Inhibitors of Src-family tyrosine kinases favour Th2 differentiation.

Two potent pyrazolo-pyrimidine inhibitors of Src-family kinases were found to enhance interleukin 4 (IL-4) and reduce interferon gamma(IFN-gamma) production in cultures of splenocytes from ovalbumin-specific TCR-transgenic BALB/c mice. The effect was increased by addition of a monoclonal antibody binding to domains 3/4 of CD4, while other antibodies binding to domain 1 had the opposite effect. The inhibitors suppressed CD40 ligand (CD40L) expression on activated CD4 T cells, which may explain this Th2 differentiating effect. More generally, the effect fits within the overall framework of signal attenuation in T cells driving this form of differentiation. The inhibitors did not revert previously induced Th1 differentiation in serial cultures of the TCR-transgenic cells. Drugs with this activity are of obvious interest as probes and potential therapeutic agents in autoimmune disease.

The importance of the back-signal from T cells into antigen-presenting cells in determining susceptibility to parasites.

It has long been known that certain MHC class II genes can dominantly suppress immune responses and so increase susceptibility to parasite infections, but the mechanism has been unclear. Recent work has revealed one way in which this form of suppression may operate, through gating by MHC class II molecules of the back-signal from activated T cells into macrophages. The two known suppressive genes of the mouse are expressed in macrophages more extensively than are other class II genes. This is associated with suppression of IL-4 production resulting, we infer, from overproduction in the macrophages of IL-12, the counter-cytokine to IL-4. The lack of IL-4 may itself be immunosuppressive, even for Th2 responses, and excess IL-12 can overinduce the antiproliferative cytokine IFN-gamma. Although this mechanism requires further substantiation, we believe that it offers a reasonable answer to an old conundrum.

Polymorphic MHC class II promoters exhibit distinct expression pattern in various antigen-presenting cell lines.

The promoter regions of MHC class II genes are characterized by the presence of conserved sequence motifs called S,X and Y boxes, which are crucial for regulation of transcription of these genes. In humans, promoter polymorphism is known to result in differential transcriptional activity at both inter-locus and inter-allelic levels, but it is not yet known how this relates to tissue-specific expression of MHC class II molecules. We sequenced the 5' regulatory regions of alpha and beta genes of I-A and I-E molecules from four mouse haplotypes and found allelic polymorphisms which were mainly confined to the X box. The promoter sequences of I-Ea genes were non-polymorphic. Transfection of four antigen-presenting cell types with promoter-reporter gene constructs revealed that the promoter sequence polymorphisms result in distinct allele- and tissue-specific activity patterns. Mutagenesis experiments in which the X2 box was reshuffled between I-A beta alleles demonstrated that this box contributes to regulation of differential MHC class II expression in the four cell types. The possibility is discussed that tissue-specific MHC class II expression may control differentiation of T-cell subsets.

Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins.

In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unusually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics.

Protective major histocompatibility complex genes and the role of interleukin-4 in collagen-induced arthritis.

To investigate the role of interleukin (IL)-4 during the triggering of collagen-induced arthritis, we examined the effects of the I-A(b) and I-E protective/suppressive genes and passively administered anti-IL-4 monoclonal antibody. In contrast to the action of I-E expression on its own, which has mainly a suppressive effect post-triggering, the combination of I-A(b) and I-E had a marked protective effect. Assuming, on the basis of previous experience with the I-A(b) allele, that it might act through suppressing early IL-4 production, we treated mice with the 11B11 IL-4-neutralizing antibody around the time of initial immunization with collagen. Treatment over a period extending to 6 days post-immunization exacerbated the arthritis, but when curtailed to 2 days post-immunization (and tested in pristane-primed animals), the disease was reduced. We conclude that IL-4 plays an essential role in triggering the disease.

Non-classical-MHC genetics of immunological disease in man and mouse. The key role of pro-inflammatory cytokine genes.

For a series of immunological diseases including asthma, inflammatory arthritis and experimental allergic encephalomyelitis the non-classical major histocompatibility complex (MHC) genetics of man and mouse has been making rapid progress. Information is available not only for the disease associations of individual candidate genes but also from the first genome scans. In both species the proinflammatory cytokine genes and/or their related receptors and inhibitors (IL-1, IL-1r, IL-1ra, IL-2, IL-6r, TNF-alpha), and to a lesser extent the anti-inflammatory cytokine IL-4 are implicated as candidate control elements. In contrast, genes for the signalling and adhesion CD molecules have so far been inconspicuous. Most of the polymorphisms so far detected have been in the regulatory sequences of these genes, rather than in the exons. It is suggested that the benefit conferred on an individual by greater flexibility in its immunoregulatory machinery may be responsible for maintaining this form of polymorphism.

Unrepresentative behavior of T cell receptor-transgenic CD4+ T cells upon adoptive transfer: lack of need for priming and an extended booster dose-response.

The response of CD4+ T cells taken from DO11.10 alpha beta TCR-transgenic mice to their specific antigen, ovalbumin, was examined in an adoptive transfer system. Read out was the % frequency of KJI-26.1+ (clonotype positive) cells within the Thy-1.2+ (T cell) population in lymph nodes. Control experiments indicated that these cells were uniformly CD4+. Immunizing the transgenic mice had no detectable effect on this frequency. Furthermore, the frequency in recipients of adoptively transferred lymph node cells was not affected by priming of the donors with ovalbumin by various procedures. Transfers were into syngeneic SCID recipients, except in one experiment, where irradiated recipients were shown to behave in the same way. Examining the effect of varying the amount of booster antigen, the response increased slowly with dose, up to a plateau in the range of 10-100 mg ovalbumin. The lack of need for priming is unusual, in comparison with an adoptive transfer of non-transgenic cells, as is the extended dose response range with such a high optimum dose. This enhanced responsiveness is interpreted in terms of a lack of down-immunoregulation in these transgenic mice.

Altered Th1/Th2 balance associated with the immunosuppressive/protective effect of the H-2Ab allele on the response to allo-4-hydroxyphenylpyruvate dioxygenase.

The H-2Ab allele exerts a dominant down-regulatory effect on the anti-allo-HPPD (4-hydroxyphenylpyruvate dioxygenase) antibody response, through a hitherto unknown mechanism. In the present study, the allo-variable peptide bound to responder H-2Ak molecules with higher affinity than to H-2Ab ones, arguing against the operation of an affinity hierarchy. Quantitative polymerase chain reaction revealed differences in cytokine mRNA expression between suppressed and high-responder mice. Lymph node cells of responder but not suppressed mice contained high levels of interleukin (IL)-4 mRNA as early as 11 h post-immunization and continued to do so for at least 8 days; this early burst was paralleled by a small burst in transforming growth factor (TGF)-beta mRNA level. Differences in IL-12 mRNA were not detected, although an early IL-12 effect could not be excluded. Interferon (IFN)-gamma appeared to contribute to the suppression at later time points. Early treatment of responder mice with anti-IL-4 monoclonal antibody (11B11) down-regulated the antibody response. The proliferative T cell response from hyperimmunized mice was reduced but still detectable in the presence of an H-2Ab allele. Thus, in the presence of this allele, the Th1 response is enhanced and that of Th2 cells suppressed, apparently as a result of the bias of H-2Ab-restricted T cells in favor of the Th1 subset.

Immunological basis of oral tolerance.

Oral tolerance may be defined as a specific reduction in the immune response brought about by feeding an antigen. It has been reviewed by us recently as a possible treatment of rheumatoid arthritis. It has a respectably long history as an experimental phenomenon, in the course of which a variety of modes of action have been proposed. More recently the following mode of action has been proposed: An antigen, for instance foreign type II collagen, passes from the lumen of the gut across multifold-cells (M-cells) lying under Peyer's patches, and thence into antigen-presenting cells within the patches. These cells then activate a local population of T cells which specializes in the secretion of transforming growth factor-beta (TGF beta) and IL-4. Following activation a few of these cells wander out through the lymphatics and blood stream, and thence through tissue, until they again find type II collagen, their recall antigen. What they find in a patient with inflammatory arthritis, it is believed, is self-type II collagen exposed within the inflamed joints, which they recognize via its cross-reaction with the foreign collagen which had originally activated them. The specialized T cells are then stimulated by their recall antigen to secrete TGF beta and IL-4. These inhibitory cytokines suppress the activity of neighboring disease-inducing Th1 cells ("bystander suppression"). The latter cells presumably recognize one or more autoantigens, the nature of which is unknown. It need not be type II collagen, which figures in the whole story only as an organ-specific antigen, which lures the suppressive T cells to the right place.

[Therapy with oral type II collagen as a new possibility of selective immunosuppression in therapy of rheumatoid arthritis]. / Die Therapie mit oralem Kollagen Typ II als neue Möglichkeit der selektiven Immunsuppression in der Therapie der rheumatoiden Arthritis.

Suppressor T cells in the mucosa of the gut are activated by absorbed antigen in order to avoid a systemic immune response to this antigen. This long known phenomenon of oral tolerance is now used in the treatment of rheumatoid arthritis with oral collagen type II which is the most important protein of cartilage. Although the role of collagen II in initiating and maintaining the immune response in the joint is not clear, these suppressor CD8+T cells can be stimulated in a trigger-specific and effector-nonspecific way by contact with collagen II in the joint. It is assumed that a local immunosuppression then takes place through the secretion of inhibitory cytokines, mainly TGF beta. Clinical studies in the treatment of rheumatoid arthritis are presently being conducted in Boston and Berlin.

Hyper-reactivity of mouse CD45RA- T cells.

Mouse CD4 T cells have been partitioned into CD45RA and CD45RA- subpopulations by means of the monoclonal antibody 14.8. The CD45RA- subpopulation proliferated more actively and generated more interleukin-4 (IL-4) in response to stimulation with anti-CD3 antibody and phytohemagglutinin, and more IL-2 in response to anti-CD3. This subpopulation is therefore hyper-reactive to these polyclonal stimulators, but does not show the bias towards T helper type 2 activity that has been found in studies with other related CD45 isoforms. No evidence of suppression was obtained by comparing proliferation of CD45RA- cells in the presence and absence of CD45RA cells. Thus mouse CD4 T cells behave in these respects similarly to those of man, as is evident in a brief review of the quiescence-activation-quiescence cycle in the two species.

Latent help to and from H-2 antigens.

An optimized system for probing allo-immunity to major histocompatibility complex (MHC) antigens by means of adoptive transfer is used to confirm and extend previous work showing that naturally occurring class I MHC antigens, while capable of inducing Th activity when presented in combination with other allo-antigens, fail to do so on their own. The Th activity which they do induce develops slowly, after repeated immunizations, and can properly be described as "latent". Latency, or "cripticity" as it is also termed, may help explain how autoimmune disease is initiated.

Memory in helper T cells of minor histocompatibility antigens, revealed in vivo by alloimmunizations in combination with Thy-1 antigen.

A cooperative antibody response in which T helper (Th) cells recognize minor histocompatibility antigens (mha) and B cells recognize Thy-1 antigen, is used to explore memory in the T cell compartment. In contrast to B cell memory, Th memory reaches a plateau rapidly, although Th memory of Thy-1 itself (or an associated antigen) behaves exceptionally in this respect. The plateau then extends over several weeks at least. Single mha, among them H-Y, generate detectable memory. Incompatible H-2 antigens, including class I antigens on their own, inhibit this response through what appears to be a mechanism of intracellular antigenic competition. Antigen presentation in this system is by host cells, as judged by lack of donor-specific restriction. Memory resides in both the CD45RA+ and CD45RA- compartments, although the majority of memory Th cells have the latter phenotype.

Tolerance of self induced in thymus organ culture.

F liver protein occurs in serum at low concentration, and therefore induces tolerance of self only in T cells. T cells which mature in cultured thymus lobes in the absence of this protein become reactive towards it but can be prevented from doing so by exposure to the protein while in culture. The threshold of tolerance induction for this soluble antigen is estimated in this way at approximately 1 microgram/ml, which is slightly less than the threshold of response of primed T cells in a proliferation assay. Freshly isolated thymocytes do not display reactivity to self-F protein, indicating that T cells normally become tolerant while still within the thymus.

Culture of specimens other than sputum for Mycobacteria.

A comparison has been made of Lowenstein-Jensen medium, with or without added pyruvate, 7H11 oleic acid-albumin agar slopes made selective by the addition of antibiotics, and selective Kirchner medium in the isolation of tubercle bacilli from specimens other than sputum. A combination of a standard mild preliminary treatment with sulphuric acid and selective medium reduced the contamination rate to 0.1%. Neither acid treatment nor antibiotics appeared to reduce the isolation rate. An effective procedure is to treat all specimens (except cerebrospinal fluid) with acid and culture on two Lowenstein-Jensen slopes (one with pyruvate), a 7H11 selective slope, and in two bottles of selective Kirchner medium.