CD4− CD8− C.B-17 SCID Thymocytes Enter the CD4+ CD8+ Stage in the Presence of Neonatally Grafted T Cells

The aim of this work was to study the selection of donor T cells and their influence on thymic development in C.B-17 scid/scid (severe combined immunodeficient; SCID) mice during chronic graft-versus-host disease (GVHD). Recipient SCID mice (H-2^d), neonatally grafted with allogeneic peripheral T cells from CBA/J strain (H-2^k) of mice, only developed a mild acute GVHD, and were, at the chronic stage, devoid of pathological symptoms. Thymic cell numbers of injected mice differed from 10⁵ to 1.2 × 10⁷ at 2–3 weeks post-injection (p.i.), and from 4 × 10⁵ to 8.5 × 10⁷ at 2 months p.i. In these mice, the thymus size was correlated to the CD4^? CD8^? (double negative; DN) to CD4⁺ CD8⁺ (double positive; DP) cell ratio, where at 2 months p.i, 8 out of 16 treated SCID mice contained 5 × 10⁶ cells or more and also possessed the highest frequencies of endogenous DP cells (25–95%). In contrast to previous findings, peripheral donor T cells from allogeneic and syngeneic mice, infiltrating the host thymus, had a positive effect on the development of endogenous DP thymocytes. Furthermore, these thymocytes were developmentally blocked at the DP stage, occasionally in combination with the expression of CD25, CD44 and CD117 but in the absence of T-cell receptor (TCR) expression. Also, at this time-point, the CBA/J donor TCR Vβ repertoire was equal to that of normal CBA/J mice, but purified responding donor cells were proliferatively inhibited against H-2^d stimulators in ex vivo mixed lymphocyte cultures. In contrast, the same responders showed a pronounced proliferation against syngeneic H-2K^k stimulators, suggesting either a reversion from anergy of autoreactive CBA/J T cells or a vast expansion of multiple self-reactive T-cell clones, when parked in a milieu with a lower concentration of self-antigens.     

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.     

Thymic differentiation of TCRαβ+ CD8αα+ IELs

Summary:  Intraepithelial lymphocytes (IELs) contain several subsets, but the origin of the T-cell receptor (TCR)αβ⁺ CD8αα⁺ IELs has been particularly controversial. Here we provide a synthesis, based on recent work, that attempts to unify the divergent views. The intestine has a primordial function in lymphopoiesis, and precursors with the potential to differentiate into T cells are found both in the epithelium and underlying lamina propria. Moreover, the thymus has been reported to export cells to the intestine that are not fully differentiated. TCRαβ⁺ CD8αα⁺ IELs can differentiate in the intestine from each of these sources, but in normal euthymic mice, the thymus appears to be the major source for TCRαβ⁺ CD8αα⁺ IELs. This unique IEL subset is a self-reactive population that requires exposure to self-agonists for selection in the thymus, similar to other regulatory T-cell populations. IELs transition through a double-positive (DP) intermediate in the thymus, but they originate from a subset of the DP cells that can be identified by its expression of CD8αα homodimers. The agonist-selected cells in the thymus are TCRβ⁺ but CD4 and CD8 double negative. The evidence suggests that reacquired expression of CD8αα and downregulation of CD5 occur after thymus export, perhaps in the intestine under the influence of interleukin-15. As a result of agonist exposure, a new gene expression program is activated. Therefore, the increased understanding of the developmental origin of TCRαβ⁺ CD8αα⁺ IELs may help us to understand how they participate in immune regulation and protection in the intestine.     

Acute Graft-versus-Host Reaction in SCID Mice Leads to an Abnormal Expansion of CD8+ Vβ14+ and a Broad Inactivation of Donor T Cells Followed by a Host-Restricted Tolerance and a Normalization of the TCR Vβ Repertoire in the Chronic Phase

The persistence and selection of allogeneic CBA/J T lymphocytes were studied during graft-versus-host (GvH) reaction in immunodeficient C. B-17 SCID (SCID) mice. After neonatal injection the donor cells primarily migrated to the spleen plus lymph nodes (SL) and the thymus of the recipients. Thirteen days post engraftment, CD8⁺ cells in SL had increased five times in cell number with an 18-fold increase of CD8⁺ Vβ14⁺ cells, paralleled by clinical signs of GvH disease (GvHD). Donor lymphocytes from these mice were proliferative unresponsive to allogeneic Balb/c or C57B1/6 SL cells, whereas 8 weeks post injection the tolerance was confined to H-2^d specific donor cells. Here, spleens had a total cell content similar to untreated SCID mice but the average percentage of donor cells had reached 25%. Moreover, the CD4/CD8 cell ratio in the donor population in SL and thymus had changed to normal and the TCR Vβ repertoire was similar to that of the originally injected cells. Following secondary transfer into syngeneic CBA/Ca nu/nu recipients donor cells regained a significant but reduced response to H-2^d stimulators indicating that the antigen specific tolerance of allogeneic donor cells in the SCID mice was due, at least in part, to a reversible state of anergy.     

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.     

Cyclosporine A Prevents the Generation of Single Positive (Lyt2+ L3T4−, Lyt2− L3T4+) Mature T Cells, but not Single Positive (Lyt2+ T3−) Immature Thymocytes, in Newborn Mice

The influence of cyclosporine A (CsA) on T-cell maturation was investigated in newborn mice. CsA treatment during the pre- and postnatal periods resulted in a hypoplasia of peripheral lymphatic organs, and absence of mature T3+ T cells in lymph nodes and spleens; no functional T-cell reactivity was observed. In thymuses of CsA-treated mice, no T3+ single positive Lyt2+ or T3+L3T4+ thymocytes could be found, but double positive (DP) cells were readily detected. A thymocyte subset with the phenotype Lyt2+L3T4-T3- was still discernible; this population was non-functional in vitro. The data show that the maturation of single positive (SP) T cells is critically influenced by CsA; under the conditions used here we found no evidence that 'leaky' autoreactive SP T cells develop in CsA-treated newborn mice.     

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.     

Induction of T ''regulatory'' cells by standardized house dust mite immunotherapy: an increase in CD4+CD25+ interleukin-10+ T cells expressing peripheral tissue trafficking markers

BACKGROUND: Clinically effective subcutaneous allergen-specific immunotherapy (SIT) is associated with altered circulating T cell cytokine production and altered local cytokine responses with increased IL-10 following allergen challenge in target organs. OBJECTIVE: This study aimed to elucidate mechanisms for these T cell changes, by examining surface expression of markers for peripheral tissue trafficking on circulating cytokine-positive T cells following standardized house dust mite- (HDM-) SIT. METHODS: A randomized conventional HDM immunotherapy study was performed on a panel of 12 HDM-allergic subjects. Nine subjects received treatment with conventional HDM immunotherapy using a standardized extract and three subjects were treated by standard pharmacotherapy alone. Symptom and medication scores and allergen-induced cutaneous late-phase responses were assessed before and 9 months after institution of therapy. Before and at 3 and 9 months of SIT, peripheral blood mononuclear cells were cultured for 14 days with HDM extract and CD4+ and CD8+ T cell expression of CD62L, CD49d and CCR5 and production of IL-10, IFN-gamma and IL-4 were analysed by flow cytometry. Allergen-specific T cell proliferation was assessed by 3H-thymidine incorporation. RESULTS: At 9 months, all SIT-treated patients showed reduced symptom scores and late-phase cutaneous responses to HDM compared with baseline levels. The proportions of CD4+ T cells which were IL-10+ were increased (P < 0.01), and the proportions of CD4+ and CD8+ T cells which were IL-4+ decreased (P < 0.05) compared with baseline. CD4+ and CD8+ T cell IFN-gamma production, expression of surface markers for peripheral tissue trafficking and allergen-specific proliferation remained unchanged during SIT treatment. However, increased proportions of CD4+CD62L(-), CD4+CD49d(hi), CD4+CCR5+ T cells expressing IL-10 were detected at 9 months of SIT compared with baseline (P < 0.05). IL-10 staining co-localized with CD4+CD25+ T cells. CONCLUSION: Clinically effective subcutaneous immunotherapy with a standardized HDM Dermatophagoides pteronyssinus preparation results in decreased numbers of IL-4+ T cells and expansion of CD4+IL-10+ T cells expressing a peripheral tissue trafficking phenotype. The co-localization of IL-10+ staining to CD4+CD25+ T cells is consistent with the induction of a T regulatory cell population by SIT.     

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.     

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.     

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.     

Murine Stress-triggered Abortion is Mediated by Increase of CD8+ TNF-α+ Decidual Cells via Substance P

PROBLEM: Stress is known to induce abortions in mice and humans. Increased levels of abortogenic type 1 helper T-cell cytokines and decreased levels of pregnancy protective cytokines could be linked to stress-triggered embryonic loss. Stress promotes neurotransmitter substance P (SP) release in tissues. SP increases the production of decidual tumor necrosis factor (TNF)-alpha, whereby the phenotype of these TNF-alpha-producing cells is hypothetical. The objective of the present study was to identify decidual TNF-alpha-producing cell populations that are involved in stress-induced murine abortion. METHOD: DBA/2J-mated CBA/J female mice were exposed to ultrasonic sound stress on day 5.5 of pregnancy. The mice were randomized and half were treated with the SP NK1-receptor antagonist (SP-RA) RP 67580 (200 microg/mouse). Frequency and cytokine profile of CD8+ cells were evaluated by immunohistochemistry and flow cytometry. Degranulation of uterine mast cells was examined histologically. RESULTS: On day 13.5 of pregnancy, the uteri were removed and the resorption rate was calculated. A mean resorption rate of 38.4% was detected in stressed mice (n = 10) compared to 13.1% in non-stressed control mice (n = 11, P < 0.01). Injection of SP-RA decreased the abortion rate to 18.4% in stressed mice (n = 19, P < 0.01). Flow cytometry revealed a stress-related increase of TNF-alpha+/CD8+ decidual T cells, which could be abrogated by SP-RA (P < 0.05). No significant differences could be observed in numbers of mast cells and total CD8+ cells in situ. CONCLUSION: Our data suggest that stress-triggered abortion is mediated by SP, and SP receptor blockade abrogates stress-triggered abortion via reduced production of TNF-alpha by CD8+ T cells.     

Restricted and Individual Usage of T-cell Receptor β-Gene Variables in Nickel-Induced CD4+ and CD8+ Cells

Nickel allergy is manifested as contact allergic eczema elicited by delayed-type hypersensitivity, the reaction being mediated by T lymphocytes. We examined the T-cell receptor (TCR) β-chain variable gene segment (Vβ) use of nickel-induced CD4⁺ and CD8⁺ T cells in the peripheral blood of nickel-sensitive and non-sensitized subjects. The results show that each patient had an individual Vβ repertoire overexpressed, these being in CD4⁺ cells Vβ10 and Vβ13 (in subject A); Vβ1, Vβ2, Vβ13 and Vβ21 (subject B); Vβ1 and Vβ10 (subject C); Vβ9 and Vβ19 (subject D). Thus, no single Vβ gene dominated in a majority of the CD4⁺ samples. The Vβ genes overexpressed in patient CD8⁺ nickel-induced T cells were Vβ1 (in subject A), Vβ1 (subject B), Vβ1 and Vβ2 (subject C) and Vβ7 (subject D), domination of Vβ1 being seen in most of the CD8⁺ samples (75%). No specific overexpression of any Vβ genes in the nickel-allergic subjects was found in comparison with the non-sensitized subjects. In conclusion, an individual pattern of restricted Vβ genes was induced with nickel in CD4⁺ and CD8⁺ T cells in each nickel allergy patient.     

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.     

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.     

Selective Expansion of a CD3+CD4-CD8- Subpopulation in Clinical Groups Associated with Human Immunodeficiency Virus Infection

T lymphocytes (CD3+) without expression of CD4/CD8 surface antigens have recently been described in the thymus and peripheral lymphoid organs. We have conducted a retrospective analysis of the literature, seeking quantitative variations in this T-cell subset in normal heterosexual controls, and in risk, pre-AIDS, and AIDS groups, by means of the subtraction [CD3-(CD4+CD8]) and the ratio 100 X [CD3-(CD4+CD8])/CD3. Dramatic T lymphocytopaenia in AIDS patients and the progressive decay of CD4+ lymphocytes and increase of CD8+ lymphocytes throughout the clinical spectrum of HIV infection have been confirmed. Furthermore, we hereby demonstrate the selective expansion of CD3+CD4-CD8- lymphocytes, directly related to the clinical state in different clinical groups of infected people when compared with controls (P less than 0.05). The inverse relationship between the CD3+CD4-CD8- cell subset and other mature T-cell subsets, mainly CD4+ (r = -0.49; P less than 0.01), suggests the existence of mutual regulatory interactions. These in vivo results, which are in agreement with those obtained in long-term infected cultures, cannot be explained by direct cytopathic effects of the virus on the very few infected cells. Thus, the implication of the expansion of these functional precursors on the prognosis for infected people, and the paradoxes of the immunodeficiency, such as lymphoproliferation and autoimmune features, are discussed.     

Expression of Activation Markers on CD4+ and CD8+ Cells from Synovial Fluid, Synovial Tissue, and Peripheral Blood of Patients with Inflammatory Arthritides

We studied the expression of the Tac antigen, the transferrin receptor (Tfr-R), HLA class II antigens (DR, DQ, DP), CD30, and Act 1 on purified CD4+ and CD8+ cells isolated from synovial fluid (SF), synovial tissue (ST), and peripheral blood (PB) of patients with rheumatoid arthritis (RA) and with non-RA inflammatory arthritides (not ST). Subfractionated T cells of PB from healthy individuals served as controls. SF CD4+ cells from RA and non-RA arthritides expressed the Tac antigen much more frequently than corresponding CD8+ cells (54 and 58% versus 16 and 17%). In contrast, SF CD8+ cells of both patient groups expressed the HLA class II antigens rather more frequently than the corresponding CD4+ cells (88 and 68% versus 72 and 40%). Tfr-R expression was low on CD4+ and CD8+ SF T cells from both patient groups. SF T cells did not express CD30, and their expression of Act 1 did not differ from that of normal PB T cells. The RA ST findings were similar to those of RA SF. The overall expression of activation markers on PB T cells of patients was slightly higher than on those of normal controls, and the RA group was slightly higher than the non-RA group. The results show that intra-articular T cells in arthritis are activated and that CD4+ and CD8+ subsets differ in their expression of Tac antigen and HLA class II antigens. There were also similar patterns of activation markers on both CD4+ and CD8+ SF cells from RA and non-RA arthritis patients, suggesting that several types of arthritis display a similar immunopathogenesis in the joints.     

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.     

Peripheral blood CD4+ and CD8+ T cell type 1 and type 2 cytokine production in atopic asthmatic and normal subjects

BACKGROUND: Increased production of IL-4 and IL-5 and decreased production of IFN-gamma by CD4+ T cells has been implicated in asthma pathogenesis. However, CD8+ T cells also produce type 1 and type 2 cytokines and the relative roles of CD4+ and CD8+ T cell cytokine production in asthma have not been previously studied. OBJECTIVE: To determine the production of the type 1 and type 2 cytokines by CD4+ and CD8+ T cell subsets in asthmatic and normal subjects. METHODS: Intracellular cytokine staining for IL-4, -5, -10, -13 and IFN-gamma was analysed in peripheral blood CD4+ and CD8+ T cells from 24 atopic asthmatic and 20 normal subjects. RESULTS: Both subsets of T cells produced all cytokines studied and there were no significant differences between CD4+ and CD8+ T cells in their capacity to produce either type 1 or type 2 cytokines. There were significantly increased frequencies of IFN-gamma-positive CD4+ (13.1 +/- 2.4%, vs. 7.3 +/- 1.4%) and CD8+ (20.0 +/- 2.9%, vs. 9.6 +/- 2.1%) T cells in asthmatic subjects compared with normal subjects (P < 0.05), but not in frequencies of CD4+ or CD8+ T cells staining positively for IL-4, -5, -10 or -13. CONCLUSION: The frequencies of peripheral blood CD8+ T cells producing type 1 and type 2 cytokines are comparable with the frequencies of CD4+ T cells. There was an increased frequency of IFN-gamma producing CD4+ and CD8+ T cells in asthmatic compared with normal subjects. Further studies investigating T cells derived from the airways and investigating various stages within the disease process are required to further elucidate the importance of type 2 and type 1 T cell cytokine production in the pathogenesis of human allergic disease.