A co-stimulatory role for CD28 in the activation of CD4+ T lymphocytes by staphylococcal enterotoxin B.

In this study we investigated the differential effect of the co-stimulatory receptor ligand molecules CD2/LFA-3, LFA-1/ICAM-1, and CD28/B7 on microbial superantigen mediated activation of CD4+ T cells. Highly purified CD4+ T cells, depleted of antigen presenting cells (APCs), do not proliferate in response to the superantigen, staphylococcal enterotoxin B (SEB). However, CD4+ T cells do respond to SEB in the presence of the LFA-3, ICAM-1, and B7 positive erythroleukemic cell line K562, murine L cells, human B7 transfected L cells or CD28 mAb. The K562 plus SEB induced response can be inhibited by combinations of mAbs to CD2 and LFA-1, and to LFA-3, ICAM-1, and B7. Addition of CD28 mAb to the CD2 and LFA-1 inhibited cultures could restore the response. Furthermore, soluble CD28 mAb alone is able to synergize with SEB to induce a proliferative CD4+ T cell response. CD4+ T cells depleted of APCs could also be activated by a pool of four mAbs directed to the V beta 5, V beta 6, V beta 8, and V beta 12 region of the TCR when a co-stimulatory signal was provided by the CD28 mAb, while the V beta mAbs alone or in combination are unable to activate CD4+ T cells in the absence of APCs. In contrast, addition of soluble mAbs to CD2 and LFA-1 molecules failed to co-stimulate SEB activated CD4+ T lymphocytes. The kinetics of the different modes of activation are distinct. SEB induced proliferation is most efficient in the presence of autologous APCs with maximal proliferation at a log4 lower SEB concentration than when CD28 mAbs were used. SEB plus K562 activation peaks on day 7, while SEB plus CD28 mAb induced proliferative responses do not peak until day 9. Thus, superantigen mediated activation of CD4+ T cells requires co-stimulatory signals, among which CD28 has distinct and unique effects.     

In vivo and in vitro death of mature T cells induced by separate signals to CD4 and alpha beta TCR.

To investigate whether a clonal deletion mechanism is responsible for the mature T cell tolerance that may be induced in vivo by TCR signal to anti-CD4 (H129.19 mAb) coated cells, we analyzed the T cell repertoire in anti-CD4 mAb treated BALB/c mice by flow cytometry following TCR signals through anti-alpha beta TCR mAb or SEB superantigen. Lymph nodes showed a strong reduction in the CD4+/CD8+ cell ratio, and a selective clonal loss of CD4+ V beta 8+ cells 4d following anti-alpha beta TCR or SEB injection, respectively. Following lymph node cell activation in a short-term in vitro assay with SEB or anti-V beta 8 mAb, a selective elimination of CD4+ V beta 8+ cells was again detected, and DNA fragmentation analysis disclosed a cell death by apoptosis. These findings suggest that TCR triggering transduces an apoptotic signal into CD4+ mAb saturated cells that in turn leads to specific holes in the mature T cell repertoire.     

Balance of Th1/Th2 Cytokines Associated with the Preventive Effect of Incomplete Freund's Adjuvant on the Development of Adjuvant Arthritis in LEW Rats

Complete Freund's adjuvant (CFA) could induce adjuvant arthritis (AA) in LEW rats and incomplete Freund's adjuvant (IFA) could induce oil induced arthritis (OIA) in DA but not in LEW rats. Lymph node cells (LNCs) from these AA and OIA rats showed increased mRNA expression of IFN-gamma, IL-2 and TNF-alpha but not IL-4. LNCs from IFA immunized LEW rats showed increased expression of IL-4, reduced expression of IFN-gamma and TNF-alpha and no IL-2, in contrast to IFA immunized DA rats. The pretreatment of IFA before CFA challenge could completely prevent AA in LEW rats and their LNCs showed increased expression of IL-4 and IFN-gamma but not IL-2 and TNF-alpha. In F1 (LEW x DA) rats, IFA could not induce OIA but the pretreatment of IFA before CFA challenge could induce very mild AA with 80% incidence, LNCs showing an elevated expression of all the above cytokines. These findings suggest that increased Th1 cytokine expression is associated with disease development and that increased IL-4 expression or the balance of Th2 over Th1 cytokine expression plays an important regulatory role in disease development.     

Massive production of Th2 cytokines by human CD4+ effector T cells transiently expressing the natural killer cell marker CD57/HNK1.

We have reported previously that uncommitted human CD4+ CD45RO- T cells default to the T-helper type 1 (Th1) pathway, if they are costimulated by anti-CD3 plus anti-CD28 monoclonal antibodies (mAb). In contrast, 5% of the uncommitted T cells differentiate into Th2 cells, if they are stimulated by anti-CD28 plus interleukin-2 (IL-2) in the absence of T-cell receptor (TCR) signals. The anti-CD28/IL-2-induced proliferation (and the resulting Th2 commitment) was not affected by neutralizing anti-IL-4 mAb, suggesting a non-conventional IL-4-independent Th2 differentiation pathway. Here we report that the respective CD4+ Th2 cells (but not the Th1 cells) coexpressed the natural killer (NK) cell marker HNK1/CD57. Expression of CD57 on Th2 cells required CD28 stimulation, and was suppressed by CD3/TCR signals. However, Th2 effector cells displayed a TCR V beta-chain usage comparable to that of committed Th1 cells (with V beta 8 dominating). Our data suggest that expression of CD57 on human CD4 T cells may be associated with defined stages of Th2 cell activation/differentiation, and may not necessarily characterize a separate T-cell lineage. The induction of cytokine production and B-cell helper function in both Th1 and Th2 populations required CD3/TCR signalling in costimulation with anti-CD28 or IL-2. Importantly, anti-CD28/IL-2-primed Th2 cells readily secreted IL-4 and induced IgE production by surface IgE- B cells in response to the first TCR signal and independent of previous contact with IL-4. Therefore, CD4+ CD57+ T cells responded comparably to murine CD4+ NK1.1+ T cells, which are critical for the development of Th2/IgE immune responses in vivo. The possible role of human CD4+ CD57/HNK1+ Th2-like cells in cancer, infection and allergy is discussed.     

TCR repertoire of suppressor CD8+CD28- T cell populations.

The cellular basis of graft rejection and the development of strategies for specific suppression of T cell responses against allogeneic and xenogeneic transplants represents an area of active investigation. Recently, a population of MHC-class I restricted CD8+CD28- T suppressor cells (Ts) which are able to inhibit specifically the proliferative response of allospecific, xenospecific and nominal-antigen specific CD4+ T helper cells (Th) has been identified. We have studied the TCR V beta gene repertoire expressed by CD8+CD28- Ts isolated from allospecific, xenospecific, and nominal antigen-specific T cell lines (TCL). A limited V beta repertoire has been found in all TCLs studied. The most restricted TCR V beta usage was observed within the population of Ts from xenospecific TCLs. The TCR V beta usage within the Ts subset of TCL differs from the TCR repertoire expressed by the CD4+ Th subset of the same TCL. This is consistent with the fact that Ts and Th cells recognize distinct MHC/ antigen complexes. The finding that the TCR repertoire used by Ts is limited opens new avenues for studying the mechanisms of transplant rejection.     

Responses of the rat immune system to arthritogenic adjuvant oil

T-cell mediated inflammatory joint diseases with similarities to rheumatoid arthritis (RA) can be triggered in arthritis-prone rat strains by intradermal injection of adjuvant oils. The pathogenesis of oil-induced arthritis (OIA) remains elusive, and a largely unresolved question is how the rat immune system responds to arthritogenic oils such as incomplete Freund's adjuvant (IFA). Here we report that IFA already induces increased plasma levels of the acute-phase reactants (APR) fibrinogen and alpha1-acid glycoprotein at day 4 postinjection (p.i). In contrast, no early responses were detected in the joints before infiltration of the T cells, which coincided with arthritis onset at 11-14 days post injection (d.p.i.) The infiltrating cells were possibly derived from draining lymph nodes (LN), which were hyperplastic and contained increased cell numbers from 4 days p.i. and onwards. The magnitude of the early increase in cell numbers and APR was regulated by non-major histocompatibility complex (MHC) genes, as determined by comparison between arthritis-susceptible DA rats and arthritis-resistant but MHC-identical LEW.lAV1 and PVG.1AV1 rats. Arthritisprone DA rats developed a weak acute-phase response, suggesting that this systemic response may be counteracting disease. The DA rats also had the largest early increase in LN-cell numbers, suggesting that the LN hyperplasia is part of a disease pathway. The analysis of hyperplastic LN after in vivo labelling with bromodeoxyuridine (BrdU) revealed increased numbers and proportions of proliferating lymphocytes, including T cells. Furthermore, polymerase chain reaction (PCR)-analysis of LN cytokine mRNA revealed upregulation of interleukin (IL)-1beta at 4 d.p.i. We conclude that adjuvant oil exposure triggers both systemic acute phase reactions and local activation of the peripheral lymphoid system. These responses are genetically regulated and may determine arthritis development and susceptibility.     

Recruitment and proliferation of CD4+ T cells in synovium following adoptive transfer of adjuvant-induced arthritis

Adjuvant-induced arthritis can be transferred to naive Dark Agouti (DA) strain (DA.CD45.1) rats by thoracic duct (TD) lymphocytes. Disease can be re-induced in convalescent rats by further transfer of arthritogenic cells, suggesting that resolution of the adoptive disease is not due to active regulation. To examine whether resolution is due to exhaustion of effector cells, we transferred the disease to DA.CD45.1 recipients, using CD4+ T cells from DA.CD45.2 donors. At the height of the adoptively transferred disease, donor cells comprised only 5-10% of recirculating CD4+ T cells but they accounted for approximately 40% of the CD4+ T cells in synovium-rich tissues of the hind paws. Approximately 65% of the donor cells in the synovium expressed a marker of proliferation (Ki-67 antigen). Division of CD4+ T cells continued in shielded paws after suppression of the recirculating pool of lymphocytes by selective irradiation. Intravenously injected CD4+ TD T lymphoblasts from arthritic donors were recruited to normal paws and, in greater numbers, to paws of animals with existing arthritis. Survival of the [125I]iodo-deoxyuridine-labeled lymphoblasts was greater in animals with existing arthritis. We conclude that effector CD4+ T cells in target tissues can proliferate in response to autoantigens and exhibit enhanced survival. However, without a continuous supply, adoptively transferred effector cells do not produce autonomous local disease, due to limits to their lifespan and ability to replicate indefinitely.     

Similar co-stimulation requirements of CD4+ and CD8+ primary T helper cells: role of IL-1 and IL-6 in inducing IL-2 secretion and subsequent proliferation.

Primary murine CD4+ and CD8+ T helper (Th) cells provide help for various immune responses by secreting lymphokines which activate effector cells. The purpose of the present study was to investigate the co-stimulatory signals that, together with T cell receptor (TCR) cross-linking, induce phenotypically distinct primary Th cells to secrete IL-2 and proliferate. We isolated highly purified populations of primary CD4+ or CD8+ T cells and stimulated them in vitro with platebound anti-CD3 mAb. TCR cross-linking by anti-CD3 mAb induced both IL-2 receptor expression and responsiveness to exogenous IL-2, but was not sufficient to induce either IL-2 secretion or T cell proliferation. Rather, for both CD4+ and CD8+ primary Th cells, IL-2 secretion and proliferation required both TCR cross-linking and antigen presenting cell (APC)-derived co-stimulatory signals. Based on G-10 adherence and sensitivity to gamma-irradiation, the APC populations able to induce primary CD4+ Th cells and primary CD8+ Th cells to secrete IL-2 were indistinguishable. In addition, we found that either IL-1 or IL-6 could replace the requirement for APC-derived co-stimulatory signals for IL-2 secretion and proliferation by both primary CD4+ Th cells and primary CD8+ Th cells. Thus, the present study has examined and compared the co-stimulatory requirements of rigorously purified subsets of IL-2-secreting primary CD4+ and primary CD8+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resistance to collagen-induced arthritis in DBA/1 mice by intraperitoneal administration of soluble type II collagen involves both CD4+ and CD8+ T lymphocytes

In this paper we report that intraperitoneal administration of type II collagen in a soluble form protects DBA/1 mice against collagen-induced arthritis (CIA) on subsequent arthritogenic challenge with soluble type II collagen in adjuvant. The degree of arthritis suppression, which is expressed in terms of reduced incidence of arthritis, delayed onset and reduced anti-collagen antibody titres, depends on the dose and timing of the pre-immunization collagen injection. In order to elucidate the r?les of CD4+ and CD8+ T lymphocytes subsets in arthritis resistance we administered monoclonal antibodies (mAb) to these antigenic determinants around the time of immunization with soluble type II collagen. Anti-CD4 mAb caused abrogation of arthritis resistance while anti-CD8 mAb was less effective. However, administration of anti-CD8 mAb two weeks after pre-immunization with soluble collagen was very effective in reversing arthritis resistance. From these findings we conclude that CD4+ and CD8+ T cells are involved in resistance to arthritis though the relative importance of each subset changes during the course of the process leading to resistance to CIA.     

An analysis of alpha/beta TCR V gene expression in the human thymus.

We have previously analysed alpha/beta T cell receptor (TCR) V gene usage in CD4+ and CD8+ subpopulations from human peripheral blood lymphocytes (PBL) and umbilical cord blood, and described a biased usage of some of the TCR V beta genes towards the CD4+ subpopulation. In this report, the TCR V gene usage in single positive (SP) CD4+ or CD8+ human thymocytes was analysed. Three previously described mAb with a biased usage in PBL and umbilical cord blood also had a skewed reactivity towards the CD4+ subpopulation in SP human thymocytes. Thus, in all 12 cases V beta 5.1 and V beta 6.7, and in 11/12 cases V beta 12 were preferentially used in the CD4+CD8-, compared to the CD4-CD8+ thymic subpopulation. Altogether, these results suggest a selection process in the thymus, supposedly through the positive influence of MHC class II determinants, to be responsible for this non-random, skewed TCR V gene usage.     

Arthritis induced in rats with adjuvant oil is a genetically restricted, alpha beta T-cell dependent autoimmune disease.

Adjuvant arthritis in rats is usually induced by injection of mycobacterium tubercle cell walls suspended in various adjuvant oils such as Freund's incomplete adjuvant (FIA) or pristane. We have recently shown that injection of adjuvant oils without inclusion of mycobacterium tubercle cell walls triggers arthritis [oil adjuvant-induced arthritis (OIA)] in the DA rat strain. The OIA is a genetically restricted disease since only DA rats are susceptible while Lewis, DA-fostered Lewis and F1 (Lew x DA) rats are relatively resistant. Activated alpha beta T cells infiltrate the affected joints of adjuvant oil-injected DA rats and treatment with monoclonal antibodies to the alpha beta T-cell receptor abrogates development of arthritis. These findings show that alpha beta T-cell activation is a critical event in the development of OIA.     

Characterization of thoracic duct cells that transfer polyarthritis.

Polyarthritis may result from the haematogenous distribution of arthritogenic effector lymphocytes that emerge in the efferent lymph and pass through the thoracic duct (TD) to the circulation. We therefore examined whether TD cells collected from rats in the late prodrome of adjuvant-induced arthritis (AA) could transfer polyarthritis adoptively and whether these cells included a subpopulation of arthritogenic cells that could be identified phenotypically. Unfractionated TD cells collected from donor rats 9 days after adjuvant inoculation were injected intravenously into normal syngeneic recipients in numbers equivalent to the overnight harvest from a single donor. TD cell subpopulations, equivalent in number to proportions in the same inoculum, were prepared by negative selection. Unfractionated TD cells transferred polyarthritis without in vitro stimulation or conditioning of recipient animals. Abrogation of arthritogenicity by depletion of alpha/beta TCR(+) cells showed that the polyarthritis was transferred by T cells. Negatively selected CD4(+) but not CD8(+) TD cells transferred AA. An arthritogenic subpopulation of CD4(+) T cells, enriched by either negative or positive selection, expressed the activation markers CD25 (IL-2 receptor alpha), CD71 (transferrin receptor), CD134 (OX40 antigen) and MHC class II. Cells expressing these markers were more numerous in TD lymph from arthritic rats than in lymph from normal rats and they included the majority of large CD4(+) T cells. Thus, arthritogenic effector T cells bearing activation markers are released into the central efferent lymph in the late prodrome of AA. Recruitment of these arthritogenic cells to synovium probably determines the polyarticular pattern of AA.     

H-2 class I Ld antigen positively selects different TCR V beta gene products in CD8+ and CD4+ T cells.

Expression of CD4+ or CD8+ determinants and ten different TCR V beta genes was analysed by two-colour flow cytometry in lymph node cells (LNC) of BALB/c and dm2 (a BALB/c Ld loss mutant) mice. TCR V beta 14 expression of CD8+ T cells and TCR V beta 7 and 14 of CD4+ T Cells were significantly more frequent in BALB/c than in dm2 LNC, whereas no differences were observed in the frequency distribution of TCR V beta+ CD4-CD8- double negative LNC from BALB/c versus dm2 mice. These results represent the first published evidence for positive selection of particular TCR V beta genes by individual MHC class I molecules in non-manipulated mice.     

Cross-talk between V beta 8+ and gamma delta+ T lymphocytes in contact sensitivity.

We have previously reported that T lymphocytes proliferating in vitro to the hapten trinitrochlorobenzene (TNCB) exhibit a very restricted V beta gene usage and response to TNCB is limited to T-cell receptors (TCR) composed of V beta 8.2 in combination with V alpha 3.2, V alpha 8 and V alpha 10. This paper investigates the role played by T lymphocytes expressing the V beta 8.2 gene segment in the contact sensitivity (CS) reaction to TNCB in the intact mouse and in its passive transfer into naive recipient mice. Mice injected with monoclonal antibodies to V beta 8 are unable to develop CS upon immunization with TNCB and 4-day TNCB-immune lymph node cells from mice that had been depleted in vivo or in vitro of V beta 8+ T lymphocytes fail to transfer CS. However, when separated V beta 8+ and V beta 8- cells were used for passive transfer, it was found that V beta 8+ T lymphocytes failed to transfer CS when given alone to recipient mice and a V beta 8- population was absolutely required. Further analysis revealed that within the V beta 8- population, T lymphocytes expressing the gamma delta TCR were fundamental to allow transfer of the CS reaction. These gamma delta cells were found to be antigen non-specific, genetically unrestricted and to rearrange the V gamma 3 gene segment. This indicates that transfer of the CS reaction requires cross-talk between V beta 8+ and gamma delta+ T lymphocytes, thus confirming our previous results obtained using TNCB-specific T-cell lines. Time-course experiments showed that V beta 8+ lymphocytes taken 4-24 days after immunization with TNCB were able to proliferate and produce interleukin-2 (IL-2) in response to the specific antigen in vitro. Similar time-course experiments were then undertaken using the passive transfer of the CS reaction system. The results obtained confirm that TNCB-specific V beta 8+ T lymphocytes are present in the lymph nodes of immunized mice from day 4 to day 24, and reveal that gamma delta+ T lymphocytes are active for a very short period of time, i.e. days 4 and 5 after immunization. In fact, TNCB-specific V beta 8+ cells are able to transfer CS when taken 4-24 days after immunization, providing the accompanying V beta 8- or gamma delta+ T lymphocyte are obtained 4 days after immunization. In contrast, injection of V beta 8+ T lymphocytes together with V beta 8- or gamma delta+ T lymphocytes that had been taken 2 or 6 days after immunization, failed to transfer significant CS into recipient mice. Taken together, our results confirm that cross-talk between V beta 8+ and gamma delta+ T lymphocytes is necessary for full development of the CS reaction and may explain why the CS reaction in the intact mouse lasts up to 21 days after immunization while the ability of immune lymph node cells to transfer CS is limited to days 4 and 5 after immunization.     

Treatment with anti-LFA-1 alpha monoclonal antibody selectively interferes with the maturation of CD4- 8+ thymocytes.

Maturation of T lymphocytes in the thymus is driven by signals provided by soluble factors and by the direct interaction between thymocytes and stromal cells. Although the interaction between T-cell receptor (TCR) and major histocompalibility complex (MHC) molecules on stromal cells is crucial for T-cell development, other accessory molecules seem to play a role in this process. In order to better understand the role of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) molecules in thymocyte maturation, mice were treated from birth with saturating doses of non-cytolytic-specific monoclonal antibodies. The effect of this treatment on thymocyte subpopulations and the expression of CD3 and TCR-alpha beta by these cells was investigated by flow cytometry. Our data demonstrated that the effective saturation of LFA-1 alpha chain in the thymus, but not ICAM-I or LFA-I beta chain, selectively interfered with the maturation of CD8+ T cells, as manifested by a marked reduction in the frequency of CD4-8+ thymocytes expressing high levels of CD3 and TCR-alpha beta. This selective reduction was also observed in peripheral blood mononuclear cells and spleen cells. The analysis of the frequencies of various V beta TCR showed that CD4-8+ thymocytes were globally affected by the treatment. These results underline the importance of the interaction between LFA-1 and its ligands in the maturation of CD8+ T cells and document the existence of different molecular requirements for the differentiation of CD4+ and CD8+ T cells.     

TCR-mediated target cell lysis by CD4+NK1+ liver T lymphocytes

In the liver, an unusual T lymphocyte population exists with the intriguing phenotype CD4+NK1+ TCR alpha beta int. Thus far, functions of these lymphocytes remained elusive. Recently, however, CD4+NK1+ liver T lymphocytes have been shown to produce cytokines. Here we show that sorted CD4+NK1+ liver lymphocytes from naive mice lyse target cells after TCR alpha beta or CD3, but not TCR gamma delta, engagement. Liver lymphocytes from beta 2-microglobulin-deficient gene disruption mutant mice failed to express such cytolytic activities and in vivo treatment with anti-NK1.1 mAb or anti-CD4 mAb, but not anti-CD8 mAb, markedly reduced target cell lysis. In vivo administration or rIL-12 impaired TCR alpha beta-mediated target cell lysis by liver lymphocytes. A similar down-regulation of cytolytic activities was observed with liver lymphocytes from mice infected with Listeria monocytogenes or Mycobacterium bovis BCG, which are potent IL-12 inducers. We anticipate (i) that cytolytic CD4+NK1+ T lymphocytes contribute to immunosurveillance of inflammatory processes in the liver and (ii) that they are influenced by IL-12.