IL-15 trans-presentation by pulmonary dendritic cells promotes effector CD8 T cell survival during influenza virus infection

We have recently demonstrated that peripheral CD8 T cells require two separate activation hits to accumulate to high numbers in the lungs after influenza virus infection: a primary interaction with mature, antigen-bearing dendritic cells (DCs) in the lymph node, and a second, previously unrecognized interaction with MHC I–viral antigen–bearing pulmonary DCs in the lungs. We demonstrate that in the absence of lung-resident DC subsets, virus-specific CD8 T cells undergo significantly increased levels of apoptosis in the lungs; however, reconstitution with pulmonary plasmacytoid DCs and CD8α⁺ DCs promotes increased T cell survival and accumulation in the lungs. Further, our results show that the absence of DCs after influenza virus infection results in significantly reduced levels of IL-15 in the lungs and that pulmonary DC–mediated rescue of virus-specific CD8 T cell responses in the lungs requires trans-presentation of IL-15 via DC-expressed IL-15Rα. This study demonstrates a key, novel requirement for DC trans-presented IL-15 in promoting effector CD8 T cell survival in the respiratory tract after virus infection, and suggests that this trans-presentation could be an important target for the development of unique antiviral therapies and more effective vaccine strategies.Clearance of a primary influenza A virus (IAV) infection is known to require killing of virus-infected host cells by activated, antigen-specific CD8 T cells in the lungs (Topham et al., 1997). Until recently, antigen-specific CD8 T cells were thought to undergo programmed activation, whereby a single, brief interaction with a mature, antigen-bearing DC in the LN was sufficient to induce a full program of activation, division, and differentiation from naive to mature, cytotoxic CD8 T cells (Kaech and Ahmed, 2001; Wong and Pamer, 2001). Increasing evidence has suggested, however, that activation of antigen-specific CD8 T cells is not as simple as previously thought, and multiple factors, including cytokine signals such as IL-2 (Wong and Pamer, 2004), IFN-α (Marrack et al., 1999; Price et al., 2000; Kolumam et al., 2005), and IL-12 (Curtsinger et al., 2003a,b; Trinchieri, 2003), and late co-stimulatory signals such as CD70 (Dolfi and Katsikis, 2007) and 4-1BBL (Bertram et al., 2002; Lin et al., 2009), can regulate and fine tune the magnitude and duration of the effector response, as well as the nature of the ensuing memory T cell population.We have recently demonstrated in a model of IAV infection that the absence of specific pulmonary DC subsets, including plasmacytoid DC (pDCs) and CD8α⁺ DCs, from the lungs leads to a significant decrease in the number of virus-specific CD8 T cells (McGill et al., 2008). Reconstitution of the lungs with physiological numbers of pDCs or CD8α⁺ DCs is able to restore the pulmonary IAV-specific CD8 T cell response to near normal levels via a mechanism that is dependent on direct DC–T cell interactions, DC-expressed MHC I, and the presence of viral antigen. Interestingly, however, this rescue is DC subset specific, as reconstitution with purified alveolar and airway DCs (aDCs) or alveolar macrophages (aMϕs) was unable to rescue the virus-specific CD8 T cell response (McGill et al., 2008). After IAV infection there is an abundance of IAV antigen– and MHC I–expressing cells present in the lungs, including infected epithelial cells. Given this fact and the inability of all DC subsets to rescue the virus-specific CD8 T cell response, it suggested that there were additional, undefined requirements for pDC- and CD8α⁺ DC–mediated rescue of the T cell response in the lungs. Further, it remained unclear what mechanism was contributing to decreased numbers of IAV-specific CD8 T cells in the lungs of aDC-depleted mice: impaired DC migration from the lungs to the LN, impaired CD8 T cell proliferation within the lungs, or impaired CD8 T cell survival within the lungs. It was also unclear what mechanism pulmonary DC subsets were using to rescue this defect.The cytokine IL-15 has been demonstrated to play a key role in promoting lymphoid homeostasis, particularly with respect to CD8 T cells (Budagian et al., 2006; Kim et al., 2008). IL-15 was initially thought to signal similar to IL-2, whereby IL-15Rα formed a heterotrimeric complex with IL-2/IL15Rβ and common γ for high affinity signaling. Although this model appears to hold true in certain situations, recent reports have demonstrated a unique, alternative signaling mechanism, termed trans-presentation. In this model, IL-15Rα is required for the processing and presentation of active IL-15 in trans to cells expressing the IL-2/IL15Rβ–common γ chain complex (Sandau et al., 2004; Schluns et al., 2004; Kobayashi et al., 2005). At this time, it is unclear which cell types serve as the primary trans-presenting cells during an immune response; however, several lines of evidence have indicated that DCs may play an important role (Burkett et al., 2003, 2004). It is known that DCs express protein for both IL-15 and IL-15Rα, and that stimulation by IFN-αβ (Mattei et al., 2001) or IFN-γ (Doherty et al., 1996; Musso et al., 1999), or exposure to viral infection leads to further up-regulation of these molecules (Liu et al., 2000; Dubois et al., 2005; Budagian et al., 2006; Mattei et al., 2009). Interestingly, DCs matured in the presence of IL-15 have been demonstrated to promote enhanced antigen-specific CD8 T cell proliferation (Jinushi et al., 2003; Mattei et al., 2009) and a robust Th1 skewing in vivo (Pulendran et al., 2004).IL-15 has been best characterized for its role in maintaining memory CD8 T cell homeostasis, primarily through promoting enhanced basal proliferation (Becker et al., 2002; Goldrath et al., 2002; Schluns et al., 2002). More recently, however, there is accumulating evidence that IL-15 is also important for promoting primary effector CD8 T cell responses (Akbar et al., 1996; Bulfone-Paus et al., 1997; Vella et al., 1998; Schluns et al., 2002; Rausch et al., 2006; Yajima et al., 2006). Surface expression of both IL-15Rα and IL-2/IL15-Rβ is up-regulated after TCR activation (Vella et al., 1998), and IL-15 has been proposed to enhance activated CD8 T cell survival after challenge with staphylococcal enterotoxin A (Vella et al., 1998), Mycobacterium tuberculosis (Rausch et al., 2006), and vesicular stomatitis virus infection (Sandau et al., 2004). Collectively, these studies suggest a particularly important role for IL-15 in the generation and maintenance of an appropriate immune response; however, it remains unclear what role IL-15 plays during the effector phase of the immune response or in what context IL-15 contributes to activated CD8 T cell survival in vivo.In this study, we demonstrate a previously unrecognized role for pulmonary DC–mediated IL-15 trans-presentation in regulating virus-specific CD8 T cell responses in the lungs after IAV infection. The reduction in T cell numbers observed in the lungs of aDC-depleted mice after IAV challenge results not from impaired proliferation within the lungs but is caused by significantly increased levels of apoptosis of virus-specific CD8 T cells compared with nondepleted controls. Further, reconstitution with purified pDCs or CD8α⁺ DCs rescues the IAV-specific CD8 T cell response by promoting increased CD8 T cell survival in the lungs of aDC-depleted mice. Additionally, our results show that IAV infection induces up-regulation of both IL-15 mRNA and protein in the lungs and that depletion of aDCs at 48 h post infection (p.i.) results in a significant reduction in pulmonary IL-15 expression. Finally, this study demonstrates that pulmonary DCs prevent virus-specific CD8 T cell apoptosis through trans-presentation of IL-15, as blockade of IL-15 or IL-15Rα on the surface of pulmonary DCs before adoptive transfer, or transfer of IL-15^−/− pulmonary DC subsets ablates the rescue of the virus-specific CD8 T cell response in the lungs of aDC-depleted mice.     

Regulatory CD4+CD25+ T cells restrict memory CD8+ T cell responses

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.     

Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates

The adoptive transfer of antigen-specific T cells that have been expanded ex vivo is being actively pursued to treat infections and malignancy in humans. The T cell populations that are available for adoptive immunotherapy include both effector memory and central memory cells, and these differ in phenotype, function, and homing. The efficacy of adoptive immunotherapy requires that transferred T cells persist in vivo, but identifying T cells that can reproducibly survive in vivo after they have been numerically expanded by in vitro culture has proven difficult. Here we show that in macaques, antigen-specific CD8(+) T cell clones derived from central memory T cells, but not effector memory T cells, persisted long-term in vivo, reacquired phenotypic and functional properties of memory T cells, and occupied memory T cell niches. These results demonstrate that clonally derived CD8+ T cells isolated from central memory T cells are distinct from those derived from effector memory T cells and retain an intrinsic capacity that enables them to survive after adoptive transfer and revert to the memory cell pool. These results could have significant implications for the selection of T cells to expand or to engineer for adoptive immunotherapy of human infections or malignancy.     

Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4(+) CCR5(+) effector-memory T (T(EM)) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4(+) memory T cell proliferation appears to prevent collapse of effector site CD4(+) T(EM) cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4(+) T(EM) cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4(+) T(EM) cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4(+) T(EM) cells from central-memory T (T(CM)) cell precursors. The instability of effector site CD4(+) T(EM) cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5(-) CD4(+) T(CM) cells. These data suggest that although CD4(+) T(EM) cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4(+) T(CM) cells.     

mTORC1 and mTORC2 selectively regulate CD8+ T cell differentiation

Activation of mTOR-dependent pathways regulates the specification and differentiation of CD4⁺ T effector cell subsets. Herein, we show that mTOR complex 1 (mTORC1) and mTORC2 have distinct roles in the generation of CD8⁺ T cell effector and memory populations. Evaluation of mice with a T cell–specific deletion of the gene encoding the negative regulator of mTORC1, tuberous sclerosis complex 2 (TSC2), resulted in the generation of highly glycolytic and potent effector CD8⁺ T cells; however, due to constitutive mTORC1 activation, these cells retained a terminally differentiated effector phenotype and were incapable of transitioning into a memory state. In contrast, CD8⁺ T cells deficient in mTORC1 activity due to loss of RAS homolog enriched in brain (RHEB) failed to differentiate into effector cells but retained memory characteristics, such as surface marker expression, a lower metabolic rate, and increased longevity. However, these RHEB-deficient memory-like T cells failed to generate recall responses as the result of metabolic defects. While mTORC1 influenced CD8⁺ T cell effector responses, mTORC2 activity regulated CD8⁺ T cell memory. mTORC2 inhibition resulted in metabolic reprogramming, which enhanced the generation of CD8⁺ memory cells. Overall, these results define specific roles for mTORC1 and mTORC2 that link metabolism and CD8⁺ T cell effector and memory generation and suggest that these functions have the potential to be targeted for enhancing vaccine efficacy and antitumor immunity.     

CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses

Naturally occurring CD4+CD25+ regulatory T cells appear important to prevent activation of autoreactive T cells. This article demonstrates that the magnitude of a CD8+ T cell-mediated immune response to an acute viral infection is also subject to control by CD4+CD25+ T regulatory cells (Treg). Accordingly, if natural Treg were depleted with specific anti-CD25 antibody before infection with HSV, the resultant CD8+ T cell response to the immunodominant peptide SSIEFARL was significantly enhanced. This was shown by several in vitro measures of CD8+ T cell reactivity and by assays that directly determine CD8+ T cell function, such as proliferation and cytotoxicity in vivo. The enhanced responsiveness in CD25-depleted animals was between three- and fourfold with the effect evident both in the acute and memory phases of the immune response. Surprisingly, HSV infection resulted in enhanced Treg function with such cells able to suppress CD8+ T cell responses to both viral and unrelated antigens. Our results are discussed both in term of how viral infection might temporarily diminish immunity to other infectious agents and their application to vaccines. Thus, controlling suppressor effects at the time of vaccination could result in more effective immunity.     

Restoration of CD28 expression in CD28- CD8+ memory effector T cells reconstitutes antigen-induced IL-2 production

The control of many persistent viral infections by Ag-specific cytolytic CD8+ T cells requires a concurrent virus-specific CD4+ Th cell response. This reflects in part a requirement of activated effector CD8+ T cells for paracrine IL-2 production as a growth and survival factor. In human CMV and HIV infection, the majority of differentiated virus-specific CD8+ T cells notably lose the ability to produce IL-2 but also lose expression of CD28, a costimulatory molecule. Analysis of the fraction of memory CD8+ T cells that continue to express CD28 revealed these cells retain the ability to produce IL-2. Therefore, we examined if IL-2 production by CD28- CD8+ T cells could be restored by introduction of a constitutively expressed CD28 gene. Expression of CD28 in CD28- CD8+ CMV- and HIV-specific CD8+ T cells reconstituted the ability to produce IL-2, which could sustain an autocrine proliferative response after Ag recognition. These results suggest that the loss of CD28 expression during differentiation of memory/effector CD8+ T cells represents a decisive step in establishing regulation of responding CD8+ T cells, increasing the dependence on CD4+ Th for proliferation after target recognition, and has implications for the treatment of viral disease with adoptively transferred CD8+ T cells.     

Chemokine receptor CXCR3 facilitates CD8(+) T cell differentiation into short-lived effector cells leading to memory degeneration

Strength of inflammatory stimuli during the early expansion phase plays a crucial role in the effector versus memory cell fate decision of CD8(+) T cells. But it is not known how early lymphocyte distribution after infection has an impact on this process. We demonstrate that the chemokine receptor CXCR3 is involved in promoting CD8(+) T cell commitment to an effector fate rather than a memory fate by regulating T cell recruitment to an antigen/inflammation site. After systemic viral or bacterial infection, the contraction of CXCR3(-/-) antigen-specific CD8(+) T cells is significantly attenuated, resulting in massive accumulation of fully functional memory CD8(+) T cells. Early after infection, CXCR3(-/-) antigen-specific CD8(+) T cells fail to cluster at the marginal zone in the spleen where inflammatory cytokines such as IL-12 and IFN-α are abundant, thus receiving relatively weak inflammatory stimuli. Consequently, CXCR3(-/-) CD8(+) T cells exhibit transient expression of CD25 and preferentially differentiate into memory precursor effector cells as compared with wild-type CD8(+) T cells. This series of events has important implications for development of vaccination strategies to generate increased numbers of antigen-specific memory CD8(+) T cells via inhibition of CXCR3-mediated T cell migration to inflamed microenvironments.     

Hypoxia-inducible factor activity promotes antitumor effector function and tissue residency by CD8+ T cells

Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting CD8⁺ T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor-infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in CD8⁺ T cells induced HIF-1α/HIF-2α–dependent differentiation of tissue-resident memory–like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion-associated phenotype, which led to retained polyfunctionality and response to αPD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in CD8⁺ TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs.     

Functional and genomic profiling of effector CD8 T cell subsets with distinct memory fates

An important question in memory development is understanding the differences between effector CD8 T cells that die versus effector cells that survive and give rise to memory cells. In this study, we provide a comprehensive phenotypic, functional, and genomic profiling of terminal effectors and memory precursors. Using killer cell lectin-like receptor G1 as a marker to distinguish these effector subsets, we found that despite their diverse cell fates, both subsets possessed remarkably similar gene expression profiles and functioned as equally potent killer cells. However, only the memory precursors were capable of making interleukin (IL) 2, thus defining a novel effector cell that was cytotoxic, expressed granzyme B, and produced inflammatory cytokines in addition to IL-2. This effector population then differentiated into long-lived protective memory T cells capable of self-renewal and rapid recall responses. Experiments to understand the signals that regulate the generation of terminal effectors versus memory precursors showed that cells that continued to receive antigenic stimulation during the later stages of infection were more likely to become terminal effectors. Importantly, curtailing antigenic stimulation toward the tail end of the acute infection enhanced the generation of memory cells. These studies support the decreasing potential model of memory differentiation and show that the duration of antigenic stimulation is a critical regulator of memory formation.     

One naive T cell,multiple fates in CD8+ T cell differentiation

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR–pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8⁺ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets.Activation of naive antigen-specific T cells is characterized by a vigorous proliferative burst, resulting in the formation of a large pool of effector T cells. After pathogen clearance, ∼95% of activated T cells die, leaving behind a stable pool of long-lived memory cells (Williams and Bevan, 2007). Two fundamentally different mechanisms could give rise to the production of effector and memory T cells during an immune response. First, single naive T cells may be destined to produce either effector T cells or memory T cells, but not both (“one naive cell, one fate”). As an alternative, effector and memory T cells could derive from the same clonal precursors within the naive T cell pool (“one naive cell, multiple fates”). As the fate decisions that control T cell differentiation could either be taken during initial T cell priming (i.e., before the first cell division) or at later stages, at least four conceptually different models describing effector and memory T cell differentiation can be formulated (Fig. S1).A first model predicts a separate origin of effector and memory T cells as a result of differential T cell priming by APCs. In this scenario, fate decisions would be taken before the first cell division, and even though cells destined to become memory cells may transiently display traits associated with effector T cells (e.g., expression of granzyme B or IFN-γ; see the following paragraphs), their ability for long-term survival would be predetermined. In line with this model, several studies have provided evidence that the fate of CD8⁺ T cells may, to some extent, be programmed during initial activation (Kaech and Ahmed, 2001; van Stipdonk et al., 2003; Masopust et al., 2004; Williams and Bevan, 2007; Bannard et al., 2009).A second model, which relies on recent data from Chang et al. (2007), likewise suggests that the priming APC plays the crucial role in determining effector or memory T cell fate, but by a strikingly different mechanism and with an opposite prediction concerning the lineage relationship of effector and memory T cells. Specifically, analysis of T cell–APC conjugates has shown that the first division of activated T cells can be asymmetric, with the daughter T cell that is formed proximal to the APC being more likely to contribute to the effector T cell subset and the distal daughter T cell being more likely to generate memory T cells (Chang et al., 2007). Assuming that all primary daughter cells survive and yield further progeny, these data would predict that single naive T cells contribute to both the effector and the memory subset.In contrast to these two models that are based on a determining role of the priming APC, two other models predict that T cell fate is determined by the cumulative effect of signals that not only naive T cells but also their descendants receive. The first of these models, termed the “decreasing potential model,” argues that T cell progeny that receive additional stimulation after priming undergo terminal differentiation toward the effector subset, whereas descendants that do not encounter these signals may transiently display certain effector functions but will ultimately become memory T cells (Ahmed and Gray, 1996). In support of this model, it has been demonstrated that continued inflammatory signals (Badovinac et al., 2004; Joshi et al., 2007) and prolonged antigenic stimulation (Sarkar et al., 2008) can lead descendant CD8⁺ T cells to preferentially develop into effector cells.If the descendants of all individual naive T cells have an equal chance of receiving signals for terminal differentiation, the standard decreasing potential model predicts that memory and effector T cells will be derived from the same population of naive T cells. However, there is evidence that the environmental factors that promote either terminal differentiation or memory T cell development may alter over the course of infection (Sarkar et al., 2008). A fourth model therefore argues that the progeny of T cells that are activated early or late during infection will receive distinct signals and, hence, assume (partially) different fates (van Faassen et al., 2005; D’Souza and Hedrick, 2006; Quigley et al., 2007; Stemberger et al., 2007a).A large number of studies in which cell differentiation was analyzed at the population level have been informative in revealing which effector properties can be displayed by T cells that subsequently differentiate into memory T cells (for review see Jameson and Masopust, 2009). In particular, two recent studies using IFN-γ or granzyme B reporter mice have shown that memory T cells can arise from cells that have previously transcribed IFN-γ or granzyme B genes (Harrington et al., 2008; Bannard et al., 2009). However, it is important to realize that these studies reveal little with regard to the developmental potential of individual naive T cells. Specifically, the fact that T cells that have a particular effector capacity can become memory T cells does not indicate that all naive T cells yield such effector cells, nor does it indicate that all memory T cells have gone through an effector phase.To determine the developmental potential of naive T cells, it is essential to develop technologies in which T cell responses can be analyzed at the single naive T cell level. In early work that aimed to follow T cell responses at the clonal level, TCR repertoire analysis has been used to assess the kinship of T cell populations (Maryanski et al., 1996; Kedzierska et al., 2004). However, as several naive T cell clones can share the same TCR, it has been argued that such analyses do not necessarily monitor T cell fate at the single T cell level (Stemberger et al., 2007b; Obar et al., 2008). Recently, Stemberger et al. (2007a) have reported on a more elegant approach to address naive T cell potency. Using the transfer of single naive CD8⁺ T cells into mice, this study provides direct evidence that a single naive CD8⁺ T cell can form both effector and memory cell subsets. However, the statistical power of single-cell transfer studies obviously has limitations. In addition, if homeostatic proliferation would occur before antigen-driven proliferation in this system, this would limit the conclusions that can be drawn with regard to the pluripotency of a single naive T cell.In this study, we have developed a technology that allows the generation of naive T cells that carry unique genetic tags (barcodes), and we describe how this technology can be used for the large-scale assessment of the developmental potential of single naive T cells. Using physiological frequencies of barcode-labeled naive CD8⁺ T cells of different functional avidities, we demonstrate that in both systemic and local infection models, effector and memory CD8⁺ T cell subsets share the same precursors in the naive T cell pool. These data demonstrate that under all conditions analyzed, single naive T cells do not selectively yield effector or memory T cells. Rather, T cell differentiation into effector and memory T cell subsets occurs by a one naive cell, multiple fates principle.     

Immediate cytotoxicity but not degranulation distinguishes effector and memory subsets of CD8+ T cells

CD8+ T cells play a central role in the resolution and containment of viral infections. A key effector function of CD8+ T cells is their cytolytic activity toward infected cells. Here, we studied the regulation of cytolytic activity in naive, effector, and central versus effector memory CD8+ T cells specific for the same glycoprotein-derived epitope of lymphocytic choriomeningitis virus. Our results show that the kinetics of degranulation, assessed by a novel flow cytometric based assay, were identical in effector and both subsets of memory CD8+ T cells, but absent in naive CD8+ T cells. However, immediate cytolytic activity was most pronounced in effector T cells, low in effector memory T cells, and absent in central memory T cells, correlating with the respective levels of cytolytic effector molecules present in lytic granules. These results indicate that an inherent program of degranulation is a feature of antigen-experienced cells as opposed to naive CD8+ T cells and that the ability of CD8+ T cells to induce target cell apoptosis/death is dependent on granule protein content rather than on the act of degranulation itself. Furthermore, these results provide a potential mechanism by which central memory CD8+ T cell-mediated death of antigen-presenting cells within the lymph node is avoided.     

Differentiating between memory and effector CD8 T cells by altered expression of cell surface O-glycans

Currently there are few reliable cell surface markers that can clearly discriminate effector from memory T cells. To determine if there are changes in O-glycosylation between these two cell types, we analyzed virus-specific CD8 T cells at various time points after lymphocytic choriomeningitis virus infection of mice. Antigen-specific CD8 T cells were identified using major histocompatibility complex class I tetramers, and glycosylation changes were monitored with a monoclonal antibody (1B11) that recognizes O-glycans on mucin-type glycoproteins. We observed a striking upregulation of a specific cell surface O-glycan epitope on virus-specific CD8 T cells during the effector phase of the primary cytotoxic T lymphocyte (CTL) response. This upregulation showed a strong correlation with the acquisition of effector function and was downregulated on memory CD8 T cells. Upon reinfection, there was again increased expression of this specific O-glycan epitope on secondary CTL effectors, followed once more by decreased expression on memory cells. Thus, this study identifies a new cell surface marker to distinguish between effector and memory CD8 T cells. This marker can be used to isolate pure populations of effector CTLs and also to determine the proportion of memory CD8 T cells that are recruited into the secondary response upon reencounter with antigen. This latter information will be of value in optimizing immunization strategies for boosting CD8 T cell responses.