T Helper-Independent Activation of Human CD8+ Cells: the Role of CD28 Costimulation

The concept that activation of MHC class I-restricted CD8⁺ cells entirely depends on help from MHC class II-restricted CD4⁺ T cells has recently been supplemented with an alternative model in which CD8⁺ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8⁺ T cells in two different in vitro models. Freshly isolated CD8⁺ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting FcγR⁺ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8⁺ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8⁺ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8⁺ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8⁺ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance.     

Induction of CD8+ CTL Recognizing Mycobacterial Peptides

Mycobacterium tuberculosis is the single, most important cause of morbidity attributable to a single infectious organism. CD8⁺ T cells play an important role in anti-tuberculous immune responses in both mice and humans. Data concerning the identity of mycobacterial antigens recognized by CD8⁺ T cells is limited; consequently, few CTL epitopes have been characterized. The authors identified allele-specific (H-2^{b and d}) MHC class I binding motifs in six prominent M. tuberculosis protein antigens (the 19 and 38 kDa lipoglycoproteins and the 10, 16, 65 and 70 kDa stress proteins). These predicted epitopes were tested for MHC binding as well as their ability to elicit peptide-specific CTL following in vivo priming. The authors were able to identify eight previously undescribed mycobacterial CTL epitopes by using spleen cells from peptide-immunized mice. In addition, CTL specific for at least one of these epitopes also recognized the naturally processed epitope presented on transfected EL4 target cells. These mycobacteria-derived CTL epitopes could be important for future analysis of the involvement of CD8⁺ T cells in M. tuberculosis infection, pathogenesis and vaccine development.     

Anti CD5 Sustains the Proliferative Response of IgM-Activated Human CD5+B Cells

The CD5 molecule is expressed by most T cells but it is present on a minor B cell subset. Whilst several studies have provided information on the physiological role of T cell CD5, the functional role of CD5 on B lymphocytes remains unclear. To address this question, tonsillar CD5⁺ B cells were sorted by dual-colour fluorescence and FACS. Sorted cells were stimulated with polyclonal anti-IgM antibodies (Ab), and monoclonal (MoAb) F(ab')₂ fragments of anti-CD5. Proliferative responses were evaluated by enumeration of Ki-67 positive cells using quantitative flow cytometry. Co-stimulation with anti-CD5 MoAb for 3 days did not affect the anti-IgM and IL2-induced proliferation of CD5⁺ B cells. This was seen under conditions where the anti-CD5 was soluble, adsorbed to the microwells or cross-linked by anti-mouse antibodies. Fewer CD25⁺ cells were detected, however, in the presence of anti-CD5. In contrast, the proliferative response of CD5⁺ B cells prestimulated for 3 days with IL-2 and anti-IgM, was sustained in a further 3-day culture period when anti-CD5 was added. It is concluded that CD5 occupancy might provide an additional signal to activated CD5⁺ B cells favouring their proliferation and differentiation into autoantibody secreting cells.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

The CD4+CD8+ and CD4+ Subsets of FOXP3+ Thymocytes Differ in their Response to Growth Factor Deprivation or Stimulation

The timing of thymic regulatory T (Treg) cell commitment remains unclear. Specifically, there is disagreement as to whether the CD4⁺CD8⁺ FOXP3⁺ thymocytes are precursors of mature CD4⁺ FOXP3⁺ Treg cells, or an independent Treg cell lineage. We reasoned that precursors should be more susceptible to apoptosis than mature Treg cells, and tested this by growth factor removal and anti-CD3 stimulation. Both treatments resulted in an increase of CD4⁺ FOXP3⁺ thymocytes, whereas the frequency of CD4⁺CD8⁺ FOXP3⁺ thymocytes decreased significantly. These changes were accompanied by an increase of annexin⁺ apoptotic cells. Both of these FOXP3⁺ subsets expressed higher levels of Bcl-2 and BIM than other thymocytes, and while in our setting expression of BIM seemed to predispose the cells to apoptosis, Bcl-2 had no apparent protective effect. These results indicate that CD4⁺CD8⁺ FOXP3⁺ thymocytes are more susceptible to apoptosis than mature CD4⁺ FOXP3⁺ Treg cells. This is consistent with the view that they are still immature and thus likely to represent a precursor population.     

Effects of Anti-CD 18 and LPS on CD 14 Expression on Human Monocytes

In this study we show that the cytokine stimulatory effect of LPS on human monocytes is enhanced by addition of monoclonal antibodies against CD18 (aCD18 MoAbs). Incubation of monocytes with αCD18 MoAbs overnight increased the CD 14 expression as detected by Leu-M3, but not with My-4. These results indicate that CD18 participates in LPS-induced TNF-o production as well as in regulating CD14 expression on monocytes. Addition of LPS to monocytes resulted in a reduction in the CD14 expression after 1/2, 1, 2 and 4h, but increased CD 14 expression was seen after LPS stimulation overnight. By doing double labelling of the monocyte population for CD 14 and CD16 it was found that the reduction in CD 14 expression occurred in the CD14⁺/CD16⁺ sub-population, while the increase in CD14 expression was seen in both the CD14⁺/CD16⁺ and the CD14⁺/CD16⁺ cells. αCDU MoAbs that were able to inhibit LPS-induced cytokine production from monocytes (3C10 and My-4) were considerably less able to detect the increase in CD14 expression after LPS stimulation than aCD14 MoAbs that did not inhibit LPS-induced cytokine production (Leu-M3 and αCD14_serva)- Our data indicate that My-4 and Leu-M3 define two populations of CD14⁺ cells on LPS stimulated human monocytes.     

Characterization of T-Cell Receptor Vβ Repertoire in Ovarian Tumour-Reacting CD3+ CD8+ CD4− CTL Lines

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T-cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3⁺ CD8⁺ CD4⁻ cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3⁺ CD8⁺ CD4⁻ lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8⁺ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour-reactive CD3⁺ CD8⁺ CD4⁻ CTL from different individuals of known HLA types.     

CD4+CD25+ regulatory cells in acquired MHC tolerance

Summary: Tolerance to self-antigens is an ongoing process that begins centrally during T-cell maturation in the thymus and continues throughout the cell's life in the periphery by a network of regulated restraints. Remaining self-reactive T-cells that escape intrathymic deletion may be silenced within the peripheral immune system by specialized regulatory CD4⁺ cells. By analogy, regulatory CD4⁺ cells that control immunity to "acquired self" should arise in circumstances where the immune system acquires tolerance to foreign MHC, such as the tolerance that develops following the exposure to foreign MHC antigens during the neonatal period. We have used this classic model of neonatal tolerance to examine the role of regulatory CD4⁺ cells in acquired tolerance to disparate class I and class II MHC. Adoptive transfer of unfractionated but not CD4⁺-depleted spleen cells from neonatal tolerant mice into SCID recipients inhibited skin graft rejection by immunocompetent CD8⁺ T cells. Using 5-bromo-2'-deoxyuridine incorporation, standard cytotoxic T-lymphocyte assays, short-term interferon-γ ELISPOT, and intracellular FACS analysis to study CD8⁺ T-cell effector function, we demonstrated that neonatal tolerant mice contain CD4⁺CD25⁺ cells that suppress the development of anti-donor CD8⁺ T-cell responses in vitro . We conclude that regulatory CD4⁺CD25⁺ cells initiate and/or maintain tolerance by preventing the development of CD8⁺ T-cell alloreactivity.     

Use of Immobilized HLA-A2:Ig Dimeric Proteins to Determine the Level of Epitope-Specific, HLA-Restricted CD8+ T-Cell Response

A novel assay to assess antigen-specific cytokine release from stimulated CD8⁺ T cells derived from the mucosal and peripheral blood compartments has been developed and standardized using the influenza A virus matrix protein (MP) peptide, GILGFVFTL. This technology is based on the capacity for the human leucocyte antigen (HLA)-A2:Ig dimeric protein to stimulate CD8⁺ T cells in a major histocompatibility complex (MHC) class I-restricted fashion without the necessity for antigen presenting cells (APC). This assay has been optimized utilizing a 9-amino acid residue (9mer) peptide, the optimal peptide length for presenting an epitope to CD8⁺ T cells. Compared to existing assays, this more sensitive and specific methodology requires fewer cells, enabling easier and more accurate monitoring of the CD8⁺ T-cell response in biological compartments, such as the mucosa during the course of viral infection and may be utilized to assess epitope-specific CD8⁺ T-cell responses in vaccine trials.     

Expression of CD28 on CD8+ and CD4+ Lymphocytes During HIV Infection

CD28 interaction with B7 molecules, expressed on the membranes of antigen-presenting cells, costimulates cytokine production, T-cell proliferation and generation of cytotoxic lymphocytes. The expression of CD28 markers on CD4⁺ and CD8⁺ lymphocytes was studied in a group of subjects at various stages of HIV infection. A reduction in the percentage of CD28-bearing CD4⁺ and CD8⁺ cell subsets was observed during the asymptomatic stage of the disease. This reduction was more pronounced in AIDS than in non-AIDS patients. At the same time, an increase in the absolute CD8⁺CD28⁻ cell number (greater in stage A than in stage B and C subjects) was observed in HIV-infected patients. The finding of an altered pattern of CD28 expression on T cells might per se explain certain early defects in the cytokine pattern and in the immune response peculiar to HIV-infected patients.     

Activation Signal Induces the Expression of B Cell-Specific CD45R Epitope (6B2) on Murine T Cells

Tlymphocytes express multiple forms of the leukocyte-common antigen CD45, transcribed by alternative usage of leukocyte-common antigen exon 4 6. The various isoforms of CD45R expressed differentially on T cells are involved in different stages of development and activation. The monoclonal antibody (MoAb) RA3-6B2 is established as a B cell-type isoform (B220)-specific marker. However, it reacts with certain activated T cells although the relationship between 6B2 expression and T-cell activation is unclear. We have examined the 6B2 expression on activated T cells and found that concanavalin A, anti-CD3 antibody and staphylococcal enterotoxin B (SEB) induced 6B2 expression on T cells. The expression was found on both CD4⁺ and CD8⁺ T cells and also was induced by SEB in vivo predominantly on CD8⁺ T cells. The 6B2⁺ T cells are IL-2R⁺ and blasted cells according to flow cytometry analysis. Therefore, the 6B2⁺ T cells are supposed to be in an activated stage. Enzymatic analysis demonstrated that trypsin treatment decreased the 6B2 expression, whereas neuraminidase increased the intensity on activated T cells. Neither endo-D or endo-H have any effect on the expression and there are no differences, in the results of immunoprecipitation and RT-PCR analysis, between control T cells and activated T celts. Taken together, the 6B2 epitope is presumed to be the product of CD45R modification and is expressed on activated T cells. These results illustrate a novel classification of a T-cell subpopuiation bearing a 6B2 epitope.     

CD26 Surface Antigen Expression on Peripheral Blood T Lymphocytes from Children with Down's Syndrome (Trisomy 21)

A phenotypical analysis carried out by two-colour flow cytometry showed that the proportion of circulating CD4⁺ T lymphocytes co-expressing the membrane-associated ectoenzyme dipeptidyl peptidase IV (CD26 antigen), a functional collagen receptor involved in T-cell triggering through its interaction with the CD45 protein tyrosine phosphatase, was significantly lower in 28 children with non-translocated trisomy 21 (Down's syndrome) (DS) than that calculated in the bloodstream of 27 age- and sex-matched healthy controls. Agonist anti-CD26 monoclonal antibodies (MoAbs), such as anti-lF7, not only modulate the surface expression of this molecule, but also enhance the proliferative activity of normal human T cells via the CD3- and CD2-mediated activation pathways. T-lymphocyte proliferation induced by antigen or polyclonal T-cell activators, including anti-CD3 or -CD2 MoAbs, is severely impaired in DS. Although the physiological ligand of CD26 surface structure is unknown, the fact that CD4⁺ T lymphocytes found in the blood of trisomic subjects are mostly CD26⁻ (anti-lF7⁻) suggests that their faulty mitogenic response may be due to phenotypical and, perhaps, strictly correlated functional abnormalities.     

CD8+ Cells are the Main Producers of IL10 and IFNγ after Superantigen Stimulation

Purified CD8⁺ T cells were recently shown to produce TH1 as well as TH2 types of cytokines upon restimulation, indicating an important role for these cells in regulation of immune responses. However, it is not known if the CD8⁺ cells would contribute to cytokine production in the presence of cytokine secreting CD4⁺ cells. In the present study the authors have investigated the proportion of cytokine-producing CD4⁺ and CD8⁺ cells in the spleen after in vitro or in vivo stimulation. They found that stimulation of spleen cells with the superantigen Staphylococcal Enterotoxin B (SEB) in the presence of IL4 promoted production of IL10 and IFNγ predominately by CD8⁺ cells. In contrast, the production of IL4 was almost exclusively confined to the CD4⁺ subset. When priming with SEB in vivo before subsequent restimulation in vitro , a protocol previously shown to induce anergy, up to 80% of the IL10 and IFNγ positive cell expressed the CD8 marker. Taken together, these results emphasize the important role of cytokine-producing CD8⁺ cells and indicate that CD4⁺ and CD8⁺ T cells may, in a given situation, produce distinct cytokines.     

Population Dynamics of CD4+ T Cells Lacking Thy-1 in Murine Retrovirus-Induced Immunodeficiency Syndrome (MAIDS)

Increased numbers of CD4⁺ Thy-1 cells have been described in the spleen (SP) of mice with retrovirusinduced immunodeliciency (MAIDS). Since this phenotypic abnormality might have considerable functional importance, the expansion of the CD4⁺ Thy-1 subset in MAIDS was characterized further. CD4⁺ Thy-1⁻ and Thy-1⁺ T-cell is from infected mice expressed similar densities of CD3 and TCR γ/β. In contrast, the Thy-I⁻ subset was uniformly CD44^hi, even early in the disease when part of Thy-I⁺ cells were still CD44¹⁰. The emergence of CD4⁺ Thy-1⁻cells occurred first in SP and lymph nodes and was observed later in thymus. The important fraction ofCD4⁺ cells lacking Thy-1 normally present in Peyer's patches was only weakly modified. Despite the major expansion of the CD4⁺ Thy-1⁻ phenotype. the proliferating fraction was not higher in this subset than in CD4⁺ Thy-1⁺ cells from infeeted miee. Persistence after hydroxyurea administration was identical in both subsets, indicating similar mean cell lifespans. Taken together, these results show that the major expansion of CD4⁺ Thy-I⁻ T-cells in MAIDS is not ascribable solely to increased proliferation within this subset. Phenotypic analysis suggests that CD4⁺ Thy-I⁻ cells result from the differentiation of Thy-I⁺ cells induced by activation signals related to retroviral infection.     

Intrathymic maturation of CD4+ T-lymphocytes in an MHC class II deficient transplant model

Major histocompatibility complex (MHC) class II knockout (class II^-) mice fail to generate CD4⁺ CD8^- T-lymphocytes. We were interested in determining whether these class II^- mice could be reconstituted with CD4⁺ CD8^- T-lymphocytes following marrow transplantation from normal (class II⁺) donors. Transplantation of class II⁺ marrow into lethally irradiated class II^- recipients failed to generate peripheral CD4⁺ CD8^- T-lymphocytes. Unexpectedly, however, transplantation of class II^- marrow into class II⁺ recipients also resulted in a deficiency of CD4⁺ CD8^- cells. Analysis of intrathymic T cells showed normal distribution of CD4 and CD8 single and double positive or negative thymocytes in normal recipients, while class II^- recipients always lacked CD4⁺ CD8^- T cells intrathymically. These results suggest, therefore, that T-cell maturation in mice requires the presence of MHC class II antigens not only in the thymus but also on immature, marrow-derived pre-thymocytes.     

CD4/CD8 lineage commitment: matching fate with competence

Summary: The outcome of positive selection of T lymphocytes is that there is a close match between the lineage adopted by a particular cell (CD4⁺ or CD8⁺) and the specificity of the T-cell receptor for the class of Major Histocompatibility Complex molecule recognized. How this match is obtained has been a matter of debate. We review the evidence, from recent and older experiments, that indicates that the process follows a selective logic, rather than an instructive one.