Migration and differentiation of CD8+ T cells

Summary: Antigen-specific responses by CD8⁺ T cells require direct cell–cell interactions between T cells and antigen-presenting cells (APC). Initially, naïve T cells must communicate with APC in lymphoid organs. Once stimulated, the resulting effector cells interact with APC in peripheral tissues. To this end, T cells must migrate to discrete sites throughout the body where antigen may be found. Recent progress in the field has revealed that the migratory abilities of T cells are critically dependent on their differentiation state, which is shaped by a multitude of factors. Thus, naïve T cells are normally restricted to recirculate between the blood and secondary lymphoid tissues, although in some autoimmune diseases they may also accumulate in chronically inflamed tissues. When CD8⁺ T cells encounter antigen and differentiate into short-lived effector CTL, they lose the ability to home to lymph nodes but gain access to peripheral tissues and sites of inflammation. Long-lived memory cells exist in (at least) two flavors: central memory cells that migrate to both lymphoid organs and peripheral sites of inflammation, and effector memory cells that are preferentially localized in non-lymphoid tissues. Our current understanding of the interplay of T cell differentiation and migration has been boosted by the development of T-GFP mice, in which transgenic green fluorescent protein is expressed selectively in naïve and central memory T cells, but not in effector cytotoxic T cells (CTL). This review will focus on recent studies in which T-GFP mice were used to dissect the traffic signals for naïve T cell homing to secondary lymphoid organs, the factors that influence the differentiation of naïve CD8⁺ T cells into cytotoxic and memory cells, as well as the in vivo trafficking routes of antigen-experienced subsets.     

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.     

JNK and p38 MAP kinases in CD4+ and CD8+ T cells

Summary: The c‐Jun aminoterminal kinase (JNK) and p38 mitogen‐activated protein (MAP) kinase signaling pathways have been associated with cell death, differentiation and proliferation. CD4⁺ and CD8⁺ T cells have different effector functions after antigen stimulation and control specific aspects of the immune response. The studies carried out in our group indicate that the role of JNK and p38 MAP kinases in CD4⁺ T cells is different from their role in CD8⁺ T cells. Moreover, these two pathways are not redundant in either T cell population. We have also shown that p38 MAP kinase regulates early stages of T cell development in the thymus. It is therefore important to consider the specific function of these kinases in each T cell population when pharmacological inhibitors of JNK and p38 MAP kinases are used for therapeutic purposes to control the immune response.     

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.     

Allergen-induced cytokine phenotypes in mice: role of CD4+ and CD8+ T cell populations

BACKGROUND: CD4+ T cells expressing type 2 cytokines have been implicated in the pathogenesis of asthma to high-molecular-weight allergens. Topical exposure of BALB/c strain mice to low-molecular-weight chemical contact and respiratory allergens stimulates type 1 and type 2 cytokine secretion phenotypes, respectively. OBJECTIVE: To examine the relative frequencies of cytokine-positive CD4+ and CD8+ T cells and their contributions to these cytokine secretion profiles. Methods Draining auricular lymph nodes were isolated 13 days after initiation of topical exposure of female BALB/c strain mice to chemical allergen, or to vehicle alone. The frequency of intracellular cytokine (IL-4 and IFN-gamma)-positive CD4+ and CD8+ lymphocytes was enumerated by flow cytometry. The relative contribution of CD4+ and CD8+ cells to cytokine secretion profiles was assessed by negative selection. RESULTS: Exposure to allergen resulted in an increased frequency of both IFN-gamma+ CD4+ and CD8+ lymphocytes, although there were no marked differences between trimellitic anhydride (TMA)- and 2,4-dinitrochlorobenzene (DNCB)-activated lymph node cells. Treatment with TMA induced approximately five times as many IL-4+ CD4+ cells as did exposure to DNCB. This pattern of cytokine staining was also observed for a further pair of contact and respiratory allergens; respectively, formalin and fluorescein isothiocyanate. CONCLUSION: These data demonstrate that the divergent immune responses induced in mice by different classes of chemical allergen are independent of changes in the frequency of IFN-gamma+ cells, but are associated with differential frequencies of IL-4-expressing CD4+ T cells.     

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.     

The role of CD45+CD4+CD3– cells in lymphoid organ development

Summary: In the last 10 years the continuing search for gene function has yielded many mutant mice that unexpectedly showed a complete lack of lymph nodes and/or Peyer's patches. With the realization that all these functionally highly diverse genes are involved at some point in the development of lymphoid organs, the challenge now is to assign a function to the molecules involved in lymphoid organ development. It will be important to determine the sequence of molecular events and assign this to the cellular events that lead to an accumulation of hematopoietic cells in one location, ultimately forming an organized lymphoid organ. Here we will focus on CD45⁺CD4⁺CD3^– cells that are the early colonizing cells in lymph nodes and Peyer's patches and develop a hypothetical model of their contribution to the creation of organized lymphoid structures.     

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.     

CD4+ and CD8+ Distribution Profile in Individuals Infected by Schistosoma mansoni

Rationale: Patients with chronic Schistosoma mansoni infection show lower anti‐soluble egg antigen (SEA) proliferation responses and higher responses to soluble worm antigen preparation (SWAP). Objective: To compare the activation status and proliferation response of peripheral blood mononuclear cells (PBMC) of infected (XTO) and egg‐negative individuals (NI) living in the same endemic area. Methods: XTO (n = 51) and NI individuals from the same geographical area (n = 37) and healthy blood donors (n = 22) were evaluated before and after stimulation with SEA and SWAP. The expression of activation markers (CD4⁺ HLADR⁺, CD8^high+HLA‐DR⁺ and CD8⁺ CD28⁺) and proliferation assay was assessed by flow cytometry. Findings: PBMC from infected patients showed lower frequency of CD4⁺ but no change in CD8⁺ T cells when compared with the healthy donor group. The ratio CD4⁺/CD8⁺ was 1.3, 0.6 and 0.5 in healthy donors, infected and non‐infected individuals, respectively. The HLA‐DR⁺ expression on CD8⁺ was higher in PBMC from infected and non‐infected individuals than from healthy donors, but similar in both total lymphocytes and CD4⁺ populations. No intergroup proliferation response differences were observed in CD4⁺ and CD8⁺ PBMC unstimulated and stimulated with SEA and SWAP. The SEA but not SWAP‐stimulated cells showed a decrease in the expression of phosphorylated extracellular signal‐regulated kinase (ERK1/2). Conclusions: XTO and NI individuals living in the same area presented a smaller per cent of CD4⁺ and a higher per cent of CD8⁺ cells. The activation by either CD8^high+HLA‐DR⁺ or CD8^high+HLA‐DR⁺/CD8⁺ was enhanced and decreased in XTO and NI by CD8⁺ CD28⁺ and CD8⁺ CD28⁺/CD8⁺ when compared with healthy donor. ERK phosphorylation was attenuated in XTO and NI individuals when stimulated with SEA but not SWAP.     

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.     

CD4+ memory T cells: functional differentiation and homeostasis

Summary: CD4⁺ T cells are central regulators of both humoral and cellular immune responses. There are many subsets of CD4⁺ T cells, the most prominent being T‐helper 1 (Th1), Th2, Th‐17, and regulatory T cells, specialized in regulating different aspects of immunity. Without participation by these CD4⁺ T‐cell subsets, B cells cannot undergo isotype switching to generate high‐affinity antibodies, the microbicidal activity of macrophages is reduced, the efficiency of CD8⁺ T‐cell responses and CD8⁺ T‐cell memory are compromised, and downregulation of effector responses is impaired. It therefore stands to reason that memory CD4⁺ T cells are likely to fulfill an important facilitator role in the maintenance and control of protective immune responses. This review discusses some issues of importance for the generation of memory CD4⁺ T cells and focuses in particular on their heterogeneity and plasticity, with respect to both phenotypic characteristics and function. Finally, we discuss a number of factors that affect long‐term maintenance of memory CD4⁺ T cells.     

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.     

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.     

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.     

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.     

Cyclic nucleotide phosphodiesterases from purified human CD4+ and CD8+ T lymphocytes

Background CD4⁺ and CD8⁺ T-lymphocytes are suggested to differentially affect airway inflammation in asthma. Agents which increase intracellular cAMP levels, such as PDE inhibitors, have been shown to diminish lymphocyte growth and differentiation, and to affect cytokine expression. Differences in the PDE isoenzyme profile between CD4⁺ and CD8⁺ cells might form a basis to differentially modify their functions by PDE inhibitors. Objective The study investigates and compares the PDE isoenzyme activity profiles of human peripheral blood CD4⁺ and CD8⁺ T-lymphocytes. Methods CD4⁺ and CD8⁺ T-lymphocytes were purified (>98%) from peripheral blood mononuclear cells by negative selection. PDE isoenzyme activity profiles were investigated using PDE isoenzyme selective inhibitors and activators. Results In CD4⁺ and CD8+ T-lymphocyte homogenates, PDE IV and PDE III activities were the predominant PDE isoenzyme activities at 0.5μM cyclic nucleotide substrate concentrations. PDE IV was localized in the soluble fraction whereas PDE III was membrane bound. Low PDE I, II and V activities were detected. About 20% of total eAMP hydrolysing capacity at 0.5 μM cAMP was insensitive to PDE isoenzyme selective inhibitors and activators and therefore could not be assigned to PDE I-IV. The PDE isoenzyme pattern was not different between CD4⁺ and CDS⁺ T-lymphocytes. Moreover, representative inhibitors of PDE HI and IV activity inhibited cAMP hydrolysis in soluble fractions of both T-lymphocyte subsets with similar potency. Enzyme kinetic analysis similarly did not reveal differences between CD4^h and CD8⁺ T-lymphocytes. Conclusion Normal CD4⁺ and CD8⁺ T-lymphocytes are likely to be equally sensitive targets for the effects of PDE inhibitors.