Divergent molecular networks program functionally distinct CD8+ skin-resident memory T cells.

Skin-resident CD8+ T cells include distinct interferon-γ-producing [tissue-resident memory T type 1 (TRM1)] and interleukin-17 (IL-17)-producing (TRM17) subsets that differentially contribute to immune responses. However, whether these populations use common mechanisms to establish tissue residence is unknown. In this work, we show that TRM1 and TRM17 cells navigate divergent trajectories to acquire tissue residency in the skin. TRM1 cells depend on a T-bet-Hobit-IL-15 axis, whereas TRM17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in TRM17 cells parallel to that induced by Hobit in TRM1 cells, with an ICOS-c-Maf-IL-7 axis pivotal to TRM17 cell commitment. Accordingly, by targeting this pathway, skin TRM17 cells can be ablated without compromising their TRM1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.

Development of a Novel CD4+ TCR Transgenic Line That Reveals a Dominant Role for CD8+ Dendritic Cells and CD40 Signaling in the Generation of Helper and CTL Responses to Blood-Stage Malaria.

We describe an MHC class II (I-Ab)-restricted TCR transgenic mouse line that produces CD4+ T cells specific for Plasmodium species. This line, termed PbT-II, was derived from a CD4+ T cell hybridoma generated to blood-stage Plasmodium berghei ANKA (PbA). PbT-II cells responded to all Plasmodium species and stages tested so far, including rodent (PbA, P. berghei NK65, Plasmodium chabaudi AS, and Plasmodium yoelii 17XNL) and human (Plasmodium falciparum) blood-stage parasites as well as irradiated PbA sporozoites. PbT-II cells can provide help for generation of Ab to P. chabaudi infection and can control this otherwise lethal infection in CD40L-deficient mice. PbT-II cells can also provide help for development of CD8+ T cell-mediated experimental cerebral malaria (ECM) during PbA infection. Using PbT-II CD4+ T cells and the previously described PbT-I CD8+ T cells, we determined the dendritic cell (DC) subsets responsible for immunity to PbA blood-stage infection. CD8+ DC (a subset of XCR1+ DC) were the major APC responsible for activation of both T cell subsets, although other DC also contributed to CD4+ T cell responses. Depletion of CD8+ DC at the beginning of infection prevented ECM development and impaired both Th1 and follicular Th cell responses; in contrast, late depletion did not affect ECM. This study describes a novel and versatile tool for examining CD4+ T cell immunity during malaria and provides evidence that CD4+ T cell help, acting via CD40L signaling, can promote immunity or pathology to blood-stage malaria largely through Ag presentation by CD8+ DC.

Sustained accumulation of antigen-presenting cells after infection promotes local T-cell immunity.

Antigen-presenting cells (APC), such as dendritic cells (DC) and macrophages, are critical for T-cell-mediated immunity. Although it is established that memory T cells accumulate and persist in peripheral tissues after the resolution of infection, whether this is also the case for APC remains unclear. Here, we report that CCR2-dependent cells infiltrate skin during acute infection with herpes simplex virus (HSV)-1 and subsequently give rise to localized populations of DCs and macrophages. These APC are found at elevated numbers at sites of resolved infection or inflammation compared with unaffected regions of skin. Importantly, this local accumulation of APC is sustained for prolonged periods of time and has important functional consequences, as it promotes interferon-γ responses by virus-specific CD4+ T cells upon localized challenge infection with HSV-1. Thus, our results highlight how infection history determines long-term changes in immune cell composition in skin and how different types of immune cells accumulate, persist and co-operate to provide optimal immunity at this critical barrier site.

Identification of a MHC I-restricted epitope of DsRed in C57BL/6 mice.

Fluorescent proteins can be used to visualize cells and their constituents by various imaging techniques. Adoptive transfer of T cells from C57Bl/6 (B6) mice that expressed DsRed.T3 under the actin promoter lead to frequent rejection of transferred cells. In short term in vivo cytotoxicity assays these mice showed detectable, but weak lysis of DsRed spleen cells but their responses could be boosted by re-challenge with DsRed spleen cells. To determine whether DsRed protein may contain an H-2(b) MHC I-restricted T cell epitope, B6 mice immune to DsRed spleen cells were examined for in vivo lysis of target cells coated with various DsRed-derived peptides selected by the SYFPEITHI epitope prediction program. This analysis identified one D(b)-restricted peptide sequence within DsRed (SSLQDGCFI) that acted as an epitope for B6 target lysis. Knowledge of this epitope could allow DsRed to be used as a model antigen in B6 mice and cautions against using this fluorochrome, as well as several others containing the immunogenic sequence, in adoptive transfer studies where rejection is not desirable.

Memory T cell subsets, migration patterns, and tissue residence.

Tissues such as the skin and mucosae are frequently exposed to microbial pathogens. Infectious agents must be quickly and efficiently controlled by our immune system, but the low frequency of naive T cells specific for any one pathogen means dependence on primary responses initiated in draining lymph nodes, often allowing time for serious infection to develop. These responses imprint effectors with the capacity to home to infected tissues; this process, combined with inflammatory signals, ensures the effective targeting of primary immunity. Upon vaccination or previous pathogen exposure, increased pathogen-specific T cell numbers together with altered migratory patterns of memory T cells can greatly improve immune efficacy, ensuring infections are prevented or at least remain subclinical. Until recently, memory T cell populations were considered to comprise central memory T cells (TCM), which are restricted to the secondary lymphoid tissues and blood, and effector memory T cells (TEM), which broadly migrate between peripheral tissues, the blood, and the spleen. Here we review evidence for these two memory populations, highlight a relatively new player, the tissue-resident memory T cell (TRM), and emphasize the potential differences between the migratory patterns of CD4(+) and CD8(+) T cells. This new understanding raises important considerations for vaccine design and for the measurement of immune parameters critical to the control of infectious disease, autoimmunity, and cancer.

Maintenance of T cell function in the face of chronic antigen stimulation and repeated reactivation for a latent virus infection.

Persisting infections are often associated with chronic T cell activation. For certain pathogens, this can lead to T cell exhaustion and survival of what is otherwise a cleared infection. In contrast, for herpesviruses, T cells never eliminate infection once it is established. Instead, effective immunity appears to maintain these pathogens in a state of latency. We used infection with HSV to examine whether effector-type T cells undergoing chronic stimulation retained functional and proliferative capacity during latency and subsequent reactivation. We found that latency-associated T cells exhibited a polyfunctional phenotype and could secrete a range of effector cytokines. These T cells were also capable of mounting a recall proliferative response on HSV reactivation and could do so repeatedly. Thus, for this latent infection, T cells subjected to chronic Ag stimulation and periodic reactivation retain the ability to respond to local virus challenge.

Blood-stage Plasmodium berghei infection generates a potent, specific CD8+ T-cell response despite residence largely in cells lacking MHC I processing machinery.

Murine cerebral malaria is a complex disease caused by Plasmodium berghei ANKA infection. Several cell types, including CD8(+) T cells, are essential effectors of disease. Although the use of transgenic parasites expressing model antigens has revealed the induction of cytotoxic T lymphocytes (CTL) specific for these model antigens, there is no direct evidence for a response to authentic blood-stage parasite antigens, nor any knowledge of its magnitude. Our studies show that there is a dramatic primary parasite-specific CTL response, akin to viral immunity, reaching approximately 30% of splenic CD8(+) T cells, with many producing interferon-γ and tumor necrosis factor-α. These cells express granzyme B and other markers of specific responders, are cytolytic, and respond to a broad array of major histocompatibility complex (MHC) I-restricted epitopes, 5 of which are identified here. Our studies indicate that vigorous CTL responses can be induced to pathogens even when they largely reside in red blood cells, which lack MHC I processing machinery.

Aire regulates the transfer of antigen from mTECs to dendritic cells for induction of thymic tolerance.

To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter. Aire deficiency reduced the number of mature single-positive OVA-specific CD4(+) or CD8(+) T cells in the thymus, independent of OVA expression. Importantly, it also contributed in 2 ways to OVA-dependent negative selection depending on the T-cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to medullary thymic epithelial cell (mTEC)-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTECs can mediate tolerance by direct presentation of Aire-regulated antigens to both CD4 and CD8 T cells.

Fixing an irrelevant TCR alpha chain reveals the importance of TCR beta diversity for optimal TCR alpha beta pairing and function of virus-specific CD8+ T cells.

TCR repertoire diversity can influence the efficacy of CD8(+) T-cell populations, with greater breadth eliciting better protection. We analyzed TCR beta diversity and functional capacity for influenza-specific CD8(+) T cells expressing a single TCR alpha chain. Mice (A7) transgenic for the H2K(b)OVA(257-264)-specific V alpha 2.7 TCR were challenged with influenza to determine how fixing this "irrelevant" TCR alpha affects the "public" and restricted D(b)NP(366) (+)CD8(+) versus the "private" and diverse D(b)PA(224) (+)CD8(+) responses. Though both D(b)NP(366) (+)CD8(+) and D(b)PA(224) (+)CD8(+) sets are generated in virus-primed A7 mice, the constrained D(b)NP(366) (+)CD8(+) population lacked the characteristic, public TCRV beta 8.3, and consequently was reduced in magnitude and pMHC-I avidity. For the more diverse D(b)PA(224) (+)CD8(+) T cells, this particular forcing led to a narrowing and higher TCR beta conservation of the dominant V beta 7, though the responses were of comparable magnitude to C57BL/6J controls. Interestingly, although both the TCR beta diversity and the cytokine profiles were reduced for the D(b)NP(366) (+)CD8(+) and D(b)PA(224) (+)CD8(+) sets in spleen, the latter measure of polyfunctionality was comparable for T cells recovered from the infected lungs of A7 and control mice. Even "sub-optimal" TCR alpha beta pairs can operate effectively when exposed in a milieu of high virus load. Thus, TCR beta diversity is important for optimal TCR alpha beta pairing and function when TCR alpha is limiting.

Cutting edge: priming of CD8 T cell immunity to herpes simplex virus type 1 requires cognate TLR3 expression in vivo.

Despite its potential for involvement in viral immunity, little evidence links TLR3 to adaptive antiviral responses. Here we show that TLR3 is required for the generation of CD8 T cell immunity to HSV-1. The magnitude of the gB-specific CD8 T cell response after flank infection by HSV-1 was significantly reduced in mice lacking TIR domain-containing adaptor-inducing IFN-beta or TLR3, but not MyD88. Impaired CTL induction was evident in chimeric mice lacking TLR3 in bone marrow (BM)-derived cells. Among the dendritic cell subsets, TLR3 was expressed by CD8alpha(+) dendritic cells, known to be involved in priming HSV-1-specific CD8 T cells. Use of mixed BM chimeras revealed that TLR3 and the MHC class I-restriction element must be expressed by the same BM-derived cell for effective priming. These data imply that a cognate linkage between TLR3 and MHC class I is required for efficient CTL priming to HSV-1.

Tissue destruction caused by cytotoxic T lymphocytes induces deletional tolerance.

Autoimmune diseases tend to be chronic and progressive, but how these responses are sustained is not clear. One cell type that might contribute to autoimmunity is the cytotoxic T lymphocyte (CTL), which, as a consequence of causing tissue destruction and production of cytokines, could provide a sustained supply of antigen and inflammatory signals for dendritic cells to maintain immune stimulation. Here we examined whether such CTL-mediated tissue damage alone could provide antigen in the right context to recruit immune effectors and sustain autoimmunity. We show that while CTL-mediated tissue damage caused the release of self-antigens that stimulated the proliferation of naive autoreactive CD8(+) T cells, such responses failed to precipitate disease and, instead, led to deletional tolerance. These findings indicate that despite the capacity of CTLs to produce inflammatory cytokines and to cause tissue damage, their responses are not sustaining, but instead favor induction of self-tolerance.

Blood-stage Plasmodium infection induces CD8+ T lymphocytes to parasite-expressed antigens, largely regulated by CD8alpha+ dendritic cells.

Although CD8(+) T cells do not contribute to protection against the blood stage of Plasmodium infection, there is mounting evidence that they are principal mediators of murine experimental cerebral malaria (ECM). At present, there is no direct evidence that the CD8(+) T cells mediating ECM are parasite-specific or, for that matter, whether parasite-specific CD8(+) T cells are generated in response to blood-stage infection. To resolve this and to define the cellular requirements for such priming, we generated transgenic P. berghei parasites expressing model T cell epitopes. This approach was necessary as MHC class I-restricted antigens to blood-stage infection have not been defined. Here, we show that blood-stage infection leads to parasite-specific CD8(+) and CD4(+) T cell responses. Furthermore, we show that P. berghei-expressed antigens are cross-presented by the CD8alpha(+) subset of dendritic cells (DC), and that this induces pathogen-specific cytotoxic T lymphocytes (CTL) capable of lysing cells presenting antigens expressed by blood-stage parasites. Finally, using three different experimental approaches, we provide evidence that CTL specific for parasite-expressed antigens contribute to ECM.

Dendritic cell-induced memory T cell activation in nonlymphoid tissues.

Secondary lymphoid organs are dominant sites of T cell activation, although many T cells are subsequently retained within peripheral tissues. Currently, these nonlymphoid compartments are viewed as sites only of effector T cell function, without the involvement of renewed induction of immunity via the interactions with professional antigen-presenting cells. We describe a method of reactivation of herpes simplex virus to examine the stimulation of tissue-resident T cells during secondary challenge. The results revealed that memory CD8+ T cell responses can be initiated within peripheral tissues through a tripartite interaction that includes CD4+ T cells and recruited dendritic cells. These findings lend evidence for the existence of a sophisticated T cell response mechanism in extra-lymphoid tissues that can act to control localized infection.

Cutting edge: Tissue-resident memory CTL down-regulate cytolytic molecule expression following virus clearance.

CTL express lytic proteins that mediate the cytolysis of virus-infected cells. In this study, cytolytic transcriptional profiles were determined for individual CTL responding to influenza A virus and HSV-1. During acute infection, influenza-specific CTL in the spleen and respiratory airways displayed highly activated cytolytic profiles, as did HSV-1-specific CTL localized in the spleen, skin, and dorsal root ganglia (DRG). In contrast, memory CTL dramatically down-regulated cytolytic molecule transcription. This occurred for both lymphoid (spleen) and tissue-resident (skin and/or lung) memory CTL. In contrast, HSV-1-specific CTL localized in the dorsal root ganglia in the presence latent HSV-1 Ag did not down-regulate cytolytic molecule transcription. Therefore, both lymphoid and tissue-resident memory CTL down-regulate cytolytic molecule transcription following virus clearance unless localized Ag is present.

Minimal activation of memory CD8+ T cell by tissue-derived dendritic cells favors the stimulation of naive CD8+ T cells.

Of the many dendritic cell (DC) subsets, DCs expressing the monomorphic coreceptor CD8 alpha-chain (CD8alpha) are localized permanently in lymphoid organs, whereas 'tissue-derived DCs' remain in nonlymphoid tissues until they 'capture' antigen and then move to local lymph nodes. Here we show that after lung infection, both naive and memory CD8+ 'killer' T cells responded to influenza virus antigens presented by lymph node-resident CD8alpha+ DCs, but only naive cells responded to antigens presented by lung-derived DCs. This difference provides a mechanism for priming naive T cell responses in conditions in which robust memory predominates. Our findings have implications for immunity to pathogens that can mutate their T cell epitopes, such as influenza virus and human immunodeficiency virus, and challenge the long-held view that memory T cells have less-stringent requirements for activation than naive T cells have.

CTL response compensation for the loss of an immunodominant class I-restricted HSV-1 determinant.

The T-cell response to even complex pathogens is often focused on only a handful of immunodominant determinants. Such narrow responses provoke a selective pressure that can drive the emergence of CTL escape variants, raising the question of whether a broader response, targeting multiple non-dominant peptides may be more beneficial. To examine the ability of the T-cell repertoire to respond to non-dominant determinants, we have investigated how mutating the dominant peptide in HSV affects the magnitude of the CD8+ T-cell response. We found that the CTL response to HSV lacking the dominant peptide was only modestly reduced compared with the wild-type virus and, surprisingly, this compensation occurred without any enhancement in the response to an established minor epitope. These findings are supportive of a malleable T-cell repertoire that can elicit strong responses to alternate, unknown determinants in the absence of the dominant response.

Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming.

Skin dendritic cells (DCs) are thought to act as key initiators of local T cell immunity. Here we show that after skin infection with herpes simplex virus (HSV), cytotoxic T lymphocyte (CTL) activation required MHC class I-restricted presentation by nonmigratory CD8(+) DCs rather than skin-derived DCs. Despite a lack of direct presentation by migratory DCs, blocking their egress from infected skin substantially inhibited class I-restricted presentation and HSV-specific CTL responses. These results support the argument for initial transport of antigen by migrating DCs, followed by its transfer to the lymphoid-resident DCs for presentation and CTL priming. Given that relatively robust CTL responses were seen with small numbers of skin-emigrant DCs, we propose that this inter-DC antigen transfer functions to amplify presentation across a larger network of lymphoid-resident DCs for efficient T cell activation.

CD4+ T cells can protect APC from CTL-mediated elimination.

Professional APC play a central role in generating antiviral CD8(+) CTL immunity. However, the fate of such APC following interaction with these same CTL remains poorly understood. We have shown previously that prolonged Ag presentation persists in the presence of a strong CTL response following HSV infection. In this study, we examined the mechanism of survival of APC in vivo when presenting an immunodominant determinant from HSV. We show that transferred peptide-labeled dendritic cells were eliminated from draining lymph nodes in the presence of HSV-specific CTL. Maturation of dendritic cells with LPS or anti-CD40 before injection protected against CTL lysis in vivo. Furthermore, endogenous APC could be eliminated from draining lymph nodes early after HSV infection by adoptive transfer of HSV-specific CTL, yet the cotransfer of significant virus-specific CD4(+) T cell help promoted prolonged Ag presentation. This suggests that Th cells may assist in prolonging class I-restricted Ag presentation, potentially enhancing CTL recruitment and allowing more efficient T cell priming.