Enhancement of helper and suppressor T cell activities by thymosin alpha 1 injection in old mice

A single injection of immunodeficient old (15-24 month old) BDF1 mice with 1-100 micrograms of synthetic thymosin alpha 1 (T alpha 1), a 28-amino acid residue peptide, shortly before horse erythrocyte (HRBC)-priming, enhances the helper activity of their spleen cells. Helper activity of spleen cells from uninjected or T alpha 1-injected HRBC-primed old mice was titrated by adding graded numbers of these primed cells to cultures containing a constant number of normal spleen cells from unprimed young (3-month-old) mice and the conjugate 2,4,6-trinitrophenyl (TNP)-HRBC. As evaluated from the in vitro anti-TNP antibody response, spleen cells exhibit higher helper activity when derived from T alpha 1-injected, as compared to uninjected, HRBC-primed old mice. No effect is observed when T alpha 1 is injected in young mice before HRBC-priming. Old mice were also injected with the N14 (N-terminal amino acid residues 1-14) or the C14 (C-terminal amino acid residues 15-28) synthetic fragments of the T alpha 1 molecule. Injection of T alpha 1 or the N14 fragment restores the helper activity of old mice to levels comparable to that displayed by helper cells from young mice. Conversely, injection of the C14 fragment in old mice induces only a negligible increase in helper activity of their spleen cells. T cells from HRBC-primed mice were separated by nylon wool filtration into Th1 (non-adherent) and Th2 (adherent) T cells and these cell subpopulations were assayed for helper activity. Helper activity of Th1 and Th2 cells is found to be impaired by aging when they are tested either separately or upon their recombination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of T-cell responses by CNS antigen-presenting cells: different roles for microglia and astrocytes

Analysis of the mechanisms underlying CNS immune surveillance and immunopathology have provided new insights into the intracerebral regulation of immune responses. Here, Francesca Aloisi, Francesco Ria and Luciano Adorini review the role of CNS antigen presenting cells and focus on the control of Th1 and Th2 responses by microglia and astrocytes.     

Deviation of pancreas-infiltrating cells to Th2 by interleukin-12 antagonist administration inhibits autoimmune diabetes

Nonobese diabetic (NOD) mice develop spontaneous insulin-dependent diabetes mellitus (IDDM), and the pancreas-infiltrating T cells invariably show a Th1 phenotype. We demonstrated here that the interleukin (IL)-12 antagonist (p40)₂ can deviate the default Th1 development of naive T cell receptor (TCR)-transgenic CD4⁺ cells to the Th2 pathway in vitro. Although (p40)₂ does not modify the cytokine profile of polarized Th1 cells, it prevents further recruitment of CD4⁺ cells into the Th1 subset. To study the involvement of Th1 and Th2 cells in the initiation and progression of IDDM, we targeted endogenous IL-12 by administration of (p40)₂ in NOD mice. (p40)₂ administration to NOD mice inhibits interferon-γ but not IL-10 production in response to lipopolysaccharide (LPS) or to the putative autoantigen IA-2. Serum immunoglobulin isotypes determined after (p40)₂ treatment indicate an increase in Th2 and a decrease in Th1 helper activity. Administration of (p40)₂ from 3 weeks of age onwards, before the onset of insulitis, results in the deviation of pancreas-infiltrating CD4⁺ but not CD8⁺ cells to the Th2 phenotype as well as in the reduction of spontaneous and cyclophosphamide-accelerated IDDM. After treating NOD mice with (p40)₂ from 9 weeks of age, when insulitis is well established, few Th2 and a reduced percentage of Th1 cells are found in the pancreas. This is associated with a slightly decreased incidence of spontaneous IDDM, but no protection from cyclophosphamide-accelerated IDDM. In conclusion, deviation of pancreas-infiltrating CD4⁺ cells to Th2 is associated with protection from IDDM. However, targeting IL-12 after the onset of insulitis, when the pancreas contains polarized Th1 cells, is not sufficient to induce an effective immune deviation able to significantly modify the course of disease.     

Approaches toward peptide-based immunotherapy of autoimmune diseases

Conclusions MHC class II-binding peptides administered in vivo can be effective and selective inhibitors of T cell activation. To selectively interfere with the activation of autoreactive T cells immunotherapy by peptides can be essentially directed to three cellular targets: APC, autoreactive T cells and regulatory T cells. Experimental models of autoimmune diseases have demonstrated that disease can be prevented or treated by peptides acting on each cellular target.The APC can be functionally inactivated by blocking the binding site of MHC class II molecules, thus preventing antigen presentation to T cells. We have demonstrated that injection of a class II-binding peptide can induce MHC class II blockade in vivo [22], resulting in inhibition of class II-restricted T cell activation and of T cell-dependent antibody responses. This is already a validated approach to prevent experimental autoimmune diseases [3, 32] and it may become applicable to the treatment of human autoimmune diseases.The autoreactive T cells could be inactivated by peptide-specific tolerance induction [14, 24], but it is necessary to define all potential autoantigenic epitopes to ensure complete and effective tolerance. Specific inhibition of autoreactive T cells could also be induced by antigen analogues acting as TCR antagonists [15], but this again requires, at a minimum, detailed knowledge of the autoantigen. Regulatory T cells represent a potentially interesting target for selective immunointervention by peptides. The restricted TCR V gene expression by autoreactive pathogenic T cells in some animal models of autoimmune diseases, for example the shared TCR V gene usage characteristic of EAE [1], raises the possibility of controlling self reactivity at the network level, as originally proposed by Cohen and colleagues [9]. Administration of peptides corresponding to TCR sequences utilized by autoreactive T cells has indeed been reported to down-regulate EAE, presumably via induction of anti-idiotypic T cells with suppressive activity [26, 43]. However, this approach does not always result in suppression of encephalitogenic effector T cells, and it may actually lead to enhanced chronic EAE [16]. It is also possible to induce regulatory T cells with suppressive activity by administration of peptides derived from the candidate autoantigen [24, 39] and this specific peptide therapy has been used to prevent the development of autoimmune diabetes in NOD mice [17]. However, if effective tolerance induction requires knowledge of all autoantigenic epitopes, induction of suppressor T cells by antigen is further complicated by our lack of definite molecular explanations for this phenomenon.Therefore, several immunoregulatory mechanisms induced by administration of class II-binding peptides may be responsible, in vivo, for interference with the activation of antigen-specific T cells. Unfortunately, with the possible exception of MHC blockade, the mechanisms involved are far from being clearly understood. What is needed is a more detailed analysis of these mechanisms in experimental autoimmune models to discern the precise mode of action of immunoregulatory peptides. This knowledge may then be applied to the treatment of human autoimmune diseases, keeping in mind that the clinical situation will invariably be even more complex.     

A miniaturized in vitro diffusion culture system.

A miniaturized diffusion culture apparatus holding 24 individual cultures and utilizing 1/10 the cells and volume of standard Marbrook-type cultures is described. This system was evaluated by studying the anti-sheep erythrocyte response of spleen cells from unprimed or primed and boosted mice. Compared to parallel Mishell--Dutton plate cultures memory responses in mini-diffusion cultures rise more slowly, peak 1--2 days later and thereafter decline less rapidly. Moreover, the diffusion cultures give a pronounced IgG response peaking at 6 days, while the abortive IgG response evident in plate cultures at 4 days rapidly disappears. While viability in the two systems is equivalent, cell recovery on days 7 and 8 is markedly higher in the diffusion cultures. Of 4 cell concentrations tested, 2 million cells are found to produce the optimum anti-sheep erythrocyte plaque-forming cell response. A 0.2 micron nucleopore membrane is preferable to a dialysis membrane and yields up to a 2-fold higher response. Replacement of the reservoir medium on day 4 impairs rather than improves the response. The culture system was also found to support an excellent primary response. This response is dependent on the presence of 2-mercaptoethanol and does not require rocking.     

Spontaneous lymphomas in mice genetically selected for high or low phytohemagglutinin responsiveness

Biozzi mice selected for high (Hi) or low (Lo) responsiveness to phytohemagglutinin (PHA) have been followed for their entire life-span to examine their pathology at death. Spontaneous lymphomas were found to exhibit higher incidence and faster development in Lo/PHA than in Hi/PHA females, whereas a similar difference between the two lines did not attain the level of statistical significance in male mice. The incidence of solid tumors was higher in Lo/PHA than in Hi/PHA males but the same in females of the two lines, yet the probability of dying from solid tumors was slightly increased in Lo/PHA mice of both sexes. All these results indicate that T-cell-mediated immunity influences mainly the spontaneous incidence of lymphomas and, to a lesser degree, the appearance of other solid tumors.     

Antigen presentation of hen egg-white lysozyme but not of ribonuclease A is augmented by the major histocompatibility complex class II-associated invariant chain

The influence of the class II-associated invariant chain (Ii) on the presentation of the protein antigens hen egg-white lysozyme (HEL) and ribonuclease A (RNase) was investigated. For this purpose the Ii- rat-2 fibroblasts were transfected with I-Ak genes with or without Ii. Transfectants expressing Ii were superior in the presentation of the complete HEL protein to a panel of I-Ak-restricted T hybridomas characterized by distinct specificities for different HEL peptides and by different sensitivities to antigen concentration. There appeared to be a correlation between the antigen-presenting capacity and the amount of Ii, in that transfectants expressing large amounts of Ii were the best antigen presentors. The presentation of synthetic HEL peptides was not influenced by Ii. In contrast to the findings with HEL, the presentation of RNase by the same set of transfectants was clearly independent of Ii. Both antigens, HEL and RNase, required processing in the chloroquine-sensitive compartment. However, only the presentation of HEL but not of RNase could be efficiently blocked by brefeldin A. These data confirm that presentation of HEL depends on de novo synthesized class II molecules, whereas the presentation of RNase seems to be predominantly mediated by a pool of pre-existing class II molecules whose interaction with endocytosed antigen does not depend on Ii. These results suggest different mechanisms for the presentation of HEL and RNase and they raise the possibility that different antigens intersect the class II pathway at distinct intracellular locations.     

Immunoregulation of lysozyme-specific suppression. II. Hen egg-white lysozyme-specific monoclonal suppressor T cell factor suppresses the afferent phase of delayed-type hypersensitivity and induces second-order suppressor T cells

Culture supernatant from a monoclonal T cell lymphoma line (LH8-105) obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme (HEL)-specific suppressor T lymphocytes is able, when injected into mice, to specifically suppress the delayed-type hypersensitivity (DTH) reaction induced by HEL. The suppressor T cell factor (TsF) exhibits fine antigenic specificity since it suppresses the DTH response induced by HEL without affecting the DTH response induced by ring-necked pheasant egg-white lysozyme (REL), a lysozyme closely related to HEL. Conversely, LH8-105 TsF is able to suppress the DTH response induced by human lysozyme, distantly related to HEL but sharing a common epitope critical for induction of suppressive activity. The fine antigenic specificity of LH8-105 TsF for a restricted epitope on the HEL molecule is confirmed by binding to HEL but not to REL immunosorbents. This TsF also bears I-J determinants, as demonstrated by binding to monoclonal anti-I-J immunosorbents, and it suppresses the afferent but not the efferent phase of the DTH response to HEL. The afferent suppression is controlled by genes apparently mapping in the I-J subregion of the H-2 complex since I-J-incompatible mice are not suppressed by LH8-105 TsF injection. This inducer-type TsF induces second-order effector suppressor T cells only in HEL-primed mice indicating the primary role of antigen, in association with H-2 (I-J) products, in the afferent portion of this suppressive circuit.     

A microcomputer program for hydrophilicity and amphipathicity analysis of protein antigens

A simple and ready-to-use program to analyze hydrophilicity and amphipathicity characteristics of protein sequences is described. Three of the most commonly used hydrophilicity scales can be selected and the block length can also be varied. The program is written in BASIC language, can be run on any microcomputer and does not require computer experience. The program output is exemplified by a sample of lysozyme hydrophilicity and amphipathicity analysis.     

Normal B cells fail to secrete interleukin-12

Interleukin-12 is a key regulatory cytokine produced by antigen-presenting cells (APC) which drives the development of interferon-γ (IFN-γ)-producing cells and promotes cell-mediated immunity. Following subcutaneous immunization with protein antigen in adjuvant, dendritic cells (DC) but not small nor large B cells in immune lymph nodes express antigenic complexes and secrete substantial amounts of bioactive IL-12 p75 upon antigen-specific interaction with T cells. We have analyzed secretion of IL-12 p40 and p75 by cell populations enriched in DC, macrophages or B cells in response to nonspecific stimulation or to interaction with antigen-specific CD4⁺ cells. These APC populations do not produce IL-12 constitutively but, upon stimulation with heat-fixed Staphylococcus aureus and IFN-γ, IL-12 p40 and p75 are secreted by DC and macrophages, whereas B cells fail to produce IL-12. B cells also fail to secrete IL-12 in response to stimulation with LPS and IFN-γ. Co-culture with CD4⁺ T hybridoma cells and antigen induces IL-12 secretion by DC. Up-regulation of IL-12 secretion by interaction with antigen-specific CD4⁺ T cells is abrogated by anti-class II monoclonal antibodies (mAb), by soluble CD40 molecules and by anti-CD40 ligand mAb, demonstrating a positive feedback between T cells and DC mediated by TCR-peptide/class II and by CD40-CD40 ligand interactions. Expression of class II and CD40 molecules is comparable in B cells and DC, and both APC types activate CD4⁺ T cells. Yet, even upon interaction with antigen-specific T cells, B cells fail to secrete IL-12. The capacity of B cells to present antigen but not to secrete IL-12 may explain their propensity to selectively drive T helper type 2 cell development.