CD5+B Cells: Differential Capping and Modulation of IgM and CD5

CD5 is associated with the B-cell antigen receptor (BcR) complex. As an approach to understanding its role in B-cell function, the authors investigated the capping and modulation of CD5 and surface IgM (sIgM). Tonsillar B cells were treated withanti-IgM or anti-CD5 antibodies, capping examined after 1 h (by fluorescence microscopy) and modulation after 24 h (by flow cytometry). CD5 co-capped and co-modulated with sIgM. Of various drugs tested, only the protein tyrosine kinaseinhibitor (genistein) had any effect on capping and co-capping. Capping of sIgM (and co-capping of CD5) but not capping of CD5 (or co-capping of sIgM) was inhibited by genistein. None of the other drugs affecting PKC or cytoskeletal structures (colchicine and cytochalasin D) had any effect. However, the PKC inhibitors, staurosporine and H-7, inhibited the modulation of sIgM by anti-IgM but not CD5 by anti-CD5. In contrast, PKC activators, PMA and mezerein, inhibited modulation of CD5 by anti-CD5 but notsIgM by anti-IgM. This suggests that direct ligation of CD5 utilizes different signalling pathways compared with sIgM. It seems likely that in CD5⁺ B cells, interaction of CD5 with its ligand CD72 modulates signals transmitted through theBcR.     

Preferential Localization of Human CD5+ B Cells in the Peritoneal Cavity

In a transgenic mouse model of autoimmune haemolytic anaemia, CD5⁺ B lymphocytes localized in the peritoneal cavity are shown to play an important role in the onset of autoimmune disease. The authors have examined whether CD5⁺ B cells are present in the peritoneal cavity of 12 human individuals with non-invasive gastrointestinal tumours and found that in humans CD5⁺ B cells preferentially lodge in the peritoneal cavity as compared to the peripheral blood and spleen while the numbers of the peritoneal B lymphocytes in humans are much lower than in mice and vary widely between individuals.     

Selective Killing of CD4+ and CD8+ Cells with Immunotoxins Containing Saporin

Saporin, a ribosome-inactivating protein from the seeds of Saponaria officinalis, was covalently linked to an anti-CD4 monoclonal antibody. The resulting immunotoxin at 10(-9)M concentration was toxic to CD4+ lymphocytes without affecting other cells. Selective elimination of CD4+ and CD8+ cells was also obtained with murine monoclonal anti-CD4 and anti-CD8 antibodies and an immunotoxin consisting of saporin linked to an anti-mouse IgG antibody.     

Activation of Resting, Pure CD4+, and CD8+ Cells via CD3

We studied the requirements for secondary activation signals in pure CD4+ and CD8+ T cells after stimulation with anti-CD3 antibodies. Stimulation of CD4+ or CD8+ cells with anti-CD3 monoclonal antibodies (MoAb) bound to polystyrene monosized particles never resulted in a proliferative response. However, DNA synthesis was observed when recombinant interleukin 2 (IL-2) or other secondary signals, such as those provided by phorbol myristate acetate (PMA) or autologous accessory cells (AC), were also added. These secondary signals were not in themselves capable of inducing DNA synthesis in the absence of particle-bound anti-CD3. We also found that the signals provided by AC may be dependent on the activation state of these cells. Thus, the effects of accessory cells were enhanced by a factor present in fetal calf serum (FCS), most likely endotoxin or lipopolysaccharide (LPS), which alone, however, were not able to activate T cells, even in the presence of particle-bound anti-CD3. Recombinant IL-1 over a broad dose range was unable to replace PMA or activated AC after stimulation with particle-bound anti-CD3. Purified CD4+ and CD8+ T cells behaved identically in all the experiments, indicating that the basic mechanisms for activation in the two T-cell subsets are identical.     

Requirements for Phytohaemagglutinin Activation of Resting Pure CD4+ and CD8+ T Cells

We have utilized a new method for obtaining highly purified cells using positive selection by immunomagnetic separation to study the conditions required for phytohaemagglutinin (PHA) activation of pure T4 and T8 cells. In the presence of accessory cells (AC), a comparable proliferative response was obtained in the two subsets. In the absence of AC, PHA induced low levels of interleukin 2 (IL-2) receptor expression as well as responsiveness to IL-2 in both T4 and T8 cells. If AC or 12- O -tetradecanoyl-phorbol-13-acctate (TPA) were also present, IL-2 production and DNA synthesis were seen in both subsets. A short preincubation with PHA primed' T fells for subsequent responsiveness to IL-2 or TPA, while preincubation with TPA did not induce response to PHA. Thus, PHA alone is sufficient for the first step of T cell activation lending to IL-2 receptor expression. The second step leading to IL-2 production, is dependent on direct interaction with AC in the presence of PHA. While T8 cells are dependent on help by T4 cells for proliferation to occur during stimulation with antigen, in PHA stimulation the requirements for activation and proliferation seem to he identical for T4 and T8 cells.     

CD8+ T Cells Induce Medullary Thymic Epithelium and CD4+CD8+CD25+ TCRβ− Thymocytes in SCID Mice

During T-cell development the transition in the thymus of CD4-CD8- double negative (DN) progenitor T cells into CD4+CD8+ double positive (DP) cells is dependent on the expression of a T-cell receptor (TCR)-beta-chain protein. In this study purified peripheral CD4+ and CD8+ T lymphocytes from the C.B-17 strain of mice were adoptively transferred into syngeneic, neonatal SCID mice, where donor cells resided at constant numbers in thymus from 2 weeks until 10 weeks post cell transfer. In the recipient thymus the CD8+ donor cells outnumbered the CD4+ cells by a factor of three to five and both subsets contained a large fraction of activated cells. During the late phase of treatment, CD8+ T cells induced high numbers of DP thymocytes in the SCID mice, a process accompanied by the maturation of medullary epithelial cells. Such thymic development in the SCID mouse was inhibited by coresiding CD4+ donor T cells. These results indicate a regulatory role by mature peripheral T cells on medullary epithelial growth and thymocyte development in the treated SCID mice.     

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.     

Phenotypical and Functional Differentiation of CD4+ CD45RA+ Human T Cells Following Polyclonal Activation

Human CD4+ T cells differ in their expression of the leucocyte common antigen. Antibodies detecting certain forms (CD45RA and CD45RO) of this antigen have been used to identify and isolate subpopulations of the CD4+ T cells. These isolated subsets have been shown to have different abilities concerning lymphokine production and provision of help to B cells for Ig production. When these T-cell subsets were activated in vitro with polyclonal activators, the production. When these T-cell subsets were activated in vitro with polyclonal activators, the CD45RA+ cells lost this marker and gained the expression of CD45RO. This was true for all mitogens used in this report, i.e. accessory cell-dependent stimulation with SEA and accessory cell-independent activation with PMA or PHA. A correlation between proliferation and differentiation was observed, but this was probably not causative as stimulation with PMA in the absence of DNA synthesis resulted in the acquisition of CD45RO and loss of the CD45RA antigen. Moreover, cells proliferating vigorously for long periods of time expressed both markers at significant levels, which suggests that proliferation did not automatically result in complete loss of the CD45RA marker. The phenotypical differentiation was associated with a functional differentiation which induced the stimulated cells' ability to act as helper cells for Ig production and to produce gamma interferon (IFN-gamma). The results obtained in this study support the contention that the CD45RA+ cells are precursors of the CD45RO+ cells and that the two subsets represent different maturational stages of the same lineage.     

CD25 Shedding by Human Natural Occurring CD4+CD25+ Regulatory T Cells does not Inhibit the Action of IL-2

Regulatory T (Treg) cells are important for the maintenance of peripheral tolerance and inhibition of pathogenic T-cell responses. Therefore, they are important for the limitation of chronic inflammation but can also be deleterious by e.g. limiting antitumour immune responses. Natural occurring Tregs are known to inhibit CD4⁺ T cell in a contact-dependent manner, but at the same time, various suppressive factors are secreted. We, here, demonstrate that human naturally occurring CD4⁺CD25⁺ Tregs are able to shed large amounts of soluble CD25 upon activation. Secretion of sCD25 could add to the inhibitory effect of Tregs as such secretion in other settings has been proposed to act as a sink for local IL-2. However, we here demonstrate that supernatant from human Tregs containing high concentration of sCD25 does not inhibit proliferation of CD4⁺CD25⁻ T cells or inhibit the action of IL-2 in an in vitro bioassay.     

Auto- and Polyreactivity of IgM from CD5+ and CD5− Cord Blood B Cells

The presence of the CD5 (67 kDa) molecule on the surface of B cells has been considered a marker for cells producing auto- and polyreactive antibodies. Cord blood B lymphocytes (rich in CD5+ B cells) have been sorted into CD5 positive and negative populations by flow cytometry using monoclonal antibodies to CD20 and CD5. Clones of these populations were obtained by immortalization with Epstein-Barr virus. Clones derived from both CD5+ and CD5- B cells produced IgM which was auto- and polyreactive with a higher frequency of these specificities in the CD5+ population. These data indicate that expression of surface CD5 on cord blood B cells is not a definitive marker of an auto/polyreactive population.     

Distinctive Developmental Origins and Specificities of Murine CD5+ B Cells

CD5⁺ B cells constitute a small fraction of cells in the spleen of adult mice that exhibit numerous features serving to distinguish them from the bulk of IgD⁺⁺CD5^?“conventional” B cells. In this review we focus on two major questions relating to this population: 1) the relationship of CD5⁺ B cells to other B cells; and 2) the distinctive enrichment of particular autoreactive specificities in this subset. The nature of their origins is clarified by a thorough analysis of intermediate stages of early B-cell development in both fetal and adult tissues. The reactivity to bromeliad-treated mouse red blood cells serves as a prototype system for the investigation of biased specificities in CD5⁺ B cells. These lines of investigation lead us to propose that CD5⁺ B cells in the adult are the remnant of a distinct fetal B-cell differentiation pathway wherein selection of cells from this fetal/neonatal population into the adult long-lived pool results in the over-expression of certain germline-encoded autoreactivities.     

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.     

Activation of Cloned Human CD4+ Th1 and Th2 Cells by Blood Dendritic Cells

Dendritic cells (DC) have been reported to be the most potent antigen-presenting cells (APC) for the activation of naive T cells and to be 10–100-fold more potent APC than monocytes (Mφ) in the mixed lymphocyte reaction. In this study the authors compared human blood DC with Mφ and B cells for their ability to activate cloned rye grass allergen Lol p I specific CD4⁺ Th₁ and Th₂ cells. In the presence of Lol  p I, all three types of APC activated Th₁ and Th₂ cells to a similar extent, as shown by T-cell proliferation and interferon-γ, interleukin-2 (IL-2) or IL-4 secretion. However, at low APC : T cell ratios, Mφ were the most potent APC for both Th₁ and Th₂ cells followed in decreasing order by DC and B cells. This hierarchy was observed with APC preparations isolated by negative selection or highly purified by positive selection using fluorescent cell sorting for HLA-DR^high-DC, CD14^pos-Mφ and CD19^pos-B cells. The data demonstrate that, in contrast to what has been reported for naive T cells, human blood DC activate cloned memory Th₁ and Th₂ cells to a similar extent as Mφ and B cells presumably because the requirements for activation of memory type T cells are less stringent than those for naive T cells.     

The lifestyle of naturally occurring CD4+CD25+Foxp3+ regulatory T cells

Summary: Numerous studies over the past 10 years have demonstrated the importance of naturally occurring CD4⁺CD25⁺Foxp3⁺ regulatory T cells (nTregs) in immune regulation. We analyzed the mechanism of action of nTregs in a well‐characterized model of autoimmune gastritis and demonstrated that nTregs act at an early stage of disease progression to inhibit the differentiation of naïve T cells to pathogenic T‐helper 1 effectors. The effects of nTregs in this model are not antigen‐specific but are mediated by activation of the nTregs by ubiquitous self‐peptide major histocompatibility complex class II complexes together with cytokines released by activated effector cells. Studies in vitro confirmed that some nTregs exist in an activated state in vivo and can be activated to exert non‐specific suppressor effector function by stimulation with interleukin‐2 in the absence of engagement of their T‐cell receptor. Natural Tregs can differentiate in vitro to exhibit potent granzyme B‐dependent, partially perforin‐independent cytotoxic cells that are capable of specifically killing antigen‐presenting B cells. Natural Treg‐mediated killing of antigen‐presenting cells may represent one pathway by which they can induce long‐lasting suppression of autoimmune disease.     

Linomide Enhances Apoptosis in CD4+CD8+ Thymocytes

Linomide, a quinoline-3-carboxamide, has a pleiotropic immune modulating capacity and inhibits development as well as progression of disease in animal models of autoimmunity. Linomide treatment of mice resulted in a dramatic, dose-dependent decrease of the thymic cell number shortly after the start of administration. Flow cytometric analysis revealed that the major thymocyte subset, the early immature type CD4⁺CD8⁺ thymocytes, were reduced in number by 75%, mature CD4⁺CD8^? or CD4^?CD8⁺ thymocytes were less sensitive to treatment. The polyclonal T cell activator Con A (Concanavalin A) was used together with IL-2 to evaluate the potential proliferative responsiveness of ex vivo thymocytes. Thymocytes from mice treated with Linomide exhibited a more vigorous proliferation than control cultures. An effect shown to not only be due to the enrichment of mature thymocytes in the cultures from Linomide treated animals, but also when purified, mature thymocytes (CD4⁺CD8^? and CD4^?CD8⁺) were cultured with Con A and IL-2, these cells responded with a significantly enhanced proliferation. In vivo Linomide treatment did not result in increased plasma concentrations of corticosterone and treatment of adrenalectomized mice resulted in a reduction of thymocytes which was comparable to the effect in intact mice, indicating that glucocorticoids (GC) are not major mediators of Linomide-induced thymocyte deletion. In addition to this, and supporting a glucocorticoid independent mode of action, Linomide treatment of thymocytes in vitro resulted in a significant increase in the number of apoptotic cells, specifically in the CD4⁺CD8⁺ subset, implicating apopotosis as one component in the course of thymocyte reduction. In addition to this, in vivo treatment with Linomide resulted in an identical pattern to that seen in vitro in that there was significantly increased apoptosis only in the CD4⁺CD8⁺. These data indicate that Linomide modifies thymocyte development using a glucocorticoid independent pathway and results in the increased apoptosis of the CD4⁺CD8⁺ subset.     

Preferential Interaction of the CD4+29+/45RA- Subset of Human CD4+ T Lymphocytes with an Antibody against the Cell-Membrane Ganglioside GD3

This study shows that the two major subpopulations of CD4+ T lymphocytes defined on the basis of differential expression of the CD29 and CD45RA antigens, show significant differences in reactivity with a monoclonal antibody against the GD3 ganglioside. Double staining studies showed that the GD3 ganglioside is predominantly expressed on cells from the CD4+29+/45RA subsets. The preferential interaction of the CD4+29+/45RA cell subset with the anti-GD3 MoAb was further confirmed by the proliferative and calcium-flux studies. Accordingly, the reciprocal, CD4+(29-)/45RA+ subset was unable to proliferate in response to the anti-GD3 MoAb alone. Although it did show a significant mobilization of calcium ions and proliferation to IL-2 when stimulated with the anti-GD3 MoAb, these response were much less pronounced than the responses of the CD4+29+/45RA- subset. Finally, when two T-cell stimulating monokines, IL-1 and IL-6, were tested for the ability to modulate the anti-GD3 mediated proliferation, only the former, but not the latter was able to enhance the proliferation. Although the natural ligand for the GD3 ganglioside remains unknown, the data presented here provide further evidence in support of the notion that the T-cell surface molecules different from the T-cell receptor MHC-antigen complex may contribute to the preferential activation of one of the CD4+ T lymphocyte subsets.     

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.     

Ability of Pure Resting CD8+ Human T Cells to Respond to Alloantigen

The ability of highly purified resting human CD8 cells to respond to alloantigens in vitro was examined. Necessary conditions for induction of interleukin 2 receptors (IL-2R), IL-2 production, proliferative responses, and various effector functions were determined. Allogeneic non-T cells induced IL-2R expression in a high proportion of resting CD4 and CD8 cells, but only CD4 cells produced detectable amounts of IL-2. CD8 cells also became IL-2 responsive upon stimulation with purified resting allogeneic CD4 or CD8 cells, indicating that HLA class I+, II- cells alone may initiate activation of resting CD8 cells. The activated CD8 cells needed the presence of simultaneously activated CD4 cells or exogenous IL-2 to be able to synthesize DNA. Effector functions like cytotoxicity, mixed lymphocyte culture (MLC) suppression, or gamma interferon (IFN-gamma) production were also only detectable when the CD8 cells were activated in the presence of IL-2.