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.     

Frequency of Chicken CD4+ and CD8+ Cells. Genetic Control and Effect of Rous Sarcoma Virus Infection

In chickens from congenic inbred lines CB and CC that differ only in the major histocompatibility complex (MHC), we observed significantly different percentages of CD4+ and CD8+ cells in peripheral blood lymphocytes (PBL) and spleen. Positive cells were detected by indirect immunofluorescence test as analysed by flow cytometry. In both PBL and spleen cell suspensions, the number of CD4+ cells was significantly higher in CB than in CC chickens, whereas in CC birds there was a higher percentage of CD8+ cells than in CB. These statistically significant differences were under the MHC control. We found no statistically significant influence of regressions or progression of Rous sarcoma virus-induced tumours on the percentage of peripheral T cells and on the interleukin-2 production in vitro.     

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.     

Remarkable Depression of CD4+2H4+ T Cells in Severe Chronic Active Epstein-Barr Virus Infection

In order to better understand the features of chronic active Epstein-Barr (EB) virus infection, we employed two-colour immunofluorescence staining with monoclonal antibodies and flow cytometry analysis to study the lymphocyte phenotypes of two patients with severe symptoms of this disorder as well as four patients with mild symptoms. We found an increased number of activated T cells, as characterized by CD4+Ia+, CD8+Ia+, or CD4+Tac+ phenotypes, and a markedly decreased CD4+2H4+ T cell subpopulation, previously characterized as a suppressor-inducer subset, in the patients with severe symptoms. In contrast, the four patients with mild symptoms showed only a slightly elevated number of activated T cells and a normal CD4+2H4+/CD4+ ratio. These phenotypic differences may suggest heterogeneity in this disorder. Also, a failure in the suppressor-inducer population could contribute to changes in the host-virus relationship and the degree of the decrease in this population may correlate directly with the severity of the disease.     

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.     

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.     

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.     

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.     

The Therapeutic Effect of Vasoactive Intestinal Peptide on Experimental Arthritis is Associated with CD4+CD25+ T Regulatory Cells

Vasoactive intestinal peptide (VIP) has been found to act as a potent anti-inflammatory factor through regulating the production of both anti- and pro-inflammatory mediators and promoting Th2-type responses. In this study, we used Chicken collagen II-induced experimental arthritis (CIA) model in Wistar rats to investigate the potential effects of VIP on rheumatoid arthritis. Our results showed that in vivo treatment of CIA-induced rats with VIP had great protective benefit at both clinical and histological levels. Disease suppression was associated with the inhibition of T cells proliferation, shifting of the immune response toward a Th2-type response and expanded CD4⁺CD25⁺ Treg in the periphery, which inhibited autoreactive T cell activation/expansion. In conclusion, the study provides evidence that VIP had great protective effect on CIA through its inhibition actions on pathogenic T cells.     

CD4+, CD8+, and CD4− CD8− T Cells in CSF and Blood of Patients with Multiple Sclerosis and Tension Headache

Two-colour flow cytometric analysis was performed on paired samples of peripheral blood (PB) and cerebrospinal fluid (CSF) of patients with untreated multiple sclerosis (MS) and, for reference, subjects with muscular tension headache (TH) using anti-CD3, anti-CD4, anti-CD8, and anti-HLA-DR monoclonal antibodies in different combinations. CD4+/CD8+ T-cell ratio was increased in CSF compared to PB in both MS patients and TH subjects to a similar extent. This was mainly due to higher CD4+ T-cell levels in the CSF compartment. The proportion of HLA-DR+ T cells was higher in CSF than PB in both MS and TH; this increase of DR+ T cells in CSF was more prominent in MS. The level of CD4+ CD8+ T cells, which represent a subset of activated T cells, was not different between CSF and PB, either in MS or in TH. The proportion of CD4- CD8- T cells, which were found generally not to be blast cells, was lower in CSF compared to PB in both patient groups. However, their CSF level was higher and their PB level lower in MS compared to TH. Results point to an accumulation of activated T-helper cells in the CSF of both MS patients and healthy subjects. Fetal-type CD4- CD8- T cells bearing the unusual T-cell receptor gamma/delta seem to be selectively recruited to the CSF of MS patients.     

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.     

Purified Human T Cells Stimulated with Cross-Linked Anti-CD3 Monoclonal Antibody OKT3: rIL-1 is a Co-Stimulatory Factor for CD4+ CD29+ CD45RA− T Cells

Accessory cells (AC) are believed to play two major roles in T-cell activation: they cross-link certain stimuli such as monoclonal antibodies, and they provide needed cytokines. To differentiate between these roles, we cross-linked OKT3 on highly purified T cells by means of Fc-specific goat anti-mouse IgG-coated polystyrene beads and studied T-cell activation after exogenously added cytokines. Following addition of AC, rIL-2, or rIL-1, CD25 was up-regulated, and the cells proliferated and became cytotoxic. Both CD4+ and CD8+ cells were activated in the presence of AC or rIL-2. In contrast, only CD4+CD29+CD45RA- cells responded in the presence of rIL-1. Anti-IL-2R p55 (anti-TAC) monoclonal antibody inhibited the proliferative response supported by rIL-2 or rIL-1. To inhibit proliferation of cells stimulated in the presence of AC, anti-TAC needed to be supplemented with anti-IL-6 antibodies, or to be added in a 10-fold higher concentration. Cultures with AC produced larger amounts of IL-2 than those supplemented with rIL-1. Only AC-containing cultures also produced detectable amounts of IL-6. These findings combined with the observation that none of 2000 purified T cells counted in each of six independent experiments expressed MHC class II antigens strongly suggest that rIL-1 can activate T cells directly, rather than indirectly by potentiating the function of contaminating AC.     

Specific Interferon-Gamma Producing CD4+ and CD8+ T Cells After Autologous EBV-B Stimulation: The Necessity of Restimulation

The response of T cells to produce interferon-γ, to proliferate and to become cytotoxic after specific stimulation with low dose (2%) autologous EBV-B cells was investigated in 15 EBV seropositive and five seronegative patients. A significantly higher number of interferon-γ producing cells (56 ± 24 per 10⁵ T cells) were found in a spot ELISA in EBV positive than in EBV negative patients (7 ± 2 spots, P<0.01) and it was only found with restimulation after 5–12 days of primary culture. No correlation was found between the extent of interferon-γ production, cytotoxicity or profileration. Specificity of EBV-induced interferon-γ production was demonstrated by comparison of the response to allogeneic EBV-B cells or IL-2 in the reslimulation phase. The response was stronger in CDS⁺ T cells than in CD4⁺ T cells and could be blocked in the restimulation phase with HLA class I and class II antiserum, respectively.     

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.     

Accumulation of CD8+ Cells after Immunization with Soluble Antigen

Rats were immunized with ovalbumin, either subcutaneously or by aerosol inhalation. The lymphocyte distribution in lymph nodes, peripheral blood, and spleen was investigated by flow cytometry after labelling with T pan (OX19 and W3/13), T helper lymphocytes (W3/25), T cytotoxic/suppressor lymphocytes (OX8), kappa light chain (MAR 18-5), or MHC class II (OX6) monoclonal antibodies. The influence of the neurotoxic agent capsaicin on the lymphocyte distribution was also analysed. Subcutaneous immunization resulted in an increased number of OX8+ cells in mesenteric lymph nodes, spleen, and peripheral blood but not in the draining lymph nodes, axillary, brachial, and mediastinal lymph nodes. The number of positive cells for the other cell markers used were not affected by immunization. The neuromodulatory effect of capsaicin had no effect on the lymphocyte distribution. The results showed that the type of immunization used, low amounts of antigen without adjuvant given during a prolonged period, selectively induced OX8+ cells. The patterns were unaffected by neuromodulation using capsaicin.     

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.     

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.