will help further define the functional potential for both cell types.     

Recombinant interleukin-7 induces proliferation of naive macaque CD4+ and CD8+ T cells in vivo

Interleukin-7 (IL-7) regulates T-cell homeostasis, and its availability is augmented in lymphopenic hosts. Naive CD8+ T cells transferred to lymphopenic mice acquire a memory-like phenotype, raising the possibility that IL-7 is the biological mediator of this effect. Here, we provide direct evidence that IL-7 induces the acquisition of memory-cell markers not only in CD8+ T cells but also in CD4+ T-cell subsets in immune-competent Indian rhesus macaques. The increase of these memory-like populations was dependent on the dose of the cytokine, and these cells were found in the blood as well as secondary lymphoid organs. Memory-like CD4+ and CD8+ T cells acquired the ability to secrete tumor necrosis factor alpha and, to a lesser extent, gamma interferon following stimulation with a cognate antigen. The phenotypic change observed in naive T cells was promptly reversed after discontinuation of IL-7. Importantly, IL-7 induced cycling of both CD4+ and CD8+ central memory and effector memory T cells, demonstrating its contribution to the maintenance of the entire T-cell pool. Thus, IL-7 may be of benefit in the treatment of iatrogenic or virus-induced T-cell depletion.     

Transcriptional regulation of CD4 gene expression by T cell factor-1/beta-catenin pathway

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.     

Memory CD4 T cells induce selective expression of IL-27 in CD8+ dendritic cells and regulate homeostatic naive T cell proliferation

Naive T cells undergo robust proliferation in lymphopenic conditions, whereas they remain quiescent in steady-state conditions. However, a mechanism by which naive T cells are kept from proliferating under steady-state conditions remains unclear. In this study, we report that memory CD4 T cells are able to limit naive T cell proliferation within lymphopenic hosts by modulating stimulatory functions of dendritic cells (DC). The inhibition was mediated by IL-27, which was primarily expressed in CD8(+) DC subsets as the result of memory CD4 T cell-DC interaction. IL-27 appeared to be the major mediator of inhibition, as naive T cells deficient in IL-27R were resistant to memory CD4 T cell-mediated inhibition. Finally, IL-27-mediated regulation of T cell proliferation was also observed in steady-state conditions as well as during Ag-mediated immune responses. We propose a new model for maintaining peripheral T cell homeostasis via memory CD4 T cells and CD8(+) DC-derived IL-27 in vivo.     

The lymphoid chemokine CCL21 costimulates naive T cell expansion and Th1 polarization of non-regulatory CD4+ T cells

CCL21 (SLC/6Ckine) is constitutively expressed by secondary lymphoid tissue and attracts CCR7-expressing mature dendritic cells and naive T cells. Recent studies demonstrated that intra-tumoral delivery of CCL21 induces tumor regression in a T cell dependent manner. CCL21 is known to mediate T cell trafficking but little is known about its function as a costimulatory molecule. Herein, we demonstrate that CCL21 costimulates expansion of CD4+ and CD8+ T cells and induces Th1 polarization. These effects were specific for naive T cells, and we show that CD4+CD25+ regulatory T cells were hyporesponsive to CCL21 induced migration, and unresponsive to CCL21 costimulation. These unique functions of CCL21 to both attract naive T cells as well as costimulate their proliferation and differentiation, suggests that CCL21 is a pivotal molecule for priming T cell responses and has therapeutic implications for local delivery of CCL21. The coordinated effects of CCL21 on T cell migration and activation may also represent a more comprehensive paradigm for the activity of other chemokines as well.     

MHC class I-positive dendritic cells (DC) control CD8 T cell homeostasis in vivo: T cell lymphopenia as a prerequisite for DC-mediated homeostatic proliferation of naive CD8 T cells

The sizes of peripheral T cell pools are regulated by competition for environmental signals within a given ecological T cell niche. Cytokines and MHC molecules have been identified as resources for which naive T cells compete to proliferate homeostatically in lymphopenic hosts to fill up their respective compartments. However, it still remains unclear to what extent CD4 and CD8 T cells intercompete for these resources and which role dendritic cells (DC) play in this scenario. Using transgenic mice in which only DC express MHC class I, we demonstrate that this type of APC is sufficient to trigger complete homeostatic proliferation of CD8 T cells in vivo. However, normal numbers of endogenous naive CD4 T cells, but not CD25(+)CD4(+) T regulatory cells, efficiently suppress this expansion in vivo. These findings identify DC as a major resource and a possible target for homeostatic competition between naive CD4 and CD8 T cells.     

In vitro differentiation from naive to mature E-selectin binding CD4 T cells: acquisition of skin-homing properties occurs independently of cutaneous lymphocyte antigen expression

We previously showed that skin-homing CD4 T cells in peripheral blood can be subdivided into three populations on the basis of the expression pattern of the cutaneous lymphocyte Ag (CLA) and fucosyltransferase VII (FucT-VII): FucT-VII(+)CLA(-), FucT-VII(+)CLA(+), and FucT-VII(-)CLA(+). In view of the known late appearance of CLA during T cell differentiation, T cells programmed to attain skin-homing properties may start to generate E-selectin-binding epitopes at early stages of differentiation before induction of CLA expression. To this end, the in vitro differentiation from naive to CLA(+) memory T cells was followed after activation with anti-CD3 mAb. Here we demonstrate that naive skin-homing CD4 T cell precursors undergo a linear differentiation process from the FucT-VII(+)CLA(-) phenotype to the FucT-VII(+)CLA(+) phenotype and eventually to the FucT-VII(-)CLA(+) phenotype. The appearance of the FucT-VII(+)CLA(-) subset coincided with or could be immediately followed by the generation of E-selectin binding epitopes, and even after E-selectin-binding epitopes were no longer detectable, CLA remained expressed for prolonged periods of time, suggesting that induction of functional E-selectin ligands depends primarily on the expression of FucT-VII, but not CLA. Immunofluorescence and confocal microscopy studies of these T cells confirm that most E-selectin ligands were found independently of CLA expression.     

Leishmania antigens are presented to CD8+ T cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo

CD8+ T cells are generated in response to Leishmania major (Lm) or Toxoplasma gondii parasitic infections, indicating that exogenously delivered Ag can be processed for presentation by MHC class I molecules. We show that presentation of Lm nucleotidase (NT)-OVA is TAP independent in vivo and in vitro, and is inhibited by chloroquine, but not by proteasome inhibitors. In contrast, the presentation of T. gondii P30-OVA relies on the TAP/proteasome pathway. Presentation of OVA- or rNT-OVA-coated beads also bypassed TAP requirement above a certain Ag threshold. TAP was also dispensable for the presentation of wild-type Lm Ags to primed CD8+ T cells in vitro. Finally, in vivo priming of CD8+ T cells involved in acquired resistance to Lm was not compromised in TAP-deficient mice. Thus, Leishmania Ags appear to be confined to an intraphagosomal processing pathway that requires higher concentrations of Ags, suggesting that these parasites may have evolved strategies to impair the efficient endoplasmic reticulum-based, TAP-dependent cross-presentation pathway to avoid or delay CD8+ T cell priming.     

Prolonged antigen expression following DNA vaccination impairs effector CD8+ T cell function and memory development

After priming, naive T cells undergo a program of expansion, contraction, and memory formation. Numerous studies have indicated that only a brief period of antigenic stimulation is required to fully commit CD8+ T cells to this program. Nonetheless, the persistence of Ag may modulate the eventual fate of CD8+ T cells. Using DNA delivery, we showed previously that direct presentation primes high levels of effector CD8+ T cells as compared with cross-presentation. One explanation now revealed is that prolonged cross-presentation limits effector cell expansion and function. To analyze this, we used a drug-responsive system to regulate Ag expression after DNA injection. Reducing expression to a single burst expanded greater numbers of peptide-specific effector CD8+ T cells than sustained Ag. Consequences for memory development were assessed after boosting and showed that, although persistent Ag maintained higher numbers of tetramer-positive CD8+ T cells, these expanded less (approximately 4-fold) than those induced by transient Ag expression (approximately 35-fold). Transient expression at priming therefore led to a net higher secondary response. In terms of vaccine design, we propose that the most effective DNA-based CD8+ T cell vaccines will be those that deliver a short burst of Ag.     

A new subset of human naive CD8+ T cells defined by low expression of IL-7R alpha

Concomitant with an increased number of memory-type cells, the amount of naive T cells steadily declines with age. Although the regulatory mechanisms behind this conversion are not fully understood, the suggestion is that both alterations in thymic output and homeostatic signals mold the naive T cell pool. In this study, we identify a new subset of circulating CD27(high)CD45RA(high) CD8+ T cells characterized by low IL-7Ralpha message and protein expression. Analysis of TCR repertoire and TCR excision circle content together with ex vivo recovery of IL-7Ralpha expression indicated that these cells should be placed into the naive T cell pool. Compared with conventional IL-7Ralpha(high) naive T cells, this subset displayed significantly lower levels of CD28 and higher levels of HLA-DR. Proliferative responses to anti-CD3/CD28 mAbs were indistinguishable from conventional naive T cells, but the responsiveness to IL-7 was limited. Strikingly, IL-7Ralpha(low) naive T cells were particularly increased in circumstances of naive CD8+ T cells shortage, as in the elderly, in patients early after hemopoietic stem cell transplantation, and in HIV-infected individuals. As common gamma chain cytokines induce rapid down-regulation of IL-7Ralpha, we propose that this new subset of naive T cells may encompass cells that have recently received homeostatic signals.     

Human postnatal CD4- CD8- CD3- thymic T cell precursors differentiate in vitro into T cell receptor delta-bearing cells

The signals required for activation and the differentiation of human triple negative postnatal thymocytes were studied in vitro. Highly purified populations of CD4-, CD8-, CD3- (triple negative) thymocytes were isolated by combined panning and preparative cell sorting and the ability of triple negative thymocytes to proliferate in response to various cytokines determined. Maximal triple negative proliferation was obtained using a mitogenic combination of CD2 antibodies and either rIL-2 or the phorbol ester, PMA. Long term growth (2 to 6 wk) of postnatal triple negative thymocytes was best achieved using CD2 antibodies and rIL-2. After in vitro culture with CD2 antibodies and rIL-2, triple negative thymocytes gave rise to TCR-delta+ cells beginning on day 2 of culture (approximately 15% CD3/TCR-delta+) reaching maximum (approximately 60% CD3/TCR-delta+) on day 7 with stable number of TCR-delta+ cells observed in vitro for up to 6 wk. Analysis of 30 clones of human postnatal triple negative thymocytes demonstrated 9 of 30 (30%) were TCR-delta+, beta F1-, essentially ruling out overgrowth of the triple negative population over time by a minor pool of contaminating TCR-delta+ cells. Thus, these studies have defined an in vitro culture system for human postnatal T cell precursors and demonstrated that precursors of human TCR-gamma delta+ T cells reside in the triple negative thymocyte pool.     

Increased expression of the NK cell receptor KLRG1 by virus-specific CD8 T cells during persistent antigen stimulation

The killer cell lectin-like receptor G1 (KLRG1) is a natural killer cell receptor expressed by T cells that exhibit impaired proliferative capacity. Here, we determined the KLRG1 expression by virus-specific T cells. We found that repetitive and persistent antigen stimulation leads to an increase in KLRG1 expression of virus-specific CD8+ T cells in mice and that virus-specific CD8+ T cells are mostly KLRG1+ in chronic human viral infections (human immunodeficiency virus, cytomegalovirus, and Epstein-Barr virus) but not in resolved infection (influenza virus). Thus, by using KLRG1 as a T-cell marker, our results suggest that the differentiation status and function of virus-specific CD8+ T cells are directly influenced by persistent antigen stimulation.     

Human virus-specific CD8+ CTL clones revert from CD45ROhigh to CD45RAhigh in vivo: CD45RAhighCD8+ T cells comprise both naive and memory cells.

It has been generally believed that human CD8+ memory cells are principally found within the CD45ROhigh population. There are high frequencies of CD8+ memory CTL specific for the human CMV tegument phosphoprotein pp65 in PBMC of long-term virus carriers; the large population of memory CTL specific for a given pp65 peptide contains individual CTL clones that have greatly expanded. In this study, we found high frequencies of pp65 peptide-specific memory CTL precursors in the CD45ROhighCD45RA- population, but also appreciable frequencies in the CD45RAhigh subpopulation. Because the majority of CD8+ T cells in PBMC are CD45RAhigh, more of the total pp65-specific memory CTL pool is within the CD45RAhigh than in the CD45ROhigh compartment. Using clonotypic oligonucleotide probes to quantify the size of individual pp65-specific CTL clones in vivo, we found the CD45RAhigh population contributed 6- to 10-fold more than the CD45ROhigh population to the total virus-specific clone size in CD8+ cells. During primary CMV infection, an individual virus-specific CTL clone was initially CD45ROhigh, but after resolution of infection this clone was detected in both the CD45ROhigh and the CD45RAhigh populations. We conclude that CD45RA+ human CD8+ T cells do not solely comprise naive cells, but contain a very significant proportion of memory cells, which can revert from the CD45ROhigh to CD45RAhigh phenotype in vivo.     

Rapid G0/1 transition and cell cycle progression in CD8+ T cells compared to CD4+ T cells following in vitro stimulation

T‐cell population consists of two major subsets, CD4⁺ T cells and CD8⁺ T cells, which can be distinguished by the expression of CD4 or CD8 molecules, respectively. Although they play quite different roles in the immune system, many of their basic cellular processes such as proliferation following stimulation are presumably common. In this study, we have carefully analyzed time–course of G0/1 transition as well as cell cycle progression in the two subsets of quiescent T‐cell population following in vitro growth stimulation. We found that CD8⁺ T cells promote G0/1 transition more rapidly and drive their cell cycle progression faster compared to CD4⁺ T cells. In addition, expression of CD25 and effects of its blockade revealed that IL‐2 is implicated in the rapid progression, but not the earlier G0/1 transition, of CD8⁺ T cells.     

Dendritic cell immunization route determines integrin expression and lymphoid and nonlymphoid tissue distribution of CD8 T cells

Exogenous dendritic cells (bone marrow-derived dendritic cell (BMDC)) display restricted trafficking in vivo after injection into mice, but the route(s) by which they generate gut-homing effector cells is unclear. Mesenteric lymph nodes (LN) and spleen were differentially targeted by i.p. and i.v. administration of BMDC, respectively, whereas mediastinal LN were targeted by both routes. BMDC injected by either route activated CD8(+) T cells to up-regulate both alpha(4)beta(1) and alpha(4)beta(7) integrins. However, the lymphoid compartment in which activation occurred determined their expression kinetics, magnitude, and population distribution. Only T cells activated in mesenteric LN after i.p. immunization expressed high levels of alpha(4)beta(7), which also correlated with localization to small intestine. These alpha(4)beta(7)(high) cells also redistributed to mediastinal LN in a manner sensitive to treatment with alpha(4)beta(7) blocking Abs, but not to mucosal addressin cell adhesion molecule-1 blocking Abs. Our results demonstrate the importance of lymphoid compartment, as dictated by immunization route, in determining integrin expression on activated T cells and their distribution in lymphoid and nonlymphoid tissues.     

IL-7/STAT5 cytokine signaling pathway is essential but insufficient for maintenance of naive CD4 T cell survival in peripheral lymphoid organs

Constitutive expression of suppressors of cytokine signaling (SOCS)1 in T lineage in vivo attenuated cytokine signaling and resulted in a dramatic reduction in the number of naive CD44(low)CD62L(high) CD4 T cells in the spleen. After adoptive transfer of thymocytes from SOCS1 transgenic mice into normal recipients, naive CD4 T cells rapidly disappeared from the spleen within 1 wk. Likewise, T cell-specific deletion of STAT5a/b in vivo resulted in a similar phenotype characterized by loss of naive CD4 T cells. Thus, STAT5-mediated signaling is crucial for promoting naive T cell survival. However, forced expression of constitutively active STAT5 failed to rescue CD4 T cells in SOCS1 transgenic mice, implying that STAT5 activation is necessary but not sufficient for naive CD4 T cell survival. Although blockade of the IL-7R, a SOCS1 target, resulted in clear inhibition of naive T cell survival, the effect occurred 3 wk after anti-IL-7R Ab treatment, but not at earlier time points. These results suggest that IL-7-mediated STAT5 activation is essential for long-term survival of naive CD4 cells after export from thymus, and that another SOCS1-sensitive cytokine is critical for short-term naive T cell survival.     

Plasmacytoid dendritic cells take up opsonized antigen leading to CD4+ and CD8+ T cell activation in vivo

Plasmacytoid dendritic cells (pDC) are the body's main source of IFN-alpha, but, unlike classical myeloid DC (myDC), they lack phagocytic activity and are generally perceived as playing only a minor role in Ag processing and presentation. We show that murine pDC, as well as myDC, express Fcgamma receptors (CD16/CD32) and can use these receptors to acquire Ag from immune complexes (IC), resulting in the induction of robust Ag-specific CD4(+) and CD8(+) T cell responses. IC-loaded pDC stimulate CD4(+) T cells to proliferate and secrete a mixture of IL-4 and IFN-gamma, and they induce CD8(+) T cells to secrete IL-10 as well as IFN-gamma. In contrast, IC-loaded myDC induce both CD4(+) and CD8(+) T cells to secrete mainly IFN-gamma. These results indicate that pDC can shape an immune response by acquiring and processing opsonized Ag, leading to a predominantly Th2 response.