High frequencies of functionally impaired cytokeratin 18-specific CD8+ T cells in healthy HLA-A2+ donors

Combining cell surface phenotyping with functional analysis, human CD8+ T cells have been divided into several subsets which are being studied extensively in diverse physiological situations, such as viral infection, cancer and ageing. In particular, so-called terminally differentiated effector cells possess a CD45RA+ CCR7- CD27- CD28- phenotype, contain perforin and, in different models, have been shown to exert direct ex vivo killing and to release interleukins upon both antigen-nonspecific and -specific stimulation. Using HLA class I multimers, we have identified a high frequency of peripheral CD8+ T cells that recognize a peptide derived from the self protein cytokeratin 18 presented by the HLA-A*0201 molecule. These cells can be detected in approximately 15% of the HLA-A2-positive healthy donors tested. A detailed analysis revealed that they must have divided extensively in vivo, have an effector cell phenotype and express various natural killer cell-associated receptors. Interestingly, however, they remained unresponsive to antigen-specific stimulation in vitro in terms of cytotoxicity and cytokine secretion. Thus, cytokeratin 18-specific cells constitute a frequently encountered, new CD8+ T lymphocyte subpopulation without classical effector status and with so far unknown function.     

A CD3- subset of CD4-8+ thymocytes: a rapidly cycling intermediate in the generation of CD4+8+ cells

T lymphocytes with the surface phenotype CD4+8- and CD4-8+ are considered to be representative of functionally mature cells. We show here that adult murine thymus contains a subpopulation of CD4-8+ cells that differ from CD4-8+ cells found in the periphery in that they do not express the T cell receptor-associated CD3 molecular complex. Such CD3-4-8+ thymocytes are cortisone sensitive and rapidly cycling in situ. Furthermore, in contrast to mature T cells, most CD3-4-8+ thymocytes express low levels of CD5 and high levels of the B2A2 antigen. CD3-4-8+ thymocytes fail to respond to a variety of mitogenic stimuli in vitro but do give rise upon short-term culture to CD4+8+ cells. It is suggested that CD3-4-8+ thymocytes represent a transitional stage of thymus differentiation between the CD4-8- and CD4+8+ compartments.     

'Radioresistant' CD4-CD8- intrathymic T cell precursors differentiate into mature CD4+CD8- and CD4-CD8+ T cells. Development of 'radioresistant' CD4-CD8- intrathymic T cell precursors

Using lectin (PNA) and monoclonal antibodies for Pgp-1, IL-2R, H-2k, CD3, and F23.1 (T cell receptor V beta 8), we characterized the 'radioresistant' CD4-CD8- double negative thymocytes at an early stage after 800 rad irradiation. Most of the CD4-CD8- cells on day 8 after irradiation expressed a high level of Thy-1, H-2k, and PNA, while a small proportion of these cells were CD3+ and/or F23.1+. The appearance of Pgp-1 and IL-2R on the 'radioresistant' double negative precursors was also sequentially examined from day 5 to day 9 after irradiation. The double negative thymocytes at day 5 expressed the highest level of Pgp-1 antigens and these cells gradually decreased in number from day 7 to day 9. By contrast, IL-2R was transiently expressed on the double negative cells on the day 7 and 8 after irradiation. These results indicate that progression of thymocyte development occurred within the CD4-CD8- thymocytes after irradiation. We further examined the homing ability of the double negative 'radioresistant' intrathymic T cell precursors to the periphery by intrathymic cell transplantation method. The double negative thymocytes proliferate and differentiate into CD4+CD8+ cells and CD4+CD8- cells but few CD4-CD8+ cells in the thymus, while only CD4-CD8+ cells were detected in the peripheral lymphoid organs 14 days after intrathymic transplantation of the double negative cells in the H-2 compatible Thy-1 congenic mice. These results suggest that the 'radioresistant' intrathymic precursors differentiate and mature in the thymus and migrate to the periphery.     

Requirement of CD4+ T cells and antigen-presenting cells for primary in vitro generation of CD8+ cytotoxic T cells against Ld-binding self-peptide p2Ca.

We investigated the cellular requirement for primary in vitro generation of cytotoxic T-lymphocytes (CTL) in BALB/c spleen cells against Ld-binding self-peptide p2Ca. Depletion of CD4+ T-cells in vitro by pretreatment with anti-CD4 monoclonal antibody (mAb) and complement or in vivo by administration of anti-CD4 mAb abrogated generation of CTL. Depletion of adherent cells by passing spleen cells through a nylon wool (NW) column also abrogated generation of CTL. Addition of peritoneal exudate cells (PEC) to spleen cells passed through the NW column restored CTL generation. These findings indicate that both CD4+ T-cells and antigen-presenting cells (APC) were necessary for CTL generation. Treatment of PEC with paraformaldehyde (PFA), but not mitomycin-C (MMC) abrogated their ability to restore CTL generation when mixed with spleen cells from the NW column, suggesting that an endocytic pathway could be involved in presentation of p2Ca on APC.     

IL-2 and IL-4 can co-modulate the generation of cytotoxic T cells through CD8- CD4- splenic lymphocytes.

Following activation with concanavalin A (Con A), murine T cells are able to suppress the generation of allospecific cytotoxic T lymphocytes (CTL). We have analysed the phenotype, tissue distribution, and mode of action of these cells in an effort to understand further the regulation of CTL-mediated immunity. The precursors of such cells are rare (1 cell per 70,000 spleen cells being able to suppress the generation of a particular allospecific response), but are much more abundant in the spleen than in the thymus. By the use of cytotoxic antibodies, we have been able to demonstrate that the splenic precursors of such cells are Thy-1.2+, CD4-, CD8- but, following activation with Con A, these cells acquire the CD8 marker. Cellular suppression by these lymphocytes is dramatically increased in the presence of the Th2-derived lymphokine, IL-4, whereas IL-2, the Th1-derived lymphokine, significantly augments the generation of CTL in a mixed lymphocyte culture even though relative suppression is still evident in the presence of Con A-activated lymphocytes. Suppression is not due to overcrowding of a cell culture since adding Con A-activated cells to an A anti-B + C culture often resulted in the suppression of the A anti-B response but not the A anti-C response, or vice versa. Suppression appears to require cellular interaction since supernatants from Con A-activated lymphocytes are unable to mediate suppression. Such cells may play an important intermediate role in homeostasis.     

The generation of protective memory-like CD8+ T cells during homeostatic proliferation requires CD4+ T cells

Antigen-specific memory T cells are a critical component of protective immunity because of their increased frequency and enhanced reactivity after restimulation. However, it is unclear whether 'memory-like' T cells generated during lymphopenia-induced homeostatic proliferation can also offer protection against pathogens. Here we show that homeostatic proliferation-induced memory (HP-memory) CD8(+) T cells controlled bacterial infection as effectively as 'true' memory CD8(+) T cells, but their protective capacity required the presence of CD4(+) T cells during homeostatic proliferation. The necessity for CD4 help was overcome, however, if the HP-memory CD8(+) T cells lacked expression of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand; also called Apo-2L). Thus, like conventional CD8(+) memory T cells, the protective function of HP-memory CD8(+) T cells shows dependence on CD4(+) T cell help.     

A role for dendritic cells in the priming of antigen-specific CD4+ and CD8+ T lymphocytes by immune-stimulating complexes in vivo

Immune-stimulating complexes (ISCOMS) are adjuvant vectors which are unusual in being able to prime both CD4(+) and CD8(+) T cells by parenteral and mucosal routes. However, their mode of action is unclear and to define better the cellular interactions involved we have studied the ability of ISCOMS containing ovalbumin (OVA) to prime TCR transgenic CD4(+) or CD8(+) T cells in vivo. Immunization with OVA ISCOMS caused activation and clonal expansion of CD4(+) and CD8(+) T cells in the T cell areas of the draining lymph nodes, followed by the migration of both CD4(+) and CD8(+) T cells into the B cell follicle. The T cells were primed to proliferate and secrete IFN-gamma after re-stimulation in vitro with the appropriate OVA peptide and CD8(+) T cell priming occurred in the absence of CD4(+) T cells. Increasing the number of dendritic cells (DC) in vivo with flt3 ligand augmented the expansion and activation of the OVA-specific T cells, particularly CD8(+) T cells. These studies indicate DC play a central role in the priming of both CD4(+) and CD8(+) T cells in vivo, and suggest that an ability to target DC may allow ISCOMS to be powerful vaccine vectors for stimulating protective immunity.     

Definition of the HLA-A2 restricted peptides recognized by human CD8+ effector T cells by flow-assisted sorting of the CD8+ CD45RA+ CD28- T cell subpopulation

In response to antigenic stimulation, naive MHC-class I restricted and antigen-specific CD8+ CD45RA+ CD28+ T cells undergo clonal expansion, differentiate into CD8+ CD45RO+ memory T cells and convert to CD8+ CD45RA+ CD28- T cells displaying potent immune effector functions upon re-encounter with the nominal antigen. We show that the effector CD8+ CD45RA+ CD28- T cell subset is expanded in peripheral blood lymphocytes (PBL) from patients with human papilloma virus (HPV)+ cervical lesions as well as in PBL from patients with pulmonary tuberculosis. Flow-cytometric cell sorted CD8+ CD45RA+ CD28- and CD8+ CD45RA+ CD28- T cells were tested for recognition of HLA-A2 restricted peptides derived either from the human papillomavirus (HPV)16-E7 gene product, or from M. tuberculosis antigens. Mostly CD8+ CD45+ CD28- T cells define antigen/peptide-specific and MHC-restricted responses. These data were confirmed in PBL from patients with tuberculosis using HLA-A2 tetramer-complexes loaded with a peptide from the M. tuberculosis Ag85b antigen by flow cytometry. The sorting of this T cell subset enables to determine the fine specificity of CD8+ effector T cells without the need for in vitro manipulation.     

Tolerogenic dendritic cells impede priming of naïve CD8+ T cells and deplete memory CD8+ T cells

Type 1 diabetes is a T‐cell‐mediated autoimmune disease in which autoreactive CD8⁺ T cells destroy the insulin‐producing pancreatic beta cells. Vitamin D3 and dexamethasone‐modulated dendritic cells (Combi‐DCs) loaded with islet antigens inducing islet‐specific regulatory CD4⁺ T cells may offer a tissue‐specific intervention therapy. The effect of Combi‐DCs on CD8⁺ T cells, however, remains unknown. To investigate the interaction of CD8⁺ T cells with Combi‐DCs presenting epitopes on HLA class I, naive, and memory CD8⁺ T cells were co‐cultured with DCs and proliferation and function of peptide‐specific T cells were analyzed. Antigen‐loaded Combi‐DCs were unable to prime naïve CD8⁺ T cells to proliferate, although a proportion of T cells converted to a memory phenotype. Moreover, expansion of CD8⁺ T cells that had been primed by mature monocyte‐derived DCs (moDCs) was curtailed by Combi‐DCs in co‐cultures. Combi‐DCs expanded memory T cells once, but CD8⁺ T‐cell numbers collapsed by subsequent re‐stimulation with Combi‐DCs. Our data point that (re)activation of CD8⁺ T cells by antigen‐pulsed Combi‐DCs does not promote, but rather deteriorates, CD8⁺ T‐cell immunity. Yet, Combi‐DCs pulsed with CD8⁺ T‐cell epitopes also act as targets of cytotoxicity, which is undesirable for survival of Combi‐DCs infused into patients in therapeutic immune intervention strategies.     

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells

Aside from an intermediate stage in thymic T-cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T-cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti-CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA-mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA-mediated effect. Autologous CD4+ and CD8+ T-cell co-cultures in the presence of PHA induced this CD4dimCD8bright T-cell expression by 33%, demonstrating a role for CD4 cells in the PHA-mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4- counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominantly T-cell receptor (TCR) alphabeta cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

MHC class II-independent CD25+ CD4+ CD8alpha beta+ alpha beta T cells attenuate CD4+ T cell-induced transfer colitis

CD4+ alpha beta T cell populations that develop in mice deficient in MHC class II (through 'knockout' of either the Aalpha, or the Abeta chain of the I-A(b) molecule) comprise a major 'single-positive' (SP) CD4+ CD8- subset (60-90%) and a minor 'double-positive' (DP) CD4+ CD8alpha beta+ subset (10-40%). Many DP T cells found in spleen, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) express CD25, CD103 and Foxp3. Adoptive transfer of SP but not DP T cells from Aalpha(-/-) or Abeta(-/-) B6 mice into congenic RAG(-/-) hosts induces colitis. Transfer of SP T cells repopulates the host with only SP T cells; transfer of DP T cells repopulates the host with DP and SP T cells. Anti-CD25 antibody treatment of mice transplanted with DP T cells induces severe, lethal colitis; anti-CD25 antibody treatment of mice transplanted with SP T cells further aggravates the course of severe colitis. Hence, regulatory CD25+ T cells within (or developing from) the DP T cell population of MHC class II-deficient mice control the colitogenic potential of CD25- CD4+ T cells.     

CD4+ T cell-independent maintenance and expansion of memory CD8+ T cells derived from in vitro dendritic cell activation

CD4+ T cells are essential for the maintenance of CD8+ memory T (Tm) cells following acute infection, but the importance of CD4+ T cells for the maintenance and expansion of CD8+ Tm cells to non-infectious antigens remains mostly unknown. Here, we showed that ovalbumin (OVA)-specific CD8+ Tm cell precursors derived from in vitro stimulation of TCR transgenic OT I CD8+ T cells with OVA protein-pulsed bone marrow-derived dendritic cells (DCOVA) can give rise to functional CD8+ Tm cells after adoptively transferred into mice. These CD8+ Tm cells can be maintained and remain fully functional in CD4+ T cell-absent environments in vivo. Furthermore, CD4+ T cells are not essential for the expansion of these CD8+ Tm cells. Finally, these in vitro DCOVA-activated CD8+ Tm cells maintained in CD4-deficient mice are also able to confer fully protective immunity against a later challenge of OVA-expressing tumor cells. Collectively, these findings demonstrate that in contrast to acute infections, maintenance and expansion of CD8+ Tm cells after priming with OVA protein-pulsed dendritic cells are independent of CD4+ T cells.     

Differential effect of CD8(+) and CD8(-) dendritic cells in the stimulation of secondary CD4(+) T cells

Dendritic cells (DC), in their role in initiation of the adaptive immune response, have been extensively studied for their capacity to interact and stimulate naive T cells. Subsets of mature murine DC isolated directly from the spleen have been shown to differ in their ability to induce proliferative responses in both primary CD4(+) and primary CD8(+) T cells; the myeloid-related CD8alpha(-) DC induce a more intense or prolonged proliferation of naive T cells than do the lymphoid-related DC bearing CD8alpha despite similar expression of MHC and co-stimulatory molecules. Here we examine the interaction of these DC subpopulations with T cells already in the activated or memory state which are known to have greater sensitivity to antigen stimulation and bear receptors with increased capacity for signal transduction. We show that influenza virus-specific CD4(+) T cell clones and splenic T cells from peptide-primed animals proliferated in response to antigen presented by separated splenic CD8(-) DC. In contrast, these T cells showed only weak, if any, proliferation in response to CD8(+) DC despite observable cluster formation in the cultures. The differential between the two DC types in inducing proliferation was even more pronounced than previously seen with primary T cells and did not reflect differential longevity of the DC in culture, altered response kinetics or deviation from IL-2 to IL-4 induction with CD8(+) DC, but was related to the levels of IL-2 induced. The deficiency in the CD8(+) DC was not overcome by using infectious virus rather than synthetic peptide as the antigen source. These results show that lymphoid-related CD8(+) splenic DC, despite their mature phenotype, fail to provide appropriate signals to secondary CD4(+) T cells to sustain their proliferation.     

Mini-review CD4 T cells are required for CD8 T cell memory generation

Whereas the role of CD4 T cells in B cell memory generation is well established and unequivocal, the role that CD4 T cells play in CD8 responses was until recently far more elusive and controversial. A series of recent reports, however, have re-assessed the role of CD4 help on CD8 responses and have given rise to surprisingly unambiguous conclusions. While studying very different systems, they demonstrated that CD4 T cells are absolutely required for the generation of bona fide CD8 memory cells; the reports allow, for the first time, strong analogies to be made between B and CD8 memory cell generation. These data invite us to drastically change our idea of CD4 help on CD8 responses because they show that the old dichotomy - Th-dependent versus Th-independent CD8 responses - is no longer accurate.     

Instruction of naive CD4+ T cells by polarized CD4+ T cells within dendritic cell clusters

Cooperation between CD4(+) T cells can enhance the response and modulate the cytokine profile, and defining these parameters has become a major issue for multivalent-vaccine strategies.We explored cooperation using adoptive transfer of two populations of TCR transgenic T cells of different specificity. One was transferred without prior activation, whereas the second was activated for five days by antigen stimulation under polarizing culture conditions. Both populations were transferred into a single adoptive host and then primed by particle-mediated DNA delivery. Polarized Th1 cells (inducers) raised the frequency of IFN-gamma(+) cells within a naive (target) population, whereas Th2 inducers raised the frequency of IL-4(+) and reduced that of IL-2(+) cells. These effects were obtained when the genes for both antigens were on the same particle, favoring presentation by the same dendritic cell, but not when on different particles delivered to different dendritic cells. Autonomy of DC clusters allows linked sets of antigens (e.g. from a single pathogen) to maintain cytokine bias, but allows other independent responses, each with their own set of autonomous clusters.     

Characterization of CD4- CD8- CD3+ T-cell receptor-alphabeta+ T cells in murine cytomegalovirus infection

In this study, we have investigated that after the intraperitoneal infection with murine cytomegalovirus (MCMV), the CD3+ CD4- CD8-(double negative; DN) T-cell receptor (TCR)alphabeta+ T cells increased in peritoneal cavity, liver and spleen in both resistant C57BL/6 and susceptible BALB/c mice. The total cellular population of these cells showed peak levels around day 5 after infection in all the three investigated organs and the following phenotypical and functional characteristics emerged. The peritoneal DN TCRalphabeta+ T cells expressed highly skewed TCRVbeta8 on day 5 after infection compared with the uninfected mice, but those in spleen and liver showed moderate and low skewed TCRVbeta8, respectively. The percentages of NK1.1+ DN TCRalphabeta+ T cells gradually decreased as did modulation of some of their activation markers consistent with an activated cell phenotype. The peritoneal DN TCRalphabeta+ T cells on day 5 after infection expressed the genes of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha, Eta-1 (early T-cell activation-1) and MCP-1 (monocyte chemoattractant protein 1) but lacked expression of interleukin-4 (IL-4). After in vitro stimulation with phorbol 12-myristate 13-acetate and calcium ionophore in the presence of Brefeldin A, higher frequencies of intracellular IFN-gamma+ DN TCRalphabeta+ T cells were detected in all three investigated organs of infected mice compared with those of uninfected mice. Stimulation of peritoneal DN TCRalphabeta+ T cells with plate-bound anti-TCRbeta monoclonal antibodies showed proliferation and also produced IFN-gamma but not IL-4. These results suggest that DN TCRalphabeta+ T cells were activated and may have an antiviral effect through producing IFN-gamma and some macrophage-activating factors during an early phase of MCMV infection.     

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.     

Cross-linking of the T cell antigen receptor interferes with the generation of CD4+8+ thymocytes from their immediate CD4-8+ precursors

The majority of thymocytes are immature cells co-expressing the surface markers CD4 and CD8. About two thirds of these cells also express the T cell antigen receptor (TcR), though at a level distinctly lower than found on mature T cells. The direct precursors of these "double-positive" thymocytes are cycling cortical blast cells of the CD4-8+ phenotype. Using a new monoclonal antibody to a constant determinant of the rat TcR alpha/beta, it is shown here that (a) about 50% of these CD8 "single-positive" committed precursor cells already express the TcR alpha/beta, though at very low levels, (b) during short-term suspension culture in medium supplemented only with fetal calf serum they not only acquire CD4 but also TcR alpha/beta levels characteristic of CD4,8 "double-positive" thymocytes, and (c) cross-linking of the TcR during culture inhibits the acquisition of the CD4 antigen in the majority of these cells.