Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells requires presentation of exogenous antigen

Cytotoxic T lymphocytes (CTLs) are thought to detect viral infections by monitoring the surface of all cells for the presence of viral peptides bound to major histocompatibility complex (MHC) class I molecules. In most cells, peptides presented by MHC class I molecules are derived exclusively from proteins synthesized by the antigen-bearing cells. Macrophages and dendritic cells also have an alternative MHC class I pathway that can present peptides derived from extracellular antigens; however, the physiological role of this process is unclear. Here we show that virally infected non-haematopoietic cells are unable to stimulate primary CTL-mediated immunity directly. Instead, bone-marrow-derived cells are required as antigen-presenting cells (APCs) to initiate anti-viral CTL responses. In these APCs, the alternative (exogenous) MHC class I pathway is the obligatory mechanism for the initiation of CTL responses to viruses that infect only non-haematopoietic cells.     

Induction of cytotoxic T-cell responses in vivo in the absence of CD4 helper cells

Cytotoxic T lymphocytes (CTL) seem to provide the major line of defence against many viruses. CTL effector functions are mediated primarily by cells carrying the CD8 (Ly-2) antigen (CD8+ cells) and are triggered by interactions of the T-cell receptor with an antigenic complex, often termed 'self plus X', composed of viral determinants in association with class I molecules of the major histocompatibility complex (MHC). The mechanism(s) of induction of virus-specific CTL in vivo is poorly understood, but data from in vitro experiments suggest that their generation is strictly dependent on functions provided by CD4+ helper T cells (also referred to as L3T4+; or TH) that respond to antigens in the context of class II (Ia) MHC determinants. The prevailing opinion that induction of most functions of CD8+ cells requires help provided by CD4+ cells has recently been challenged by the observation that CD8+ cells alone can mediate a variety of responses to alloantigens in vitro and in vivo; however, the possibility that CTL to self plus X could be generated in vivo in the absence of TH cells has not been evaluated. We report here that C57BL/6J (B6) and AKR/J mice, when functionally depleted of CD4+ cells by in vivo treatment with the CD4+-specific rat monoclonal antibody GK1.5 (refs 8-14) responded to ectromelia virus infection by developing an optimal in vivo virus-specific CTL response, and subsequently recovered from the disease (mousepox) that was lethal for similarly infected nude mice (CD4-, CD8-).     

Clonal deletion of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen-presenting cells

One mechanism ensuring self tolerance of T cells is the clonal deletion of thymocytes bearing alpha beta T-cell receptors. The stage of thymocyte development at which the interaction with antigen-presenting cells (APCs) leads to deletion, however, has not been determined directly. Indirect evidence suggests that intrathymic APCs induce deletion of CD4+8+ thymocytes (which die by apoptosis) but deletion at less and more mature developmental stages has also been implied. It is also not clear if clonal elimination of thymocytes can be triggered by peripheral antigens carried on extrathymic APCs migrating through the thymus. Here we show antigen-specific induction of apoptosis in CD4+8+ thymocytes cultured in suspension, by thymic as well as splenic APCs. Thus the recognition of antigen by CD4+8+ thymocytes may lead to deletion, suggesting that this is the central mechanism of tolerance induction, which is not limited by the antigen-presenting ability of the thymic stroma.     

Human gamma delta+ T cells respond to mycobacterial heat-shock protein

Most T cells recognize antigen through the T-cell antigen receptor (TCR)alpha beta-CD3 complex on the T-cell surface. A small percentage of T cells, however, do not express alpha beta but a second type of TCR complex designated gamma delta (ref. 2). Unlike alpha beta+ lymphocytes, gamma delta+ lymphocytes do not generally express CD4 or CD8 molecules, and the nature of antigen recognition by these cells is unknown. To study antigen recognition by gamma delta+ lymphocytes we raised a gamma delta+ alpha beta- -CD4-CD8- line from an individual immune to PPD (purified protein derivative). This line showed a specific proliferative response to PPD and to a recombinant mycobacterial heat-shock protein (HSP) of relative molecular mass 65,000 (65K). The gamma delta+ line was shown to exhibit a major response to HSP in the presence of autologous antigen-presenting cells (APCs). Minor responses occurred, however, with APCs matched for some HLA class I or II antigens, whereas no response occurred with HLA-mismatched APCs. These findings, therefore, document the requirement of HSP-reactive gamma delta+ lymphocytes for histocompatible APCs.     

Cross-dressed dendritic cells drive memory CD8+ T-cell activation after viral infection

After an infection, cytotoxic T lymphocyte precursors proliferate and become effector cells by recognizing foreign peptides in the groove of major histocompatibility complex (MHC) class I molecules expressed by antigen-presenting cells (APCs). Professional APCs specialized for T-cell activation acquire viral antigen either by becoming infected themselves (direct presentation) or by phagocytosis of infected cells, followed by transfer of antigen to the cytosol, processing and MHC class I loading in a process referred to as cross-presentation. An alternative way, referred to as 'cross-dressing', by which an uninfected APC could present antigen was postulated to be by the transfer of preformed peptide-MHC complexes from the surface of an infected cell to the APC without the need of further processing. Here we show that this mechanism exists and boosts the antiviral response of mouse memory CD8(+) T cells. A number of publications have demonstrated sharing of peptide-loaded MHC molecules in vitro. Our in vitro experiments demonstrate that cross-dressing APCs do not acquire peptide-MHC complexes in the form of exosomes released by donor cells. Rather, the APCs and donor cells have to contact each other for the transfer to occur. After a viral infection, we could isolate cross-dressed APCs able to present viral antigen in vitro. Furthermore, using the diphtheria toxin system to selectively eliminate APCs that could only acquire viral peptide-MHC complexes by cross-dressing, we show that such presentation can promote the expansion of resting memory T cells. Notably, naive T cells were excluded from taking part in the response. Cross-dressing is a mechanism of antigen presentation used by dendritic cells that may have a significant role in activating previously primed CD8(+) T cells.     

Apolipoprotein-mediated pathways of lipid antigen presentation

Peptide antigens are presented to T cells by major histocompatibility complex (MHC) molecules, with endogenous peptides presented by MHC class I and exogenous peptides presented by MHC class II. In contrast to the MHC system, CD1 molecules bind lipid antigens that are presented at the antigen-presenting cell (APC) surface to lipid antigen-reactive T cells. Because CD1 molecules survey endocytic compartments, it is self-evident that they encounter antigens from extracellular sources. However, the mechanisms of exogenous lipid antigen delivery to CD1-antigen-loading compartments are not known. Serum apolipoproteins are mediators of extracellular lipid transport for metabolic needs. Here we define the pathways mediating markedly efficient exogenous lipid antigen delivery by apolipoproteins to achieve T-cell activation. Apolipoprotein E binds lipid antigens and delivers them by receptor-mediated uptake into endosomal compartments containing CD1 in APCs. Apolipoprotein E mediates the presentation of serum-borne lipid antigens and can be secreted by APCs as a mechanism to survey the local environment to capture antigens or to transfer microbial lipids from infected cells to bystander APCs. Thus, the immune system has co-opted a component of lipid metabolism to develop immunological responses to lipid antigens.     

CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes

A long-standing paradox in cellular immunology concerns the conditional requirement for CD4+ T-helper (T(H)) cells in the priming of cytotoxic CD8+ T lymphocyte (CTL) responses in vivo. Whereas CTL responses against certain viruses can be primed in the absence of CD4+ T cells, others, such as those mediated through 'cross-priming' by host antigen-presenting cells, are dependent on T(H) cells. A clearer understanding of the contribution of T(H) cells to CTL development has been hampered by the fact that most T(H)-independent responses have been demonstrated ex vivo as primary cytotoxic effectors, whereas T(H)-dependent responses generally require secondary in vitro re-stimulation for their detection. Here, we have monitored the primary and secondary responses of T(H)-dependent and T(H)-independent CTLs and find in both cases that CD4+ T cells are dispensable for primary expansion of CD8+ T cells and their differentiation into cytotoxic effectors. However, secondary CTL expansion (that is, a secondary response upon re-encounter with antigen) is wholly dependent on the presence of T(H) cells during, but not after, priming. Our results demonstrate that T-cell help is 'programmed' into CD8+ T cells during priming, conferring on these cells a hallmark of immune response memory: the capacity for functional expansion on re-encounter with antigen.     

Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction

CD8+ T cells have a crucial role in resistance to pathogens and can kill malignant cells; however, some critical functions of these lymphocytes depend on helper activity provided by a distinct population of CD4+ T cells. Cooperation between these lymphocyte subsets involves recognition of antigens co-presented by the same dendritic cell, but the frequencies of such antigen-bearing cells early in an infection and of the relevant naive T cells are both low. This suggests that an active mechanism facilitates the necessary cell-cell associations. Here we demonstrate that after immunization but before antigen recognition, naive CD8+ T cells in immunogen-draining lymph nodes upregulate the chemokine receptor CCR5, permitting these cells to be attracted to sites of antigen-specific dendritic cell-CD4+ T cell interaction where the cognate chemokines CCL3 and CCL4 (also known as MIP-1alpha and MIP-1beta) are produced. Interference with this actively guided recruitment markedly reduces the ability of CD4+ T cells to promote memory CD8+ T-cell generation, indicating that an orchestrated series of differentiation events drives nonrandom cell-cell interactions within lymph nodes, optimizing CD8+ T-cell immune responses involving the few antigen-specific precursors present in the naive repertoire.     

Toll-dependent selection of microbial antigens for presentation by dendritic cells

Dendritic cells constitutively sample the tissue microenvironment and phagocytose both microbial and host apoptotic cells. This leads to the induction of immunity against invading pathogens or tolerance to peripheral self antigens, respectively. The outcome of antigen presentation by dendritic cells depends on their activation status, such that Toll-like receptor (TLR)-induced dendritic cell activation makes them immunogenic, whereas steady-state presentation of self antigens leads to tolerance. TLR-inducible expression of co-stimulatory signals is one of the mechanisms of self/non-self discrimination. However, it is unclear whether or how the inducible expression of co-stimulatory signals would distinguish between self antigens and microbial antigens when both are encountered by dendritic cells during infection. Here we describe a new mechanism of antigen selection in dendritic cells for presentation by major histocompatibility complex class II molecules (MHC II) that is based on the origin of the antigen. We show that the efficiency of presenting antigens from phagocytosed cargo is dependent on the presence of TLR ligands within the cargo. Furthermore, we show that the generation of peptide-MHC class II complexes is controlled by TLRs in a strictly phagosome-autonomous manner.     

Viral infection switches non-plasmacytoid dendritic cells into high interferon producers

Type I interferons (IFN-I) are important cytokines linking innate and adaptive immunity. Plasmacytoid dendritic cells make high levels of IFN-I in response to viral infection and are thought to be the major source of the cytokines in vivo. Here, we show that conventional non-plasmacytoid dendritic cells taken from mice infected with a dendritic-cell-tropic strain of lymphocytic choriomeningitis virus make similarly high levels of IFN-I on subsequent culture. Similarly, non-plasmacytoid dendritic cells secrete high levels of IFN-I in response to double-stranded RNA (dsRNA), a major viral signature, when the latter is introduced into the cytoplasm to mimic direct viral infection. This response is partially dependent on the cytosolic dsRNA-binding enzyme protein kinase R and does not require signalling through toll-like receptor (TLR) 3, a surface receptor for dsRNA. Furthermore, we show that sequestration of dsRNA by viral NS1 (refs 6, 7) explains the inability of conventional dendritic cells to produce IFN-I on infection with influenza. Our results suggest that multiple dendritic cell types, not just plasmacytoid cells, can act as specialized interferon-producing cells in certain viral infections, and reveal the existence of a TLR-independent pathway for dendritic cell activation that can be the target of viral interference.     

Cytotoxic T lymphocytes against a soluble protein

Thymus-derived (T) lymphocytes recognize antigen in conjunction with surface glycoproteins encoded by major histocompatibility complex (MHC) genes. Whereas fragments of soluble antigens are presented to T helper lymphocytes (TH), which carry the CD4 antigen, in association with class II MHC molecules, CD8-bearing cytotoxic T lymphocytes (CTL) usually see cellular antigens (for instance virally-encoded proteins) in conjunction with MHC class I molecules. The different modes of antigen presentation may result from separate intracellular transport: vesicles containing class II molecules are thought to fuse with those carrying endocytosed soluble proteins. Class I molecules, in contrast, can only pick up degradation products of intracellular proteins (see refs 7 and 8). This makes biological sense; during an attack of a virus, class I-restricted CTL destroy infected cells and class II-restricted TH guide the humoural response to neutralize virus particles and toxins. But here we provide evidence that CTL specific for ovalbumin fragments can be induced with soluble protein, and that intracellular protein degradation provides epitopes recognized by these CTL. These findings suggest the existence of an antigen presenting cell that takes up soluble material and induces CTL.     

Phenotypic differences between alpha beta versus beta T-cell receptor transgenic mice undergoing negative selection

T-cell differentiation in the thymus is thought to involve a progression from the CD4-CD8- phenotype through CD4+CD8+ intermediates to mature CD4+ or CD8+ cells. There is evidence that during this process T cells bearing receptors potentially reactive to 'self' are deleted by a process termed 'negative selection' One example of this process occurs in mice carrying polymorphic Mls antigens, against which a detectable proportion of T cells are autoreactive. These mice show clonal deletion of thymic and peripheral T-cell subsets that express the autoreactive V beta 3 segment of the T-cell antigen receptor, but at most a two-fold depletion of thymic cells at the CD4+CD8+ stage. By contrast, transgenic mice bearing both alpha and beta chain genes encoding autoreactive receptors recognizing other ligands, show severe depletion of CD4+CD8+ thymocytes as well, suggesting that negative selection occurs much earlier. We report here the Mls 2a/3a mediated elimination of T cells expressing a transgene encoded V beta 3-segment, in T-cell receptor alpha/beta and beta-transgenic mice. Severe depletion of CD4+CD8+ thymocytes is seen only in the alpha/beta chain transgenic mice, whereas both strains delete mature V beta 3 bearing CD4+ and CD8+ T cells efficiently. We conclude that severe CD4+CD8+ thymocyte deletion in alpha/beta transgenic mice results from the premature expression of both receptor chains, and does not reflect a difference in the timing or mechanism of negative selection for Mls antigens as against the allo- and MHC class 1-restricted antigens used in the other studies.     

Cytotoxic T cells specific for the circumsporozoite protein of Plasmodium falciparum

Malaria is initiated by the inoculation of a susceptible host with sporozoites from an infected mosquito. The sporozoites enter hepatocytes and develop for a period as exoerythrocyte or hepatic stage parasites. Vaccination with irradiated sporozoites can provide protective immunity and a recent study shows that this can also be conferred by immunization with a recombinant salmonella expressing only the circumsporozoite protein that normally covers the sporozoites. Protection against infection is likely to be mediated by cytotoxic CD8+ cells, as depletion of CD8+ T cells in a sporozoite-immunized animal can completely abrogate immunity. Here we demonstrate directly the existence of CD8+ cytotoxic T lymphocytes (CTL) that recognize the circumsporozoite protein. B10.BR mice immunized with sporozoites or with recombinant vaccinia virus expressing the CS protein of Plasmodium falciparum contain CTL that specifically kill L cell fibroblasts transfected with the gene encoding the same CS protein. The peptide epitope from the CS protein that is recognized by CTL from this strain of mice is from a variant region of the protein.     

Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes

Human cluster-of-differentiation 1 (CD1) is a family of cell surface glycoproteins of unknown function expressed on immature thymocytes, epidermal Langerhans cells and a subset of B lymphocytes. Three homologous proteins, CD1a, b and c, have been defined serologically, and the CD1 gene locus on human chromosome 1 contains five potential CD1 genes. Analysis of the predicted amino-acid sequences of CD1 molecules reveals a low but significant level of homology to major histocompatibility complex (MHC) class I and class II molecules, and, like MHC class I molecules, CD1 molecules are associated non-covalently with beta 2-microglobulin. These structural similarities to known antigen-presenting molecules, together with the expression of CD1 on cells capable of antigen presentation, suggest a role for CD1 molecules in antigen recognition by T cells. Here we demonstrate the specific recognition of CD1a by a CD4-CD8- alpha beta T-cell receptor (TCR) expressing cytolytic T lymphocyte (CTL) line and the specific recognition of CD1c by a CD4-CD8- gamma delta TCR CTL line. The interaction of CD1-specific CTLs with CD1+ target cells appeared to involve the CD3-TCR complex, and did not show evidence of MHC restriction. These results suggest that for a subset of T cells, CD1 molecules serve a function analogous to that of MHC class I and II molecules.     

Can B cells turn on virgin T cells?

The first event in the initiation of an immune response is the capture and presentation of antigen to T cells. Such presentation involves two distinct steps: (1) display of the antigen, which requires uptake, processing and re-expression of the antigen in association with MHC molecules on the presenting cell surface; and (2) triggering, in which the presenting cell provides signals leading to the activation of the responding T cell. Two sorts of cells can capture antigens, the 'professional' antigen-presenting cells (APCs) such as dendritic cells and macrophages, and the B cells. Both types of cells can display antigens and the APCs are known to be able to trigger resting T cells. But despite in vitro evidence that certain B-cell types can reactivate previously-activated T cells, it is not yet clear whether a B cell can initiate an immune response by providing the signals necessary to activate a resting T cell. We reasoned that resting B cells should not have this capacity because of the problems this would present with tolerance to self idiotypes. By exploiting the unique properties of the avian haematopoietic system, we have examined the presenting capacity of B cells in vivo and found that resting B cells are indeed unable to activate resting T cells.     

GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells.

Dendritic cells comprise a system of highly efficient antigen-presenting cells which initiate immune responses such as the sensitization of T cells restricted by major histocompatibility complex molecules, the rejection of organ transplants and the formation of T-cell-dependent antibodies. Dendritic cells are found in many non-lymphoid tissues, such as skin (Langerhans cells) and mucosa, and they migrate after antigen capture through the afferent lymph or the bloodstream to lymphoid organs, where they efficiently present antigen to T cells. Dendritic cells are difficult to isolate and, although they originate from bone marrow their site of maturation and the conditions that direct their growth and differentiation are still poorly characterized. Granulocyte macrophage-colony stimulating factor (GM-CSF) favours the outgrowth of dendritic cells from mouse peripheral blood. Here we extend this finding to man and demonstrate that cooperation between GM-CSF and tumour necrosis factor-alpha (TNF-alpha) is crucial for the generation of human dendritic/Langerhans cells from CD34+ haematopoietic progenitors. The availability of large numbers of these cells should now facilitate the understanding of their role in immunological regulation and disorder.     

Positive selection of CD4+ T cells mediated by MHC class II-bearing stromal cell in the thymic cortex

T lymphocytes differentiate in the thymus, where functionally immature, CD4+CD8+ (double positive) thymocytes develop into functionally mature CD4+ helper cells and CD8+ cytotoxic (single positive) T cells. The thymus is the site where self-reactive T cells are negatively selected (clonally deleted) and where T cells with the capacity to recognize foreign antigens in association with self-proteins encoded by the major histocompatibility complex (MHC) are positively selected. The net result of these developmental pathways is a T-cell repertoire that is both self-tolerant and self-restricted. One unresolved issue is the identity of the thymic stromal cells that mediate the negative and positive selection of the T-cell repertoire. Previous work has pointed to a bone-marrow-derived macrophage or dendritic cell as the inducer of tolerance, whereas a radiation-resistant, deoxyguanosine-resistant thymic cell seems to mediate the positive selection of self-MHC restricted T cells. Thymic stromal cells in the cortex interact with the T-cell antigen receptor on thymocytes. Using several strains of transgenic mice that express the class II MHC molecule I-E in specific regions of the thymus, we show directly that the positive selection of T cells is mediated by an I-E-bearing cell in the thymic cortex.     

Deletion of self-reactive T cells before entry into the thymus medulla

The thymus is important in the differentiation of bone marrow-derived precursor cells into functional T cells; humoral factors, as well as physical interactions with nurse cells, dendritic cells and epithelial cells, are thought to be instrumental in this process. Thymic lymphocytes mature during their migration from the cortical to the medullary region of the thymus, when they undergo phenotypic changes that include the acquisitions of T-cell antigen receptors, hormone receptors and differentiation antigens. Cortical T cells are thus mostly CD4+CD8+, whereas medullary T cells are either CD4+CD8- or CD4-CD8+. During this period T cells are subjected to two types of repertoire selection: all T cells recognizing self-MHC with low affinity may be preferentially amplified (positive selection), and in a second step T cells with high-affinity receptors for self-MHC determinants plus self antigens are eliminated (negative selection). We have described two monoclonal antibodies specific for the V beta 6 gene segment of the alpha/beta heterodimeric T-cell antigen receptor and have shown that most CD4+/V beta 6+ T cell recognize the Mlsa antigenic determinant but not Mlsb; similar results have been reported for V beta 8.1 and Mlsa. In both situations, tolerance to Mlsa correlated in an MHC-dependent fashion with absence of V beta 6 or V beta 8.1 T-cell antigen receptor expressing T cells in the periphery. We show here by immunostaining of thymus cryosections and cytofluorometric analysis that V beta 6-expressing cortical T cells are present at high density in both Mlsa and Mlsb mice, but do not enter the medullary region of Mlsa animals.     

KIR expression on self-reactive CD8+ T cells is controlled by T-cell receptor engagement

Natural killer cell tolerance is maintained by the interaction of killer inhibitory receptors (KIRs) with self-major histocompatibility complex class I gene products. A subset of T cells also expresses inhibitory receptors, but the functional significance of these receptors on T cells is unclear. Here we show that, in the absence of T-cell receptor (TCR) engagement, KIRs expressed on CD8+ T cells are slowly downregulated by KIR ligands expressed on antigen-presenting cells. The resulting expression levels of KIR are no longer able to inhibit T-cell function. In contrast, TCR engagement sustains KIR expression, and re-induces functional levels of KIR expression after ligand-induced downregulation of KIR. Our data indicate that KIR expression on CD8+ T cells in vivo may be maintained through continuous encounters with antigen. As KIR-mediated inhibition of T-cell activation can be bypassed at high antigen concentrations, dynamic KIR expression may mediate T-cell tolerance to self-antigens by sparing self-reactive T cells, thus enabling them to mediate potentially useful immune functions to quantitatively or qualitatively different antigens.     

Evidence for a physical association of CD4 and the CD3:alpha:beta T-cell receptor

CD4 is a molecule expressed on the surface of T lymphocytes which recognize foreign protein antigens in the context of class II major histocompatibility complex (MHC) molecules. Recognition of antigen:class II MHC complexes by CD4+ T cells can be inhibited by anti-CD4 (ref. 3). Nevertheless, specific recognition of the antigen:Ia complex is clearly a function of the T-cell receptor, which is composed of CD3 and the variable polypeptides alpha and beta. Thus, it has been proposed that CD4 serves an accessory function in the interaction of CD4+ T cells and Ia-bearing antigen-presenting cells by binding to non-polymorphic portions of class II MHC molecules and stabilizing the cell interaction. Based on our observation that anti-CD4 could inhibit activation of a cloned line of CD4+ T cells by antibodies directed at a particular epitope on the variable region of the T-cell receptor, we have recently proposed that CD4 is actually part of the T-cell antigen recognition complex, physically associated with CD3:alpha:beta. But numerous studies showing that CD3 and CD4 are not stably associated on the T-cell surface would appear to contradict this model. Here we show that anti-T-cell-receptor antibodies can co-modulate expression of the T-cell receptor and CD4, and that the monovalent Fab fragment of such an anti-T-cell-receptor antibody can, in conjunction with bivalent anti-CD4 antibody, generate an activating signal for the T cell. These findings provide further evidence for a physical association of the T-cell receptor complex and CD4.