Interleukin-2 and Serum Thymic Factor Enable Autologous Rosette-forming T Lymphocytes to Generate Helper and Cytotoxic Functions

The autologous rosette-forming T cells (Tar cells) isolated by means of their ability to form rosettes with autologous erythrocytes were characterized by the use of OKT monoclonal anti-human T-cell subset antibodies and a monoclonal anti-HLA-DR antibody. We found that the phenotype of Tar cells was OKT 3⁺4⁺8⁺Dr⁻ as determined by both indirect imnrunofluorescence microscopy and complement-mediated killing of ⁵¹Cr-labelled Tar cells. In addition, we found that Tar lymphocytes were able to develop cytotoxicity against allogeneic and trinitrophenol (TNP)-conjugated autologous target cells in the presence of interleukin-2 (IL-2) or serum thymic factor. However, these cells showed little or no cytotoxicity in the absence of interleukin-2 or serum thymic factor. Tar lymphocytes generated helper function for B lymphocytes in the presence of interleukin-2 in both pokeweed mitogen (PWM)- and purified protein derivative (PPD)-stimulated cultures. Nevertheless, non-IL-2-treated Tar cells did not exhibit any helper activity on B cells. Finally, pretreatment of Tar cells with 1000–1500 rad of X ray made these cells unable to develop helper function for B lymphocytes. It is concluded that: (1) OKT 3⁺4⁺8⁺Dr⁻ Tar cells are able to generate cytotoxicity against alloantigens and TNP-labelled self structures provided they are stimulated by IL-2 or serum thymic factor; (2) these cells need both to proliferate and to receive help from IL-2 to develop helper cells capable of assisting B-lymphocyte differentiation into plasma cells in both PWM- and PPD-stimulated cultures.     

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.     

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.     

Effect of Prostaglandin E2 on Pokeweed Mitogen-Activated Human Lymphocyte Cultures

It is shown that a short incubation of peripheral human lymphocytes with PGE₂ is able to reduce the B cell differentiation induced by PWM. The target of PGE₂ action appears to belong to T lymphocytes, since the treatment of non-T cells is uneffective in reducing the immune response. Both OKT4⁺ and OKT8⁺ subsets are sensitive to PGE₂. Data concerning the role of endogenous as well as exogenous PGE₂ either on unfractionated or fractionated OKT4⁺, OKT8⁺ and non-T lymphocytes are also discussed. The PGE₂ -inhibition on immunoglobulin synthesis in PWM-stimulated cultures seems to be mediated by a complex effect on both the T cell subsets.     

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.     

Anti CD5 Sustains the Proliferative Response of IgM-Activated Human CD5+B Cells

The CD5 molecule is expressed by most T cells but it is present on a minor B cell subset. Whilst several studies have provided information on the physiological role of T cell CD5, the functional role of CD5 on B lymphocytes remains unclear. To address this question, tonsillar CD5⁺ B cells were sorted by dual-colour fluorescence and FACS. Sorted cells were stimulated with polyclonal anti-IgM antibodies (Ab), and monoclonal (MoAb) F(ab')₂ fragments of anti-CD5. Proliferative responses were evaluated by enumeration of Ki-67 positive cells using quantitative flow cytometry. Co-stimulation with anti-CD5 MoAb for 3 days did not affect the anti-IgM and IL2-induced proliferation of CD5⁺ B cells. This was seen under conditions where the anti-CD5 was soluble, adsorbed to the microwells or cross-linked by anti-mouse antibodies. Fewer CD25⁺ cells were detected, however, in the presence of anti-CD5. In contrast, the proliferative response of CD5⁺ B cells prestimulated for 3 days with IL-2 and anti-IgM, was sustained in a further 3-day culture period when anti-CD5 was added. It is concluded that CD5 occupancy might provide an additional signal to activated CD5⁺ B cells favouring their proliferation and differentiation into autoantibody secreting cells.     

Activation Signal Induces the Expression of B Cell-Specific CD45R Epitope (6B2) on Murine T Cells

Tlymphocytes express multiple forms of the leukocyte-common antigen CD45, transcribed by alternative usage of leukocyte-common antigen exon 4 6. The various isoforms of CD45R expressed differentially on T cells are involved in different stages of development and activation. The monoclonal antibody (MoAb) RA3-6B2 is established as a B cell-type isoform (B220)-specific marker. However, it reacts with certain activated T cells although the relationship between 6B2 expression and T-cell activation is unclear. We have examined the 6B2 expression on activated T cells and found that concanavalin A, anti-CD3 antibody and staphylococcal enterotoxin B (SEB) induced 6B2 expression on T cells. The expression was found on both CD4⁺ and CD8⁺ T cells and also was induced by SEB in vivo predominantly on CD8⁺ T cells. The 6B2⁺ T cells are IL-2R⁺ and blasted cells according to flow cytometry analysis. Therefore, the 6B2⁺ T cells are supposed to be in an activated stage. Enzymatic analysis demonstrated that trypsin treatment decreased the 6B2 expression, whereas neuraminidase increased the intensity on activated T cells. Neither endo-D or endo-H have any effect on the expression and there are no differences, in the results of immunoprecipitation and RT-PCR analysis, between control T cells and activated T celts. Taken together, the 6B2 epitope is presumed to be the product of CD45R modification and is expressed on activated T cells. These results illustrate a novel classification of a T-cell subpopuiation bearing a 6B2 epitope.     

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.     

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.     

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.     

Interleukin-4 and Interferon-Gamma Production by Leishmania Stimulated Peripheral Blood Mononuclear Cells from Nonexposed Individuals

Interferon-gamma (IFN-γ) and interleukin-4 (IL-4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non-exposed individuals was investigated. IFN-γ was measured in culture supernatants after antigen stimulation. For the measurement of IL-4, antigen stimulated cells were pulsed with PMA and ionomycin before IL-4 release was measured. L. donovani and L. major antigens induced IL-4 production (105–1748pg/ml) in 13 and seven cultures, and IFN-γ production (1.7- > 66IU/ml) in 14 and 11 of 20 cultures, respectively. IL-4 production rose steeply after 6 days of antigen stimulation suggesting a response due to antigen recognition. Both IL-4 and IFN-γ production was abrogated by depletion of CD2⁺ or CD4⁺ but not CD8⁺ cells. CD2⁺ or CD4⁺ but not CD8⁺ enriched cultures produced cytokines as unseparated PBMC. Thus, in non-exposed individuals circulating Leishmania reactive CD4⁺ T cells could be demonstrated. The cells from different individuals showed different patterns of IFN-γ and/or IL-4 production upon antigenic stimulation. In experimental leishmaniasis the early balance between IFN-γ and IL-4 is important for the clinical outcome. Our findings call for studies of the importance of cytokine production by cross-reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL-4 detection is useful for this purpose.     

Sequential Analysis of Distributions of Donor-derived Thymocytes Bearing T-cell Antigen Receptor (TCR) and Donor-derived la+ Cells in Thymuses of Fully Allogeneic Bone Marrow Chimera in Mice

Lethally irradiated SJL/J mice were reconstituted with B10 bone marrow cells, and the process of thymic reconstitution by donor derived cells positive for I- A or Vβ8 molecules was investigated. The donor-derived la⁺ cells appeared in the medulla on day 7 after reconstitution. The la+ cells became confluent up to day 14, and the cellularity in the medulla on day 17 was almost the same as that in the normal thymus. Dull Vβ8⁺ thymocytes were first recognized in the cortex on day 10 and were identifiable in the medulla by day 14. The Vβ8⁺ cells seemed to be mainly CD4⁺8⁺ double-positive. Furthermore, most of the Vg8'cells in the medulla of chimeras given cyclosporin A for 3 weeks after reconstitution appeared to be CD4⁺8⁺. The present findings demonstrate that CD4 8+ thymocytes which bear a low concentration of TCR exist in the thymic medulla at a relatively early stage when donor-derived la⁺ cells have already settled there. The coincidental appearance and coexistence of la⁺ cells and TCR⁺ thymocytes in the medulla suggest that these histological characteristics may be related to the selection of thymocytes in this area.     

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.     

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.     

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.     

Defective Calcium-Dependent Signal Transduction in T Lymphocytes of Ataxia-Telangiectasia

T-cell functions of two patients with ataxia-telangiectasia were investigated. Patients with ataxia-telangiectasia had reduced percentages of circulating CD3⁺ cells and CD4⁺ cells, although neither patient had a reduced percentage of circulating CD8⁺ cells. The proliferative responses and interleukin-2 production of peripheral blood mononuclear cells to T-cell mitogens were reduced in the patients. The intracellular calcium concentration in T cells or CD4⁺ cells from both patients was only slightly increased after phytohaemagglutinin stimulation. Moreover, the concentration after OKT3 stimulation was not or only slightly increased in T cells or CD4⁺ cells from both patients. Our results suggest that the functional defect of T cells is caused by defective Ca²⁺-dependent signal transduction through the CD3 complex of the surface in T cells of ataxia-telangiectasia.     

Comparative Analysis of Peripheral Natural Killer Cells in the Two Phases of the Ovarian Cycle

Changes in endometrial Natural Killer (NK) cells during the luteal phase of the ovarian cycle are important in initiating/maintaining a subsequent pregnancy. In the present study it was investigated whether during the menstrual cycle changes occur also in peripheral blood (PB) NKs.

Method of study

Blood samples during the follicular and the luteal phase were collected from 30 women without fertility problems. Samples were analyzed by flow-cytometry for: (1) NK cells (CD3⁻CD16⁺CD56⁺) and (2) intracellular production of interferon-γ (IFN-γ) by NK cells. For the comparison and correlation of the two populations between the two phases, Wilcoxon signed-rank test and Spearman's Coefficient were used.

Results

The differences in percentages of CD3⁻CD16⁺CD56⁺ cells and that of CD3⁻CD16⁺CD56⁺/IFN-γ⁺ cells between the follicular and the luteal phase were not statistically significant (10.61 ± 5.11 versus 9.76 ± 4.57 and 6.48 ± 7.90 versus 7.30 ± 6.77, respectively, P  > 0.05). The correlation between the two variables (NK% and NK/IFN-γ%) was weakly positive ( P  = 0.07) only in the follicular phase.

Conclusion

The study did not reveal menstrual cycle-depended changes in PB NK cells. Thus, a suggestion to measure these cells in a specific phase of the cycle in order to predict the outcome of a subsequent pregnancy in women with fertility problems is objected.     

Oligoclonality of TCRint Cells with a Low Diversity of TCR Complementarity-Determining Region 3 in Mice with Graft- Versus-Host Disease

Conventional T cells (i.e. TCR^high) are generated by the main stream of T-cell differentiation in the thymus. However, primordial T cells (i.e. TCR^int) are generated by extrathymic pathways and an alternative intrathymic pathway. Since TCR^int cells contain self-reactive clones, the diversity of the T-cell antigen receptor (TCR) complementarity-determining region (CDR) 3 was examined. The predominant Vβ8.2⁺ clones among TCR^int cells were selected for DNA sequencing. Thymectomized, irradiated mice subjected to bone-marrow transplantation (BMT) were used; graft-versus-host disease (GVHD), B6→(B6 × C3H/He) F ₁ and syngeneic BMT, B6→B6. In these combinations, only TCR^int cells were generated. Vβ8.2⁺ cells with a low diversity of CDR3 of V-gene expanded in GVHD mice. Vβ8.2⁺ cells of TCR^int and TCR^high cells in normal mice were polyclonal, showing that the former has a lower diversity of CDR3 than the latter. The clonality of activated TCR^high cells was examined, in which CD3^high cells (bm12 mice) were injected into 1 Gy-irradiated B6 nude mice. Some Vβ8.2⁺ clones among TCR^high cells were expanding but the diversity of CDR3 was greater than that of CD3^int cells, despite the fact that the recognition site of the H-2 difference was smaller. Taken together with invariant usage of Vα14, these results suggest that TCR^int cells have a low diversity of CDR3 of Vβ genes.