Georgette Vansanten

Anti-CTLA-4 treatment induces IL-10-producing ICOS+ regulatory T cells displaying IDO-dependent anti-inflammatory properties in a mouse model of colitis

Background and aims: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to act as a negative regulator of T cell function and has been implicated in the regulation of T helper 1 (Th1)/Th2 development and the function of regulatory T cells. Tests were carried out to determine whether anti-CTLA-4 treatment would alter the polarisation of naive T cells in vivo. Methods: Mice were treated with anti-CTLA-4 monoclonal antibody (mAb) (UC10-4F10) at the time of immunisation or colonic instillation of trinitrobenzene sulfonic acid (TNBS). The cytokines produced by lymph node cells after in vitro antigenic stimulation and the role of indoleamine 2,3 dioxygenase (IDO) and of interleukin-10 (IL-10) were tested, and the survival of mice was monitored. Results: Injection of anti-CTLA-4 mAb in mice during priming induced the development of adaptive CD4+ regulatory T cells which expressed high levels of ICOS (inducible co-stimulator), secreted IL-4 and IL-10. This treatment inhibited Th1 memory responses in vivo and repressed experimental intestinal inflammation. The anti-CTLA-4-induced amelioration of disease correlated with IDO expression and infiltration of ICOShigh Foxp3+ T cells in the intestine, suggesting that anti-CTLA-4 acted indirectly through the development of regulatory T cells producing IL-10 and inducing IDO. Conclusions: These observations emphasise the synergy between IL-10 and IDO as anti-inflammatory agents and highlight anti-CTLA-4 treatment as a potential novel immunotherapeutic approach for inducing adaptive regulatory T cells.

Injection of lipopolysaccharide induces the migration of splenic neutrophils to the T cell area of the white pulp: role of CD14 and CXC chemokines.

There is increasing evidence that neutrophils are involved in the regulation of adaptive immunity. We therefore tested whether these cells may colocalize with T lymphocytes in lymphoid organs. Our results demonstrate that administration of the microbial product LPS induces the migration of neutrophils in the spleen from the red pulp and the marginal zone to the area of the white pulp where T cells reside. This movement is CD14‐dependent, whereas the recruitment of neutrophils in the peritoneal cavity is increased in the absence of CD14. Our data further suggest the involvement of the chemokine MIP‐2 and keratinocyte‐derived chemokine and their receptor CXCR2. We conclude that neutrophils may interact with naïve T cells upon infection/inflammation and that the migration of neutrophils in the lymphoid organs and in the periphery is regulated differently by a signal transduced by CD14

Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs

Aims—To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues. Methods—This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared. Results—Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessels. Conclusions—These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur.

Distinct VH repertoires in primary and secondary B cell lymphocyte subsets in the preimmune repertoire of A/J mice: the CRI‐A idiotype is preferentially associated with the HSAlow B cell subset

The anti‐arsonate immune response of A/J mice is characterized by the occurrence of several recurrent idiotypes with a different temporal pattern of expression. The CRI‐A idiotype is typically a memory idiotype since it appears late in the primary and dominates the secondary as well as subsequent immune responses. The CRI‐C idiotype is present throughout the responses, including the primary one. Naive adult A/J mice treated repeatedly with anti‐μ or anti‐δ monoclonal antibodies exhibit a completely different balance of HSAlow and HSAhigh B cell subsets and an opposite idiotype profile after immunization with p‐azophenylarsonate coupled to hemocyanin. Anti‐μ  treatment leads to a striking enhancement of the HSAlow cell subset associated with an earlier important synthesis of CRI‐A+ antibodies, while anti‐δ treatment enhances significantly the HSAhigh compartment with a strong decrease of CRI‐A and persistence of CRI‐C1 antibodies. Semiquantitative PCR analysis reveals that the presence of CRI‐A transcripts is associated with the HSAlow compartment, while CRI‐C transcripts are mainly associated with HSAhigh B cell subsets. This has been demonstrated with spleen cells of adult A/J mice treated with anti‐μ or anti‐δ antibodies and also with purified B cell subsets of unimmunized adult A/J mice and on neonatal spleen cells. It appears that the memory (CRI‐A) idiotype is selected into the HSAlow B cell subset before antigen arrival.

PHENOTYPIC CHARACTER OF PHAGE PROTEIN ABNORMALITIES INDUCED BY BROMOURACIL.

Abstract The incorporation of bromouracil, a thymine analogue, into the DNA of bacteriophage T 2 stimulates the synthesis of significant quantities of abnormal proteins. Their abnormal character is deduced from the study of certain properties of phages produced in the presence of bromouracil: stability of structure, adsorption on a sensitive bacterial strain and immunologic specificity. The modifications studied are not transmitted to the progeny synthesized in the absence of the base analogue. In the presence of bromouracil, the synthesis of phage DNA and protein is much less affected than the synthesis of virus particles. This difference is attributed to the fact that the abnormal proteins cannot be integrated into the phage particles.

On the structural alterations of phage proteins synthesized in the presence of pancreatic ribonuclease

Abstract The addition of pancreatic ribonuclease to a lysogenic culture of Bacillus megaterium , induced by ultraviolet irradiation, leads to the formation of noninfectious phage particles of normal deoxyribonucleic acid content. The stability of the tail structure is sharply decreased. The head proteins, dispersed by a sonic oscillator, fail to react with the specific antibodies of control phage. The peptides obtained by tryptic digestion give a distinctly abnormal “fingerprint” by chromatography and electrophoresis. The hypothesis is put forward that the ribonuclease acts by modifying the specificity of the ribonucleic acids involved in the ordering of amino acids along the genetic messenger.

Altered affinity maturation in primary response to (4-hydroxy-3-nitrophenyl) acetyl (NP) after autologous reconstitution of irradiated C57BL/6 mice.

Immune responses developing in irradiated environment are profoundly altered. The memory anti-arsonate response of A/J mice is dominated by a major clonotype encoded by a single gene segment combination called CRIA. In irradiated and autoreconstituted A/J mice, the level of anti-ARS antibodies upon secondary immunization is normal but devoid of CRIA antibodies. The affinity maturation process and the somatic mutation frequency are reduced. Isotype switching and development of germinal centers (GC) are delayed. The primary antibody response of C57BL/6 mice to the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP)-Keyhole Limpet Hemocyanin (KLH) is dominated by antibodies encoded by a family of closely related VH genes associated with the expression of the λ1 light chain.We investigated the anti-NP primary response in irradiated and autoreconstituted C57BL/6 mice. We observed some splenic alterations as previously described in the irradiated A/J model. Germinal center reaction is delayed although the extrafollicular foci appearance is unchanged. Irradiated C57BL/6 mice are able to mount a primary anti-NP response dominated by λ1 positive antibodies but fail to produce high affinity NP-binding IgGl antibodies. Following a second antigenic challenge, irradiated mice develop enlarged GC and foci. Furthermore, higher affinity NP-binding IgG1 antibodies are detected.