Cytokine and chemokine expression in the skin from patients with maculopapular exanthema to drugs

Background:  Maculopapular exanthema (MPE) is the most frequent clinical manifestation of nonimmediate allergic reactions to drugs and T helper 1 (Th1) cytokines and CD4⁺ T cells have been shown to play an important role in its pathogenesis. We assessed the role of cytokines and chemokines and their receptors in the pathogenesis of MPE.
Methods:  We evaluated skin biopsies and peripheral CD4⁺ and CD8⁺ T cells from 27 patients during the acute phase of the reaction and 26 exposed controls. Semiquantitative real-time PCR was performed to determine the expression of cytokines and chemokines and their receptors and immunohistochemistry was used to determine the same chemokines and their receptor proteins in skin.
Results:  There was a high expression of the Th1 cytokines interferon-γ ( P  = 0.006) and tumor necrosis factor-α ( P  = 0.022) in skin and CD4⁺ T cells ( P  = 0.007 and P  = 0.005, respectively); and of the Th1 chemokines CXCL9 ( P  = 0.005) and CXCL10 ( P  = 0.028) in the skin, while their receptor CXCR3 was increased in skin ( P  = 0.006) and CD4⁺ T cells ( P  = 0.03). Homing chemokine receptors were also increased: CCR6 in skin ( P  = 0.026) and CD4⁺ T cells ( P  = 0.016), and CCR10 only in CD4⁺ T cells ( P  = 0.016), as well as their ligands, CCL20 and CCL27, in skin alone. Immunohistochemistry confirmed these results.
Conclusions:  These data show significant differences in the expression of chemokines and chemokine receptors, related with a Th1 profile, in both skin biopsies and peripheral CD4⁺ T cells in patients with drug-induced MPE.     

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.     

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

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

Distribution of immunocompetent cells in decidua of controlled and uncontrolled (choriocarcinoma/hydatidiform mole) trophoblast invasion

Problem:  Pregnancy has been considered as a model of successfully controlled tissue invasion where trophoblast cells infiltrate the maternal decidua without being rejected or without destroying the tissue. In choriocarcinoma (CC) and hydatidiform mole (HM), a dysregulation of invasive (malignant/benign) trophoblast cells is present. Immunocompetent cells (IC) are known to be involved in rejection pathways of malignant cells and can also be identified in early pregnancy decidua. The aim of the present study was to identify the phenotype of IC in decidua of women with normal pregnancy (NP), CC and HM.
Methods:  Immunocompetent cells were detected by immunohistochemistry in decidual tissue from first trimester NP ( n  = 10), CC ( n  = 12) and HM ( n  = 11) using antibodies against CD8⁺, CD3⁺, CD56⁺, CD68⁺ cell surface markers and mast cell tryptase (MCT). A scaled eye piece was used for cell counting to obtain semiquantitative results. Statistical analysis was performed using Wilcoxon rank/Mann–Whitney tests.
Results:  We observed a significantly increased number of lymphocytes positive for CD8, CD3 and MCT positive granulocytes in CC and HM compared with the samples from NP (all P  ≤ 0.001). Lymphocytes positive for natural killer (NK) cell marker CD56 were significantly decreased in CC and HM versus NP ( P  ≤ 0.001). The number of CD68 positive cells (macrophages) were not significantly different among the tissue pools.
Conclusion:  The increase of CD8/CD3 T cells and mast cells in CC and HM and the decrease of CD56 cells, compared with NP, suggests the necessity of a balance between T and NK cells in controlling trophoblast invasion.     

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.     

CD8alpha+beta(low) effector T cells in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by the loss of self-tolerance to nuclear antigens. Aberrant T-cell function plays a central role in lupus pathogenesis. We and others previously demonstrated that peripheral TCRαβ⁺CD3⁺ T cells express CD8β either at a high (CD8β^high) or low density (CD8β^low), thereby defining two functionally distinct subsets. CD8β^low T cells express predominantly CD8αα and less CD8αβ as a coreceptor, display a differentiated phenotype and exert effector function. CD8β^high T cells appear to be the precursors expressing predominantly the heterodimeric efficient CD8αβ coreceptor, exhibiting a naïve phenotype and high proliferative capacity. In the present study, the distribution and functional properties of CD8β^high and CD8β^low T cells of SLE patients were compared ( n  = 20) with those of healthy subjects ( n  = 16). It was found that expansion of CD8β^low T-cell subset correlated with disease activity indicating chronic antigenic stimulation leading to a major lack of naïve CD8β^high precursor T cells in SLE. Functional characteristics of CD8β^low T cells including production of cytokines and cytotoxic granules were not significantly different between patients with SLE and healthy individuals. We speculate that unbalanced CD8β^high/CD8β^low T-cell relation reflects a skewed homeostasis within the CD8⁺ T-cell compartment towards fully differentiated effector T cells possibly due to persistent antigen stimulation in SLE.     

Functional Changes of Human Peripheral B-Lymphocytes in Pre-Eclampsia

Problem  The aim of our study was to investigate the functional changes of human peripheral B-lymphocytes in healthy and pre-eclamptic pregnancies.
Method of study  Twenty patients with pre-eclampsia and 15 healthy third-trimester pregnant women were recruited in this study. Peripheral blood mononuclear cells (PBMCs) were isolated and directly stained with fluorescein isothiocyanate (FITC)-labeled anti-CD27 monoclonal antibody (mAb) and phycoerythrin (PE)-labeled anti-CD38 mAb. The percentages of the individual B-cell subsets were estimated out of total lymphocytes by flow cytometric analysis. Additionally, the enriched PBMCs were cultured with or without the stimulation of pokeweed mitogen (PWM) for 5 days. Then morphologic observation of plasma cells was analysed by Wright-Giemsa stain, and antibody-producing cells were detected by enzyme-linked immunospot assay.
Results  The percentage of CD27⁻CD38⁻ naïve B-cells and CD27⁻CD38⁺ plasma cells did not differ between study groups ( P  >   0.05). The percentage of CD27⁺CD38⁻ memory B-cells and CD27⁺CD38⁺ plasma cell pre-cursors increased in pre-eclamptic women compared with the controls ( P  <   0.05). Irrespective of whether the PBMCs were stimulated with or w/o PWM in vitro , the mean percentages of generated plasma cells were significantly higher in pre-eclamptic group than in the controls ( P  <   0.05). There were more antibody-producing cells in pre-eclamptic women following the activation of PWM than those in the controls ( P <  0.01).
Conclusion  Our findings implicate that the functional changes of human circulating B-cells might contribute to the etiology of pre-eclampsia.     

Lymphocytic Choriomeningitis Virus Infection is Associated with Long-Standing Perturbation of LFA-1 Expression on CD8+ T Cells

Flow cytometric analysis of splenocytes from mice infected with lymphocytic Choriomeningitis virus revealed marked and long-standing up-regulation of LFA-1 expression on CD8⁺, but not on CD4⁺ T cells. Appearance of CD8⁺ T cells with a changed expression of adhesion molecules reflected polyclonal activation and expansion which was demonstrated not to depend on CD4⁺ T cells or their products. Cell sorting experiments defined virus-specific CTL to be included in this population (LFA-1^hiMEL-14^lo), but since about 80% of splenic CD8⁺ T cells have a changed phenotype, extensive bystander activation must take place; this is indicated also by the finding that CD8⁺LFA-l^hi cells transiently express several markers of cellular activation, e. g. transferrin receptor, IL-2Rα and β. Analysis of cells from the cerebrospinal fluid of mice infected intracerebrally showed that virtually all T cells present belonged to the CD8LFA-lhi subset and, correspondingly, the ligand ICAM-1 was found to be up-regulated on endothelial cells in the inflamed meninges. Preincubation of LCMV-primed donor splenocytes with anti-LFA-1 markedly inhibited the transfer of virus-specific delayed-type hypersensitivity to naive recipients. Together, these findings indicate that up-regulation of LFA-1 expression is a critical factor involved in directing activated CD8⁺ T cells to sites of viral infection.     

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 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.     

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.     

Expression of inhibitory-motif killer immunoglobulin-like receptor, KIR2DL1, is increased in natural killer cells from women with pelvic endometriosis

Problem:  We investigated inhibitory and activation motif expression of killer immunoglobulin-like receptor (KIR) by natural killer (NK) cells, which may be pathogenetically involved in endometriosis.
Method of study:  We compared cells from 24 Japanese women laparoscopically diagnosed with endometriosis, to cells from 25 women with other laparoscopic diagnoses. KIR expression by NK cells was assessed in peripheral blood (PB) and peritoneal fluid (PF) by flow cytometry. Intracellular immunoreceptor tyrosine-based (IT) inhibitory and activation motifs (ITIM and ITAM) of KIR in PB was assessed by Western blotting.
Results:  ITIM-KIR expression by PB NK cells was significantly and similarly greater than ITAM-KIR expression in women with and without endometriosis. Percentages of CD56⁺ NK cells in PB and PF did not differ significantly between women with and without endometriosis; however, the percentage of CD158a⁺ cells among CD56⁺ NK cells in PB and PF was significantly higher in women with than without endometriosis.
Conclusions:  ITIM-KIR expressing NK cells might confer tolerance to peritoneal endometriotic implants.     

Low-dose cyclosporine A therapy increases the regulatory T cell population in patients with atopic dermatitis

Background:  Atopic dermatitis (AD) is a T cell dependent chronic relapsing inflammatory skin disorder successfully treated with cyclosporine A (CsA). Clinical observations indicate that even low-dose CsA therapy is successful in severely affected AD patients. We studied the impact of low-dose CsA therapy on the ability of T helper cells to be activated, and examined whether regulatory T (Treg) cells are increased in these patients.
Methods:  Peripheral T cells were activated in a whole blood sample and interleukin-2 producing cells were measured by intracellular cytokine staining. Regulatory T cells were analyzed by intracellular FoxP3 staining. Regulatory T cells (CD4⁺CD25⁺CD127^low) and effector T cells (CD4⁺CD25⁻CD127⁺) were sorted by flow cytometry and used for suppression assays.
Results:  A group of AD patients treated with low-dose CsA had a significantly larger Treg cell population than a healthy control subject group. In individual patients, onset of low-dose CsA therapy reduced the ability of T cells to be activated to 42 ± 18% ( P  <   0.005) and significantly increased Treg cells, both in absolute numbers (1.6-fold change) and frequencies (1.7-fold change). Treg cells from AD patients showed similar suppressive capacities as Treg cells from healthy donors. Furthermore, Treg cells from AD patients had skin homing properties.
Conclusion:  Our results indicate that the therapeutic effect of low-dose CsA therapy in AD patients might be not only mediated by the inhibition of T cell hyperactivity but also by an increased population of Treg cells.     

Primary Stimulation of CD4+ Cells in the Presence of IL-4 or IFN-γ Alters the Frequencies of Cytokine-Producing Cells at Restimulation

The induction of specific effector functions in naive T cells may be directed by accessory signals during activation. These could be elicited through binding to cell surface molecules or through factors secreted by antigen-presenting cells or other simultaneously activated cells. We have investigated the influence of CD8⁺ cells and of exogenousiy added cytokines (interleukin (IL)-2, IL-4 and interferon (IFN)-γ) on the cylokine production in splenic CD4⁺ T cells. IL-2, IL-4, IL-5 and IFN-γ production in CD4⁺ cells was measured at the single cell level during primary mitogen stimulation in vitro in the presence or absence of factors or CD8⁺ cells. On day 5 the cells were restimulated with mitogen alone and analysed to evaluate the short-term development of cytokine-producing cells in such cultures. Preactivation in the presence of either exogenous IL-4 or IFN-γ led to an increased production of IL-4 and IFN-γ respectively at restimtilation, and the effects of both IL-4 and IFN-γ were augmented by IL-2. After preactivation in the presence of IL-2 and IL-4, every third CD4⁺ cell could be induced to produce IL-4. Exogenous IL-4 or IFN-γ further decreased each other's production. Depletion of CD8⁺ cells before activation resulted in a slight increase of IL-4-producing cells, indicating that simultaneous activation of CD8⁺ cells will influence lymphokine production in CD4⁺ cells. The results suggest that the pattern of lymphokines induced in naive cells may be influenced by factors secreted by preactivated CD4⁺ and CD8⁺ cells, and that naive cells are preferentially 'recruited' to produce similar cytokines.     

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.     

CD3−4−8− Thymocyte Precursors with Interleukin-2 Receptors Differentiate Phenotypically in Coculture with Thymic Stromal Cells

CD3⁻4⁻8⁻ interleukin-2 receptor positive (IL-2R⁺) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL-2R⁺CD3⁻4⁻8⁻ thymocytes were obtained by positive panning of IL-2R⁺ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2½ days significant numbers of CD4⁺8⁺ and CD3⁺4⁺8⁻ cell populations developed, the latter expressing the αβ T-cell receptor (αβ-TCR). Thus heterogeneous stromal cell cultures support the development of IL-2R⁺ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal-dependent step.     

T-Lymphocyte Functions in Acute Leukaemia Patients with Severe Chemotherapy-Induced Cytopenia: Characterization of Clonogenic T-Cell Proliferation

Intensive chemotherapy for acute leukaemia is followed by a period of severe chemotherapy-induced leukopenia. We used a limiting dilution assay to investigate whether remaining CD4⁺ and CD8⁺ T lymphocytes derived from such leukopenic patients could be activated and undergo clonogenic proliferation. The activation signal in our model was accessory cells (irradiated normal peripheral blood mononuclear cells) + phytohaemagglutinin (PHA) + interleukin-2 (IL-2). During severe leukopenia a majority of circulating lymphocytes were CD4⁺ T cells. Clonogenic proliferating T lymphocytes were detected for all patients. Higher frequencies of clonogenic cells were detected in the CD8⁺ subset as compared to the CD4⁺ subset. However, for both subsets frequencies of proliferating cells were decreased compared with healthy individuals. The CD4⁺ and CD8⁺ lymphocytes were also capable of proliferation in response to alloactivation, and accessory cells mainly containing acute myelogenous leukaemia blast were efficient as accessory cells for activation. For the CD4⁺ cells, increased proliferation was detected in the presence of acute myelogenous leukaemia (AML) blasts compared with normal accessory cells. Based on our results we conclude that: (1) although acute leukaemia patients with therapy-induced leukopenia have both a quantitative and a qualitative T-cell defect, (2) the remaining T-cell population includes a subset capable of clonogenic proliferation. However, (3) proliferation of the clonogenic CD4⁺ cells can be modulated by AML blasts.     

Contrary Roles of IL-4 and IL-12 on IL-10 Production and Proliferation of Human Tumour Reactive T Cells

The cytokine profile of tumour reactive T cells is likely to play a central role in their function. However, little is known about how cytokine patterns of tumour reactive T cells can be regulated. Here, the authors investigated the influence of exogenous regulatory cytokines in addition to interleukin-2 (IL-2) on cytokine patterns and the proliferation of T cells recognizing an autologous sarcoma cell line. In this system, IL-4 and IL-12 showed the most polarizing influences on tumour reactive T cells. Exogenous IL-4 induced a predominant production of IL-4 while decreasing the interferon-γ (IFN-γ) and IL-10 production by tumour reactive T cells. It also stimulated the growth of tumour reactive CD4⁺ T cell clones. In contrast, IL-12 substantially increased the production of IL-10 and IFN-γ. This was accompanied by a growth inhibition of tumour reactive T cells. The growth of CD4⁺ tumour reactive T cells was also suppressed by exogenous IL-10. This study shows that cytokine patterns and proliferation tumour reactive T cells can be significantly influenced by exogenous cytokines and confirms the hypothesis of a negative feedback loop of IL-12 by the induction of IL-10 in the context of human tumour reactive T cells.     

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.