Autoreactive T cells that bypass negative selection are not tolerant and respond to self-antigen stimulation during infection

Le répertoire cellulaire Τ a pour but d'être tolérant aux antigènes du soi afin d'éviter l'induction de maladies autoimmunes. C'est pourquoi les lymphocytes Τ autoréactifs sont éliminés dans le thymus lors de leur développement par le processus de sélection négative. La plupart des recherches étudient les lymphocytes Τ de haute avidité. Ces lymphocytes Τ de haute avidité sont très sensibles et réagissent fortement à un antigène du soi. En conséquence, ces cellules induisent le développement de maladies autoimmunes lorsqu'elles ciblent des organes exprimant l'antigène du soi. Plusieurs études ont montré que les lymphocytes Τ qui réagissent faiblement aux antigènes spécifiques à un tissu, nommé lymphocytes Τ de faible avidité, peuvent contourner les mécanismes de tolérance centrale et périphérique. J'ai utilisé des souris Rip-mOva qui expriment l'Ovalbumine comme antigène du soi spécifique à un tissu. Dans ces souris transgéniques Rip-mOva, les lymphocytes Τ de faible avidité survivent à la sélection négative. Une fois stimulés à la périphérie, ces lymphocytes Τ CD8+ de faible avidité ont la capacité d'infiltrer les organes qui expriment l'antigène du soi chez les souris Rip-mOva et peuvent induire une destruction tissulaire. L'objectif principal de mon projet de thèse était de comprendre les caractéristiques phénotypiques et fonctionnelles de ces lymphocytes Τ dans un état d'équilibre et dans un contexte infectieux. Pour étudier ces cellules dans un modèle murin bien défini, nous avons généré des souris exprimant un récepteur de cellule Τ transgénique appelé OT-3. Ces souris transgéniques OT-3 ont des lymphocytes Τ CD8+ de faible avidité spécifiques à l'épitope SIINFEKL de l'antigène Ovalbumine. Nous avons démontré qu'un grand nombre de lymphocytes Τ CD8+ OT-3 ne sont pas éliminés lors de la sélection négative dans le thymus après avoir rencontré l'antigène du soi. Par conséquent, les lymphocytes Τ OT-3 de faible avidité sont présents dans une fenêtre de sélection comprise entre la sélection positive et négative. Cette limite se définie comme le seuil d'affinité et est impliquée dans l'échappement de certains lymphocytes Τ OT- 3 autoréactifs. A la périphérie, ces cellules sont capables d'induire une autoimmunité après stimulation au cours d'une infection, ce qui nous permet de les définir comme étant non tolérante et non dans un état anergique à la périphérie. Nous avons également étudié le seuil d'activation des lymphocytes Τ OT-3 à faible avidité à la périphérie et avons constaté que des ligands peptidiques plus faibles que l'épitope natif SIINFEKL sont capables de les activer au cours d'une infection ainsi que de les différencier en lymphocytes Τ effecteurs et mémoires. Les données illustrent une déficience lors de la sélection négative dans le thymus de lymphocytes Τ CD8+ autoréactifs de faible avidité contre un antigène du soi spécifique à tissu et montrent que ces cellules sont entièrement compétentes lors d'une infection. - The diverse Τ cell repertoire needs to be tolerant to self-antigen to avoid the induction of autoimmunity. This is why autoreactive developing Τ cells are deleted in the thymus. The deletion of self-reactive Τ cells occurs through the process of negative selection. Most studies investigated high avidity Τ cells. These high avidity Τ cells are very sensitive and strongly react to a self-antigen. As a consequence, these cells induce the development of autoimmunity when they target organs which express the self-antigen. High avidity autoreactive CD8+ Τ cells are deleted in the thymus. However, several studies have shown Τ cells that weakly respond to tissue-restricted antigen, referred to as low avidity Τ cells, can bypass central and peripheral tolerance mechanisms. I used Rip-mOva mice that expressed Ovalbumin as a neo self-antigen in a tissue-restricted fashion. In these transgenic Rip-mOva mice low avidity CD8+ Τ cells survive negative selection. Upon stimulation in the periphery, these low avidity CD8+ Τ cells have the ability to infiltrate organs that express the self-antigen in the Rip-mOva mice and can also induce the destruction of the tissue. The major aim of my PhD project was to understand the phenotypic and functionality characteristics of these Τ cells in a steady-state condition and in a context of an infection. To study these cells in a well-defined mouse model, we generated OT-3 Τ cell receptor transgenic mice that express low avidity CD8+ Τ cells that are specific for the SIINFEKL epitope of the Ovalbumin antigen. We have been able to demonstrate that a large number of OT-3 CD8+ Τ cells survive negative selection in the thymus after encountering the self-antigen. Thus, low avidity OT-3 Τ cells are present in a window of selection comprised between positive and negative selection. This boundary defined as the affinity threshold is involved in the escape of some autoreactive low avidity OT-3 Τ cells. Once they circulate in the periphery, they are able to induce autoimmunity after stimulation during an infection, allowing us to allocate these cells as being non-tolerant and not in an anergic state in the periphery. We have also looked at the threshold of activation of low avidity OT-3 CD8+ Τ cells in the periphery and found that peptide ligands that are weaker than the native SIINFEKL epitope are able to activate OT-3 Τ cells during an infection and to differentiate them into effector and memory Τ cells. The data illustrate the impairment of negatively selecting low avidity autoreactive CD8+ Τ cells against a tissue-restricted antigen in the thymus and shows that these cells are fully competent upon an infection.

Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular tumor spheroids.

The binding and penetration of two 125I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) and their F(ab')2 and Fab fragments were measured in multicellular spheroids of poorly (HT29) and moderately well differentiated (Co112) human colon adenocarcinomas which express different amounts of CEA. Spheroids cultured in vitro model tumor microenvironments where poor vascular supply may modulate antigen expression and accessibility. The two MAb studied, 202 and 35, were shown previously to react with different CEA epitopes and to have high affinities of 1.2 and 5.8 X 10(9) M-1, respectively. MAb 202 has also been shown to cross-react with antigens present on human granulocytes and normal epithelial cells from human lung and pancreas. Specific binding of intact MAb and fragments of both antibodies was demonstrated for both types of human colon carcinoma spheroids compared to mouse colon carcinoma (CL26) and mammary tumor (EMT6/Ro) spheroids. Total binding of MAb and fragments was greater (1.5- to 2.5-fold) after 4 h compared to 1 h of exposure; the amount of binding compared to control IgG1 was 5- to 30-fold greater after 1-h incubation and 15 to 200 times greater after 4 h. This binding was stable as demonstrated by short and long wash experiments at 37 degrees and 4 degrees C. The binding of F(ab')2 and Fab fragments of the anti-CEA MAb 35 to spheroids of human colon Co112 was almost 2-fold greater than that of the intact MAb. However, for MAb 202, the binding of intact MAb and F(ab')2 was greater than that of Fab fragments. In addition the binding of both intact and F(ab')2 fragments of MAb 202 was greater than that obtained with MAb 35. Specific binding of both antibodies to HT29 spheroids, which express less CEA, was decreased for MAb and fragments of both 202 and 35. Autoradiography and immunoperoxidase experiments were performed to determine the penetration of MAb and fragments after incubation with intact spheroids. Comparisons were made with labeled MAb directly applied to frozen sections of spheroids. F(ab')2 and Fab fragments of both antibodies were bound at the surface of intact spheroids and penetrated to eight to ten cells, but the intact MAb were localized mainly at the spheroid surface and the outer one to three cell layers. There was much less binding at the surfaces of HT29 compared to Co112 spheroids. An enzyme immunoassay using MAb 35 and 202 demonstrated that Co112 spheroids produced about 8-fold more CEA/mg of cell protein than did monolayer cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

T cell reactivity against mycolyl transferase antigen 85 of M. tuberculosis in HIV-TB coinfected subjects and in AIDS patients suffering from tuberculosis and nontuberculous mycobacterial infections.

The mycolyl transferase antigen 85 complex is a major secreted protein family from mycobacterial culture filtrate, demonstrating powerful T cell stimulatory properties in most HIV-negative, tuberculin-positive volunteers with latent M.tuberculosis infection and only weak responses in HIV-negative tuberculosis patients. Here, we have analyzed T cell reactivity against PPD and Ag85 in HIV-infected individuals, without or with clinical symptoms of tuberculosis, and in AIDS patients with disease caused by nontuberculous mycobacteria. Whereas responses to PPD were not significantly different in HIV-negative and HIV-positive tuberculin-positive volunteers, responses to Ag85 were significantly decreased in the HIV-positive (CDC-A and CDC-B) group. Tuberculosis patients demonstrated low T cell reactivity against Ag85, irrespective of HIV infection, and finally AIDS patients suffering from NTM infections were completely nonreactive to Ag85. A one-year follow-up of twelve HIV-positive tuberculin-positive individuals indicated a decreased reactivity against Ag85 in patients developing clinical tuberculosis, highlighting the protective potential of this antigen.

Radiolabeled fragments of monoclonal antibodies against carcinoembryonic antigen for localization of human colon carcinoma grafted into nude mice.

Four monoclonal antibodies against carcinoembryonic antigen (CEA) have been selected from 32 hybrids that produce antibodies against this antigen, by the criteria of high affinity for CEA and low cross-reactivity with granulocyte glycoprotein(s). The specificity of tumor localization in vivo of the four MAb, and their F(ab')2 and Fab fragments was compared in nude mice bearing grafts of a serially transplanted, CEA-producing, human colon carcinoma. The distribution of radiolabeled MAb and their fragments after intravenous injection was analyzed by direct measurement of radioactivity in tumor and normal organs, as well as by whole-body scanning and by autoradiography of tumor sections. Paired labeling experiments, in which 131I-labeled antibody or fragments and 125I-labeled control IgG are injected simultaneously, were undertaken to determine the relative tumor uptakes of each labeled protein. The tumor antibody uptake divided by that of control IgG defines the specificity index of localization. Tumor antibody uptakes (as compared with the whole mouse), ranging between 7 and 15, and specificity indices ranging between 3.4 and 6.8, were obtained with the four intact MAb at day 4-5 after injection. With F(ab')2 fragments of the four MAb, at day 3, the tumor antibody uptakes ranged between 12 and 24 and the specificity indices between 5.3 and 8.2. With the Fab fragments prepared from the two most promising MAb, the antibody uptakes reached values of 34 and 82 at day 2-3 and the specificity indices were as high as 12 and 19. The scanning results paralleled those obtained by direct measurement of radioactivity. With intact MAb, tumor grafts of 0.5-1 g gave very contrasted positive scans 3 d after injection. Using MAb fragments, tumors of smaller size were detectable earlier. The best results were obtained with Fab fragments of MAb 35, which gave clear detections of tumors weighing only 0.1 g as early as 48 h after injection. Autoradiographs of tumor sections from mice injected with 125I-labeled MAb demonstrated that the radioactivity was localized in the tumor tissues and not in the stromal connective tissue of mouse origin. The highest radioactivity concentration was localized in areas known to contain CEA such as the pseudolumen of glands and the apical side of carcinoma cells. The penetration of radioactivity in the central part of tumor nodules and the pseudolumen appeared to be increased with the use of MAb fragments.

Elevated inflammatory markers combined with positive pneumococcal urinary antigen are a good predictor of pneumococcal community-acquired pneumonia in children.

BACKGROUND: Our objective was to evaluate procalcitonin (PCT) and C-reactive protein (CRP) as predictors of a pneumococcal etiology in community-acquired pneumonia (CAP) in hospitalized children. METHODS: Children requiring hospitalization for CAP were prospectively enrolled. The following indices were determined: antibodies against pneumococcal surface proteins (anti-PLY, pneumococcal histidine triad D, pneumococcal histidine triad E, LytB and pneumococcal choline-binding protein A), viral serology, nasopharyngeal cultures and polymerase chain reaction for 13 respiratory viruses, blood pneumococcal polymerase chain reaction, pneumococcal urinary antigen, PCT and CRP. Presumed pneumococcal CAP (P-CAP) was defined as a positive blood culture or polymerase chain reaction for Streptococcus pneumoniae or as a pneumococcal surface protein seroresponse (≥2-fold increase). RESULTS: Seventy-five patients were included from which 37 (49%) met the criteria of P-CAP. Elevated PCT and CRP values were strongly associated with P-CAP with odds ratios of 23 (95% confidence interval: 5-117) for PCT and 19 (95% confidence interval: 5-75) for CRP in multivariate analysis. The sensitivity was 94.4% for PCT (cutoff: 1.5 ng/mL) and 91.9% for CRP (cutoff: 100 mg/L). A value≤0.5 ng/mL of PCT ruled out P-CAP in >90% of cases (negative likelihood ratio: 0.08). Conversely, a PCT value≥1.5 ng/mL associated with a positive pneumococcal urinary antigen had a diagnostic probability for P-CAP of almost 80% (positive likelihood ratio: 4.59). CONCLUSIONS: PCT and CRP are reliable predictors of P-CAP. Low cutoff values of PCT allow identification of children at low risk of P-CAP. The association of elevated PCT or CRP with a positive pneumococcal urinary antigen is a strong predictor of P-CAP.

Lentiviral delivery of the human wild-type tau protein mediates a slow and progressive neurodegenerative tau pathology in the rat brain.

Most models for tauopathy use a mutated form of the Tau gene, MAPT, that is found in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) and that leads to rapid neurofibrillary degeneration (NFD). Use of a wild-type (WT) form of human Tau protein to model the aggregation and associated neurodegenerative processes of Tau in the mouse brain has thus far been unsuccessful. In the present study, we generated an original "sporadic tauopathy-like" model in the rat hippocampus, encoding six Tau isoforms as found in humans, using lentiviral vectors (LVs) for the delivery of a human WT Tau. The overexpression of human WT Tau in pyramidal neurons resulted in NFD, the morphological characteristics and kinetics of which reflected the slow and sporadic neurodegenerative processes observed in sporadic tauopathies, unlike the rapid neurodegenerative processes leading to cell death and ghost tangles triggered by the FTDP-17 mutant Tau P301L. This new model highlights differences in the molecular and cellular mechanisms underlying the pathological processes induced by WT and mutant Tau and suggests that preference should be given to animal models using WT Tau in the quest to understand sporadic tauopathies.

Homeostasis and in vivo effector function of antigen-specific CD4+CD25+ regulatory T cells in experimental transplantation.

CD4+CD25+ regulatory T cells (Tregs) play a critical role in the prevention of autoimmune diseases as well as in the induction and maintenance of dominant tolerance in transplantation models. While their suppressive function has been extensively studied in vitro, their homeostasis and mechanisms of immunoregulation still remain to be clarifi ed in vivo. Using a murine adoptive transfer and skin allograft model, we analysed the expansion, effector function and traffi cking of effector T cells in the presence or absence of donor-specifi c Tregs. Although hyporesponsive to allogeneic and polyclonal stimulation in vitro, transferred Tregs survived and expanded, in response to an allograft in vivo. When co-transferred with naive CD4+CD25- effector T cells, they specifi cally prevented donor but not 3rd party allograft rejection by inhibiting the production of effector cytokines rather than the proliferation of effector T cells in response to alloantigens. The co-transfer of donor-specifi c Tregs did not affect the homing of effector T cells towards the graft draining lymph nodes, but it markedly reduced the infi ltration of the allograft by these pathogenic cells. Furthermore, in recipients where donor-specifi c transplantation tolerance was induced, Tregs preferentially accumulated in the allograft draining lymph nodes and within the grafted skin itself. Taken together, our results suggest that the suppression of graft rejection is an active process that involves the persistent presence of Tregs at the site of antigenic challenge.

Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles.

PURPOSE: To study the kinetics of polylactide (PLA) nanoparticle (NP) localization within the intraocular tissues and to evaluate their potential to release encapsulated material. METHODS: A single intravitreous injection (5 micro L) of an NP suspension (2.2 mg/mL) encapsulating either Rh-6G (Rh) or Nile red (Nr) was performed. Animals were killed at various times, and the NPs localization within the intraocular tissues was studied by environmental scanning electron microscopy (ESEM), confocal microscopy, light microscopy histology, fluorescence microscopy, and immunohistochemistry. Eyes injected with blank NPs, free Rh, or PBS solution were used as the control. RESULTS: ESEM showed the flow of the NPs from the site of injection into the vitreous cavity and their rapid settling on the internal limiting membrane. Histology demonstrated the anatomic integrity of the injected eyes and showed no toxic effects. A mild inflammatory cell infiltrate was observed in the ciliary body 6 hours after the injection and in the posterior vitreous and retina at 18 to 24 hours. The intensity of inflammation decreased markedly by 48 hours. Confocal and fluorescence microscopy and immunohistochemistry showed that a transretinal movement of the NPs was gradually taking place with a later localization in the RPE cells. Rh encapsulated within the injected NPs diffused and stained the retina and RPE cells. PLA NPs were still present within the RPE cells 4 months after a single intravitreous injection. CONCLUSIONS: Intravitreous injection of PLA NPs appears to result in transretinal movement, with a preferential localization in the RPE cells. Encapsulated Rh diffuses from the NPs and stains the neuroretina and the RPE cells. The findings support the idea that specific targeting of these tissues is feasible. Furthermore, the presence of the NPs within the RPE cells 4 months after a single injection shows that a steady and continuous delivery of drugs can be achieved.

In vivo imaging of T cell delivery to tumors after adoptive transfer therapy.

Adoptive transfer therapy of in vitro-expanded tumor-specific cytolytic T lymphocytes (CTLs) can mediate objective cancer regression in patients. Yet, technical limitations hamper precise monitoring of posttherapy T cell responses. Here we show in a mouse model that fused single photon emission computed tomography and x-ray computed tomography allows quantitative whole-body imaging of (111)In-oxine-labeled CTLs at tumor sites. Assessment of CTL localization is rapid, noninvasive, three-dimensional, and can be repeated for longitudinal analyses. We compared the effects of lymphodepletion before adoptive transfer on CTL recruitment and report that combined treatment increased intratumoral delivery of CTLs and improved antitumor efficacy. Because (111)In-oxine is a Food and Drug Administration-approved clinical agent, and human SPECT-CT systems are available, this approach should be clinically translatable, insofar as it may assess the efficacy of immunization procedures in individual patients and lead to development of more effective therapies.

Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.

According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions.

Large TCR diversity of virus-specific CD8 T cells provides the mechanistic basis for massive TCR renewal after antigen exposure.

Ex vivo analysis of virus-specific CD8 T cell populations by anchored PCR has shown that the CD8 TCR repertoire was less oligoclonal (seven to nine clonotypes per individual epitope) than previously thought. In the current study, TCR diversity was investigated by assessing both the overall TCR β-chain variable regions usage as well as the CDR3 regions in ex vivo-isolated CMV- and EBV-specific CD8 T cells from 27 healthy donors. The average number of clonotypes specific to most single viral epitopes comprised between 14 and 77. Changes in the CD8 TCR repertoire were also longitudinally assessed under conditions of HIV-1 chronic infection (i.e., in patients with suppressed virus replication and after treatment interruption and Ag re-exposure). The results showed that a large renewal (≤80%) of the TRB repertoire occurred after Ag re-exposure and was eventually associated with an increased T cell recognition functional avidity. These results demonstrate that the global CD8 TCR repertoire is much more diverse (≤9-fold) than previously estimated and provide the mechanistic basis for supporting massive repertoire renewal during chronic virus infection and Ag re-exposure.

Secretory component delays the conversion of secretory IgA into antigen-binding competent F(ab')2: a possible implication for mucosal defense.

Secretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at approximately 10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the alpha-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the kappa light chain was not digested at all, suggesting that the F(ab')2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the alpha-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab')2 is discussed in terms of mucosal immune defenses.

Effects of epidural analgesia on pelvic floor function after spontaneous delivery : a longitudinal retrospective study

Abstract: The aim of the study was to assess the effects of epidural analgesia on pelvic floor function. Eighty- two primiparous women (group 1, consisting of 41 given an epidural, and group 2 of 41 not given an epidural) were investigated during pregnancy and at 2 and 10 months after delivery by a questionnaire, clinical examination, and assessment of bladder neck behavior, urethral sphincter function and intravaginal/intra-anal pressures. The prevalence of stress urinary incontinence was similar in both groups at 2 months (24% vs. 17%, P = 0.6) and 10 months (22% vs. 7%, P = 0.1), as was the prevalence of decreased sexual vaginal response at 10 months (27% vs. 10%, P= 0.08). Bladder neck behavior, urethral sphincter function and intravaginal and intra-anal pressures showed no significant differences between the two groups. Ten months after spontaneous delivery, there were no significant differences in the prevalence of stress urinary incontinence and decreased sexual vaginal response, or in bladder neck behavior, urethral sphincter function and pelvic floor muscle strength between women who had or had not had epidural analgesia.

Anti-idiotypic monoclonal antibody AB3, reacting with the primary antigen (CEA), can localize in human colon-carcinoma xenografts as efficiently as AB1.

BALB/c mice were immunized with anti-idiotypic monoclonal (MAb) antibody (anti-Id or Ab2) directed against an AB1 MAb anti-carcinoembryonic (CEA) in order to obtain AB3 MAbs (anti-anti-Id). AB3 MAbs were shown to recognise the primary antigen (CEA) and one of them was tested extensively in vitro and in vivo. This AB3 MAb was shown to bind specifically to CEA on frozen sections of a human colon carcinoma by immunoperoxidase. Scatchard plot analyses showed that the affinity of this AB3 was of the same order of magnitude as the AB1. In vivo experiments, in nude mice bearing CEA-producing human colon-carcinoma xenografts showed that up to 30% of the intravenously injected dose of 125I-labelled AB3 were localized per gram of tumour tissue. Furthermore, calculation of the ratios of AB3 concentration in the tumour over those in normal organs such as lung, liver, kidney, spleen and bone gave relatively high values similar to results obtained with AB1. All together our results show that AB3 can localize as efficiently and specifically in the tumour as AB1, despite the fact that the mice from which it was derived were immunized with a mouse MAb (AB2) and had never been exposed to CEA.