CD4+ and CD8+ T cells acquire specific lymphokine secretion potentials during thymic maturation.

Peripheral CD4+ and CD8+ T lymphocytes carry out different functions during immune reactions, partly as a result of the distinct patterns of lymphokines that they secrete upon stimulation. Using thymic cells from adult and newborn mice as well as from fetal organ cultures, we show here that this functional differentiation occurs inside the thymus and is completed during the single positive stage by the time the T-cell receptor becomes fully coupled to the intracellular activation pathways leading to lymphokine secretion. Surprisingly, CD4+8- thymocytes differ from their immediate progeny, naive peripheral CD4+ cells, in that they secrete a broader range of lymphokines, including interleukins 4, 5 and 10 and gamma-interferon, and more closely resemble immunologically experienced (activated or memory) CD4+ lymphocytes.     

Development of CD4-CD8+ cytotoxic T cells requires interactions with class I MHC determinants

Differentiation of bone marrow derived precursors into mature T cells takes place in the thymus. During differentiation, T cells develop the receptor repertoire which allows them to recognize antigen in the context of self major histocompatibility complex (MHC) molecules. Mature T helper cells (mostly CD4+ CD8-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (mostly CD4-CD8+) recognize antigen in the context of class I MHC determinants. Thymic MHC-encoded determinants greatly influence the selection of the T-cell receptor repertoire. In addition to positive selection, a negative selection to eliminate self-reactive T-cell clones is thought to occur in the thymus, but how this 'education' occurs is not well understood. It has been suggested that during differentiation an interaction between the T-cell receptor (TCR) and MHC-encoded determinants occurs, leading to the selection of an MHC-restricted receptor repertoire. In support of this hypothesis, class-II-specific, CD4+ CD8- helper T cells fail to develop in mice neonatally treated with anti-class II monoclonal antibody (mAb). As CD4-CD8+ cells differ from the CD4+ CD8- lineage (in function, MHC-restriction specificity and perhaps site of education) we examined whether interactions with MHC determinants are also necessary for the development of class-I-specific T cells. Here we show that mice chronically treated with anti-class I mAb from birth lack CD4-CD8+ cells and cytotoxic T-cell precursors, indicating that most CD4-CD8+ T cells need interaction with class I MHC molecules during differentiation.     

Acquisition of specific cytotoxic activity by human T4+ T lymphocytes in culture

Mature human T lymphocytes can be separated by monoclonal antibodies OKT4 and OKT8 according to their surface phenotypes into T4+T8- and T4-T8+ subsets. From short-term experiments using bulk cultures, the helper/inducer function has been assigned to the T4+T8- subset and the cytotoxic/suppressor function to the T4-T8+ subset. Thus if T lymphocytes are separated after stimulation in a mixed lymphocyte reaction (MLR), the entire cytotoxic activity is found in the T4-T8+ fraction whereas the T4+T8- fraction shows no detectable cytotoxicity. If, however, T lymphocytes are cloned after MLR and grown in long-term culture, a surprisingly large fraction of T4+ T lymphocyte clones (TLC) shows cytotoxic activity. Here we report that T4+ TLC can acquire specific cytotoxicity during in vitro cultivation.     

Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells

In early simian immunodeficiency virus (SIV) and human immunodeficiency virus-1 (HIV-1) infections, gut-associated lymphatic tissue (GALT), the largest component of the lymphoid organ system, is a principal site of both virus production and depletion of primarily lamina propria memory CD4+ T cells; that is, CD4-expressing T cells that previously encountered antigens and microbes and homed to the lamina propria of GALT. Here, we show that peak virus production in gut tissues of SIV-infected rhesus macaques coincides with peak numbers of infected memory CD4+ T cells. Surprisingly, most of the initially infected memory cells were not, as expected, activated but were instead immunophenotypically 'resting' cells that, unlike truly resting cells, but like the first cells mainly infected at other mucosal sites and peripheral lymph nodes, are capable of supporting virus production. In addition to inducing immune activation and thereby providing activated CD4+ T-cell targets to sustain infection, virus production also triggered an immunopathologically limiting Fas-Fas-ligand-mediated apoptotic pathway in lamina propria CD4+ T cells, resulting in their preferential ablation. Thus, SIV exploits a large, resident population of resting memory CD4+ T cells in GALT to produce peak levels of virus that directly (through lytic infection) and indirectly (through apoptosis of infected and uninfected cells) deplete CD4+ T cells in the effector arm of GALT. The scale of this CD4+ T-cell depletion has adverse effects on the immune system of the host, underscoring the importance of developing countermeasures to SIV that are effective before infection of GALT.     

Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.     

HIV preferentially infects HIV-specific CD4+ T cells

HIV infection is associated with the progressive loss of CD4(+) T cells through their destruction or decreased production. A central, yet unresolved issue of HIV disease is the mechanism for this loss, and in particular whether HIV-specific CD4(+) T cells are preferentially affected. Here we show that HIV-specific memory CD4(+) T cells in infected individuals contain more HIV viral DNA than other memory CD4(+) T cells, at all stages of HIV disease. Additionally, following viral rebound during interruption of antiretroviral therapy, the frequency of HIV viral DNA in the HIV-specific pool of memory CD4(+) T cells increases to a greater extent than in memory CD4(+) T cells of other specificities. These findings show that HIV-specific CD4(+) T cells are preferentially infected by HIV in vivo. This provides a potential mechanism to explain the loss of HIV-specific CD4(+) T-cell responses, and consequently the loss of immunological control of HIV replication. Furthermore, the phenomenon of HIV specifically infecting the very cells that respond to it adds a cautionary note to the practice of structured therapy interruption.     

CD4+ murine T cells develop from CD8+ precursors in vivo

The adult murine thymus contains four subpopulations of thymocytes defined by the T-cell surface antigens CD4 (L3T4) (a marker of helper T cells) and CD8 (Lyt2) (a marker of cytotoxic/suppressor T cells): CD4+8- and CD4-8+ (single positives), CD4+8+ (double positives) and CD4-8- (double negatives). To understand how T cells develop in the thymus, it is important to determine the lineage relationships among these subpopulations. In particular, the status of double positives, which make up approximately 80% of the total thymocyte population, has long been controversial. Some purpose that double positives are 'dead-end cells' that all die in the thymus, perhaps because they have been rejected by some selection process. Others suggest that, although most double positives die in the thymus, some develop into the more mature single positives that leave the thymus. The experiments presented here show that repeated injections of anti-CD8 monoclonal antibodies block the development of CD4+ cells, demonstrating that these cells develop from CD8+ precursors, probably double positive thymocytes, in vivo.     

Circulating CD8+ cytotoxic T lymphocytes specific for HTLV-I pX in patients with HTLV-I associated neurological disease

The human T-lymphotropic virus type I (HTLV-I), the first human retrovirus to be characterized, is associated with adult T-cell leukaemia and a chronic progressive disease of the central nervous system termed tropical spastic paraparesis, or HTLV-I-associated myelopathy. Only 1% of individuals infected with HTLV-I develop clinical disease however. The various manifestations of an HTLV-I infection may be related to differences in the genetic backgrounds of individuals, infection with variant strains of HTLV-I, differences in viral tropism or host immune response to the virus. Whereas the humoral response to HTLV-I is well characterized, little is known about the human cellular immune response, such as the production of cytotoxic T lymphocytes. Here we report the presence of high levels of circulating HTLV-I-specific cytotoxic T lymphocytes in patients with HTLV-I associated neurological disease but not in HTLV-I seropositive individuals without neurological involvement. These cytotoxic T lymphocytes are CD8+, HLA class I- restricted and predominantly recognize the HTLV-I gene products encoded in the regulatory region pX. These findings suggest that HTLV-I-specific cytotoxic T lymphocytes may contribute to the pathogenesis of associated neurological disorders associated with HTLV-I.     

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.     

CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity

The long-term persistence of pathogens in a host that is also able to maintain strong resistance to reinfection, referred to as concomitant immunity, is a hallmark of certain infectious diseases, including tuberculosis and leishmaniasis. The ability of pathogens to establish latency in immune individuals often has severe consequences for disease reactivation. Here we show that the persistence of Leishmania major in the skin after healing in resistant C57BL/6 mice is controlled by an endogenous population of CD4+CD25+ regulatory T cells. These cells constitute 5-10% of peripheral CD4+ T cells in naive mice and humans, and suppress several potentially pathogenic responses in vivo, particularly T-cell responses directed against self-antigens. During infection by L. major, CD4+CD25+ T cells accumulate in the dermis, where they suppress-by both interleukin-10-dependent and interleukin-10-independent mechanisms-the ability of CD4+CD25- effector T cells to eliminate the parasite from the site. The sterilizing immunity achieved in mice with impaired IL-10 activity is followed by the loss of immunity to reinfection, indicating that the equilibrium established between effector and regulatory T cells in sites of chronic infection might reflect both parasite and host survival strategies.     

Different patterns of peripheral migration by memory CD4+ and CD8+ T cells

Infections localized to peripheral tissues such as the skin result in the priming of T-cell responses that act to control pathogens. Activated T cells undergo migrational imprinting within the draining lymph nodes, resulting in memory T cells that provide local and systemic protection. Combinations of migrating and resident memory T cells have been implicated in long-term peripheral immunity, especially at the surfaces that form pathogen entry points into the body. However, T-cell immunity consists of separate CD4(+) helper T cells and CD8(+) killer T cells, with distinct effector and memory programming requirements. Whether these subsets also differ in their ability to form a migrating pool involved in peripheral immunosurveillance or a separate resident population responsible for local infection control has not been explored. Here, using mice, we show key differences in the migration and tissue localization of memory CD4(+) and CD8(+) T cells following infection of the skin by herpes simplex virus. On resolution of infection, the skin contained two distinct virus-specific memory subsets; a slow-moving population of sequestered CD8(+) T cells that were resident in the epidermis and confined largely to the original site of infection, and a dynamic population of CD4(+) T cells that trafficked rapidly through the dermis as part of a wider recirculation pattern. Unique homing-molecule expression by recirculating CD4(+) T effector-memory cells mirrored their preferential skin-migratory capacity. Overall, these results identify a complexity in memory T-cell migration, illuminating previously unappreciated differences between the CD4(+) and CD8(+) subsets.     

Hassall's corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus

Hassall's corpuscles-first described in the human thymus over 150 years ago-are groups of epithelial cells within the thymic medulla. The physical nature of these structures differs between mammalian species. Although Hassall's corpuscles have been proposed to act in both the removal of apoptotic thymocytes and the maturation of developing thymocytes within the thymus, the function of Hassall's corpuscles has remained an enigma. Here we report that human Hassall's corpuscles express thymic stromal lymphopoietin (TSLP). Human TSLP activates thymic CD11c-positive dendritic cells to express high levels of CD80 and CD86. These TSLP-conditioned dendritic cells are then able to induce the proliferation and differentiation of CD4(+)CD8(-)CD25(-) thymic T cells into CD4(+)CD25(+)FOXP3(+) (forkhead box P3) regulatory T cells. This induction depends on peptide-major histocompatibility complex class II interactions, and the presence of CD80 and CD86, as well as interleukin 2. Immunohistochemistry studies reveal that CD25(+)CTLA4(+) (cytotoxic T-lymphocyte-associated protein 4) regulatory T cells associate in the thymic medulla with activated or mature dendritic cells and TSLP-expressing Hassall's corpuscles. These findings suggest that Hassall's corpuscles have a critical role in dendritic-cell-mediated secondary positive selection of medium-to-high affinity self-reactive T cells, leading to the generation of CD4(+)CD25(+) regulatory T cells within the thymus.     

Induction of CD8+ cytotoxic T cells by immunization with purified HIV-1 envelope protein in ISCOMs

To reduce the risks of immunization with killed or live attenuated virus vaccines, it may be advantageous to use a pure, defined antigen that contains determinants for both humoral and cellular immunity. However, although most non-living intact protein preparations induce antibodies and CD4+ major histocompatibility complex (MHC) class II-restricted helper and/or cytotoxic T lymphocytes (CTL), they do not elicit CD8+ MHC class I restricted CTL. Indeed, with a few exceptions, it has not so far been possible to induce CD8+ CTL by immunizing with intact soluble proteins. We show here that a single subcutaneous immunization in mice with immunostimulating complexes containing either purified intact gp160 envelope glycoprotein of the human immunodeficiency virus (HIV)-1 or influenza haemagglutinin results in reproducible and long-lasting priming of HIV specific or influenza-specific CD8+, MHC class I restricted CTL.     

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.     

Identical V beta T-cell receptor genes used in alloreactive cytotoxic and antigen plus I-A specific helper T cells

T lymphocytes involved in the cellular immune response carry cell-surface receptors responsible for antigen and self recognition. This T-cell receptor molecule is a heterodimeric protein consisting of disulphide-linked alpha- and beta-chains with variable (V) and constant (C) regions. Several complementary DNA and genomic DNA clones have been isolated and characterized. These analyses showed that the genomic arrangement and rearrangement of T-cell receptor genes using VT, diversity (DT), joining (JT) and CT gene segments is very similar to the structure of the known immunoglobulin genes. We have isolated two cDNA clones from an allospecific cytotoxic T cell, one of which shows a productive V beta-J beta-C beta 1 rearrangement without an intervening D beta segment. This V beta gene segment is identical to the V beta gene expressed in a helper T-cell clone specific for chicken red blood cells and H-21. The other clone carries the C beta 2 gene of the T-cell receptor, but the C beta 2 sequence is preceded by a DNA sequence that does not show any similarity to V beta or J beta sequences.     

Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells

Mice homozygous for a beta 2-microglobulin gene disruption do not express any detectable beta 2-m protein. They express little if any functional major histocompatibility complex (MHC) class I antigen on the cell surface yet are fertile and apparently healthy. They show a normal distribution of gamma delta, CD4+8+ and CD4+8- T cells, but have no mature CD4-8+ T cells and are defective in CD4-8+ T cell-mediated cytotoxicity. Our results strongly support earlier evidence that MHC class I molecules are crucial for positive selection of T cell antigen receptor alpha beta+ CD4-8+ T cells in the thymus and call into question the non-immune functions that have been ascribed to MHC class I molecules.     

Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype

The immune system must distinguish not only between self and non-self, but also between innocuous and pathological foreign antigens to prevent unnecessary or self-destructive immune responses. Unresponsiveness to harmless antigens is established through central and peripheral processes. Whereas clonal deletion and anergy are mechanisms of peripheral tolerance, active suppression by T-regulatory 1 (Tr1) cells has emerged as an essential factor in the control of autoreactive cells. Tr1 cells are CD4+ T lymphocytes that are defined by their production of interleukin 10 (IL-10) and suppression of T-helper cells; however, the physiological conditions underlying Tr1 differentiation are unknown. Here we show that co-engagement of CD3 and the complement regulator CD46 in the presence of IL-2 induces a Tr1-specific cytokine phenotype in human CD4+ T cells. These CD3/CD46-stimulated IL-10-producing CD4+ cells proliferate strongly, suppress activation of bystander T cells and acquire a memory phenotype. Our findings identify an endogenous receptor-mediated event that drives Tr1 differentiation and suggest that the complement system has a previously unappreciated role in T-cell-mediated immunity and tolerance.     

The duration of antigen receptor signalling determines CD4+ versus CD8+ T-cell lineage fate

Signals elicited by binding of the T-cell antigen receptor and the CD4/CD8 co-receptor to major histocompatibility complex (MHC) molecules control the generation of CD4+ (helper) or CD8+ (cytotoxic) T cells from thymic precursors that initially express both co-receptor proteins. These precursors have unique, clonally distributed T-cell receptors with unpredictable specificity for the self-MHC molecules involved in this differentiation process. However, the mature T cells that emerge express only the CD4 (MHC class II-binding) or CD8 (MHC class I-binding) co-receptor that complements the MHC class-specificity of the T-cell receptor. How this matching of co-receptor-defined lineage and T-cell-receptor specificity is achieved remains unknown, as does whether signalling by the T-cell receptors, co-receptors and/or general cell-fate regulators such as Notch-1 contributes to initial lineage choice, to subsequent differentiation processes or to both. Here we show that the CD4 versus CD8 lineage fate of immature thymocytes is controlled by the co-receptor-influenced duration of initial T-cell receptor-dependent signalling. Notch-1 does not appear to be essential for this fate determination, but it is selectively required for CD8+ T-cell maturation after commitment directed by T-cell receptors. This indicates that the signals constraining CD4 versus CD8 lineage decisions are distinct from those that support subsequent differentiation events such as silencing of co-receptor loci.