Chris P. M. Broeren

Costimulation Light: Activation of CD4+ T Cells with CD80 or CD86 Rather Than Anti-CD28 Leads to a Th2 Cytokine Profile

To examine the role of CD28 and CTLA-4 in Th cell differentiation, we used a novel microsphere-based system to compare the effects of CD28 ligation by Ab or CD80/CD86. One set of beads was prepared by coating with anti-CD3 and anti-CD28 Ab. Another set of beads was prepared by immobilizing anti-CD3 and murine CD80-Ig fusion protein or murine CD86-Ig fusion protein on the beads. The three sets of beads were compared in their effects on the ability to activate and differentiate splenic CD4 T cells. When purified naive CD4+ cells were stimulated in vitro, robust proliferation of similar magnitude was induced by all three sets of beads. When cytokine secretion was examined, all bead preparations induced an equivalent accumulation of IL-2. In contrast, there was a marked difference in the cytokine secretion pattern of the Th2 cytokines IL-4, IL-10, and IL-13. The B7-Ig-stimulated cultures had high concentrations of Th2 cytokines, whereas there were low or undetectable concentrations in the anti-CD28-stimulated cultures. Addition of anti-CTLA-4 Fab augmented B7-mediated IL-4 secretion. These studies demonstrate that B7 is a critical and potent stimulator of Th2 differentiation, and that anti-CD28 prevents this effect.

Autoantigen-Specific IL-10-Transduced T Cells Suppress Chronic Arthritis by Promoting the Endogenous Regulatory IL-10 Response

Deficient T cell regulation can be mechanistically associated with development of chronic autoimmune diseases. Therefore, combining the regulatory properties of IL-10 and the specificity of autoreactive CD4+ T cells through adoptive cellular gene transfer of IL-10 via autoantigen-specific CD4+ T cells seems an attractive approach to correct such deficient T cell regulation that avoids the risks of nonspecific immunosuppressive drugs. In this study, we studied how cartilage proteoglycan-specific CD4+ T cells transduced with an active IL-10 gene (TIL-10) may contribute to the amelioration of chronic and progressive proteoglycan-induced arthritis in BALB/c mice. TCR-transgenic proteoglycan-specific TIL-10 cells ameliorated arthritis, whereas TIL-10 cells with specificity for OVA had no effect, showing the impact of Ag-specific targeting of inflammation. Furthermore, proteoglycan-specific TIL-10 cells suppressed autoreactive proinflammatory T and B cells, as TIL-10 cells caused a reduced expression of IL-2, TNF-α, and IL-17 and a diminished proteoglycan-specific IgG2a Ab response. Moreover, proteoglycan-specific TIL-10 cells promoted IL-10 expression in recipients but did not ameliorate arthritis in IL-10-deficient mice, indicating that TIL-10 cells suppress inflammation by propagating the endogenous regulatory IL-10 response in treated recipients. This is the first demonstration that such targeted suppression of proinflammatory lymphocyte responses in chronic autoimmunity by IL-10-transduced T cells specific for a natural Ag can occur via the endogenous regulatory IL-10 response.

Altered) Self Peptides and the Regulation of Self Reactivity in the Peripheral T cell Pool

Tolerance for self has appeared incomplete for many self antigens. We have obtained experimental evidence that both for self heat shock proteins and T cell receptor V-gene products, reactive T cells are part of the normal immune repertoire. Furthermore, it has become apparent that stimulation of T cell responsiveness to these antigens, by using peptide immunisation or by transfer of activated T cells, raises resistance to experimentally induced autoimmune arthritis. In addition, available evidence has suggested that these reactivities may be functional during natural processes of disease remission. The observations with regard to heat-shock proteins have indicated that mechanism leading to disease resistance are most efficiently triggered by exposing the immune system to non-self antigens such as bacterial hsps, which are similar to, but not identical to, self. Experimental evidence has been obtained, that conserved bacterial hsp peptides, may trigger self hsp reactive T cells, with disease suppressive regulatory potential. It is possible that such self hsp reactive T cells, being expanded by recognising bacterial peptides as full agonists, do, in fact, perceive the self epitopes as partial agonists, and therefore have the possibility of displaying downregulatory activity at the site of inflammation. Experiments with peptide analogs of self epitopes, being variants of disease critical T cell epitopes, have indeed suggested that also their activity in modulating disease may comply with the principles of dominant immunological tolerance.

Induction of antigen specific CD4+ T cell responses by invariant chain based DNA vaccines.

In this report, the use of DNA vaccination to induce class II restricted antigen specific proliferative responses was studied. To this end, a construct encoding the invariant chain (Ii) was engineered in which the Class II associated invariant chain peptide (CLIP) sequence was replaced by an immunogenic epitope derived form Heat Shock Protein 60, HSP60 178-186. Transfection studies in vitro showed that this construct can be used to efficiently load MHC class II molecules and present epitopes to MHC class II restricted antigen specific T cells. In addition, we showed that intradermal immunisation of Lewis rats with these constructs induced antigen specific T cells in vivo. Therefore, our Ii-gene constructs can be used to immunise for defined CD4+ T cell epitope sequences.

Molecular cloning and sequencing of the cDNA for dog interleukin-4

T lymphocytes play a crucial role in the development of protective immunity against intracellular pathogens. Studies on mouse CD4 T cell clones showed that helper T (Th) cells can be divided into Th1 and Th2 subsets based on their cytokine production. Interleukin-4 (IL-4) is a cytokine produced predominantly by the Th2 subset. It is involved in stimulation and class switching of B cells, acts as a growth factor for Th2 cells, and inhibits Th1 type of reactivity (Mosmann et al. 1987). In mice resistance and susceptibility toLeishmania infection were highly correlated with predominance of Th1 and Th2 reactivity, respectively (Locksley and Scott 1991). We study the immune responses in dogs infected with Leishmania infantum. Protective immunity in canine visceral leishmaniosis (CVL) is associated with a Th1 type of response (Pinelli et al. 1994, 1995). Our knowledge of CVL would greatly benefit from the development of dog Th2 specific reagents. This paper reports the cloning and sequencing of cDNA containing the coding sequence of dog IL-4 performed by the rapid amplification of cDNA ends (RACE) protocol (Frohman 1995). For this purpose mRNA isolated from concanavalin-A-stimulated dog peripheral blood mononuclear leucocytes isolated from a Leishmania–infected dog was reverse transcribed into

ICOS Contributes to T Cell Expansion in CTLA-4 Deficient Mice

Both CD28 and ICOS are important costimulatory molecules that promote Ag-specific cellular and humoral immune reactions. Whereas CD28 is generally thought to be the most important molecule in the initiation of a T cell response, ICOS is considered to act during the effector phase. We have investigated the contribution of ICOS to T cell responses in the absence of CTLA-4-mediated inhibition. Mice lacking CTLA-4, which show spontaneous CD28-mediated CD4+ T cell activation, expansion and differentiation, were treated with antagonistic αICOS antibodies. Blocking the interaction between ICOS and its ligand B7RP-1 significantly reduced this aberrant T cell activation and caused a reduction in T cell numbers. In vitro analysis of CD4+ T cells from treated mice revealed that ICOS blockade significantly reduced Th1 differentiation, while Th2 differentiation was only moderately inhibited. Further in vitro stimulation experiments demonstrated that ICOS is able to induce proliferation of murine CD4+ and CD8+ T cells but only in the presence of IL-2. These results indicate that ICOS is not only important for T cell effector function but also contributes to the expansion phase of a T cell response in the presence of CD28 signaling.

Effector and regulatory T cells derived from the same T cell clone differ in MHC class II-peptide multimer binding.

MHC class II‐peptide multimers are a valuable tool for antigen‐specific detection of CD4+ T cells. However, it has been proposed that T cells in a hypo‐responsive state can have diminished binding of such multimers. In the present study, we investigated this phenomenon at the clonal level. We found that anergic CD4+ T cells had a reduced capacity to bind MHC class II‐peptide multimers compared to their non‐anergic counterparts. Increasing the incubation temperature, time, or MHC‐peptide valency could not equalize multimer binding by anergic and non‐anergic T cells. Neither anergic T cells nor non‐anergic T cells internalized the MHC class II‐peptide dimers efficiently, and in both cases the dimers bound to the plasma membrane at locations containing a low amount of raft‐associated lipids. Disruption of lipid rafts, however, led to decreased dimer binding by non‐anergic T cells and to a lesser extent by anergic T cells. Finally, we show that the depth of the anergic state of the T cell, which determines its ability to regulate other T cell responses, correlates with the reduced dimer binding. We here demonstrate for the first time differential MHC class II‐peptide multimer binding by regulatory (anergic) and effector T cells with identical TCR.

Induction of experimental autoimmune arthritis by a public epitope of the T cell receptor variable α domain of an arthritogenic T cell clone

T cell receptor (TCR) peptide immunizations have been demonstrated to protect against experimental autoimmune diseases. These findings have led to clinical trials employing TCR peptides in multiple sclerosis and rheumatoid arthritis patients. Previously, we identified a strongly immunogenic region of the TCR α chain of an arthritogenic T cell clone (AV11 66‐80). In this report, we show that rats immunized with AV11 66‐80 developed arthritis with clinical symptoms and histology similar to adjuvant arthritis (AA). Transfer of this disease into naive rats using AV11 66‐80‐specific T cells proved the T cell‐mediated character of the disease. The AV11 66‐80 arthritic rats developed resistance to Mycobacterium tuberculosis‐induced AA, indicating that both forms of arthritis depended on similar regulatory mechanisms. This first demonstration of TCR peptide‐induced arthritis, together with an earlier report on a polymorphism in this very same AV11 66‐80 region involved in arthritis resistance in mice, suggests a central role of the public epitope AV11 66‐80 in the control of autoimmune arthritis. Although TCR peptide immunizations can be exploited to prevent experimental autoimmunity, caution should be taken in the induction of TCR peptide‐specific T cells for immunotherapy to avoid adverse effects as shown here.

Cartilage proteoglycan-specific T cells as vectors of immunomodulatory biologicals in chronic proteoglycan-induced arthritis.

Systemic administration of agents that neutralize or antagonize Th1-mediated pro-inflammatory responses has been demonstrated to ameliorate inflammation in chronic autoimmune disease. However, systemic administration of such immunosuppressive biologicals causes serious side effects and has only limited success. To minimize these side effects, autoantigen-specific lymphocytes have been proposed as a carrier to deliver immunosuppressive agents to sites of inflammation. Here we studied the effects of primary cartilage proteoglycan-specific CD4+ T cells that were transduced using an efficient method of viral transduction with active genes encoding IL-1beta receptor antagonist, soluble TNF-alpha receptor-Ig, IL-4 or IL-10 in chronic proteoglycan-induced arthritis in mice. This is the first study describing such gene therapy using primary CD4+ T cells in a chronic arthritis. Moreover, the impact of proteoglycan-specific Th1, Th2 or naïve T cells was studied. Although proteoglycan-TCR transgenic CD4+ T cells can transfer arthritis to lymphopenic recipients, none of the proteoglycan-TCR transgenic T cell phenotypes that were tested induced worsening of arthritis in wild type hosts. Proteoglycan-specific T cells ameliorated arthritis when expressing the transduced IL-10 gene, and not when expressing the other transgenes/phenotypes. Although all of the tested biologicals can suppress in a wide range of different inflammatory disorders, especially IL-10 would therefore serve as a promising candidate to be used in cellular gene therapy for chronic arthritis.

Comparison of detection techniques for cytokine reverse transcriptase polymerase chain reaction; digoxigenin-labeled polymerase chain reaction permits sensitive detection of cytokine mRNA in rat heart allografts.

The polymerase chain reaction (PCR) is a sensitive method for the analysis of cytokine mRNA expression. The amount of specific mRNA in tissues involved in an inflammatory immune response can be low and therefore requires highly sensitive detection of the PCR products. In our study we have compared different detection techniques in order to replace the commonly used detection by means of radiolabeled probes. Besides the detection of DNA in agarose gels by ethidium bromide (EB), we used detection by digoxigenin (DIG)-labeled probes, as well as the direct incorporation of DIG-labeled nucleotides in the PCR, in comparison to detection by means of 32P-labeled probes. In vitro activated rat lymph node cells, lymph node tissue, and acutely or chronically rejected rat heart allografts were examined for expression of mRNA of the cytokines IL-2 and IFNgamma. The directly DIG-labeled PCR appeared to be the best alternative for detection of PCR products by means of radiolabeled probes. While IL-2 mRNA was not detected by means of EB and IFNgamma mRNA was only detected at the highest PCR cycle numbers in acutely and chronically rejected rat heart allografts, both cytokine mRNAs were readily detected by directly DIG-labeled PCR.