Contrasting frequencies of antitumor and anti-vaccine T cells in metastases of a melanoma patient vaccinated with a MAGE tumor antigen

Melanoma patients have high frequencies of T cells directed against antigens of their tumor. The frequency of these antitumor T cells in the blood is usually well above that of the anti-vaccine T cells observed after vaccination with tumor antigens. In a patient vaccinated with a MAGE-3 antigen presented by HLA-A1, we measured the frequencies of anti-vaccine and antitumor T cells in several metastases to evaluate their respective potential contribution to tumor rejection. The frequency of anti–MAGE-3.A1 T cells was 1.5 × 10−5 of CD8 T cells in an invaded lymph node, sixfold higher than in the blood. An antitumor cytotoxic T lymphocyte (CTL) recognizing a MAGE-C2 antigen showed a much higher enrichment with a frequency of ∼10%, 1,000 times higher than its blood frequency. Several other antitumor T clonotypes had frequencies >1%. Similar findings were made on a regressing cutaneous metastasis. Thus, antitumor T cells were ∼10,000 times more frequent than anti-vaccine T cells inside metastases, representing the majority of T cells present there. This suggests that the anti-vaccine CTLs are not the effectors that kill the bulk of the tumor cells, but that their interaction with the tumor generates conditions enabling the stimulation of large numbers of antitumor CTLs that proceed to destroy the tumor cells. Naive T cells appear to be stimulated in the course of this process as new antitumor clonotypes arise after vaccination

Выпускная квалификационная работа

Introduction: Interferon Gamma Release Assay (IGRA) has proven to be a useful test to evaluate the immune response to Mycobacterium tuberculosis antigens in children over the age of 5 years as an alternative to tuberculin skin testing (TST). Much less is known about its performance in younger children, who are at higher risk for developing tuberculosis (TB) disease after exposure. We aimed to evaluate the accuracy of using IGRA in TB screening in this population. Methods: Children below the age of 5 years at high risk for TB infection were prospectively enrolled, to compare the performance of TST and the QuantiFERON-TB Gold-In-Tube test (QFT). Children were treated in accordance with the diagnosis made at baseline and followed-up for 12 months. Results: We included a total of 60 children of which 97 blood samples were available for analysis. There was 90.72% agreement between TST and QFT (Kappa test 0.59, moderate agreement). With TST as a reference, the QFT positive predictive value was 0.72 and the negative predictive value 0.93. Discordant results were observed with 6% TST+/QFT- paired tests. When we restricted the comparison of TST and QFT to non-BCG-vaccinated children, the degree of agreement was more substantial (95%, Kappa test 0.75) and the negative predictive value was 0.99. We observed 3% discordant TST-/QFT+ results. All children with active TB disease had concordant positive QFT results, with QFT values above 4.00 IU/ml. Conclusion: In a low TB prevalence country, serial testing of QFT was found to produce a moderate agreement with TST results. False positive QFT results would have been eliminated by using a higher cutoff without misdiagnosing the children with TB disease. Some of the false negative QFT results could be explained by false positive TST results on consecutive testing. For now the most prudent approach would be to consider discordant QFT-/TST+ results as false negative QFT results, taking into account the young age of our population and the potential risk for evolution to active TB disease

Spécificité des lymphocytes T présents dans une tumeur humaine qui régresse après vaccination avec un antigène MAGE

Several small scale clinical trials of vaccination with MAGE antigens have been conducted in metastatic melanoma patients with measurable disease. Therapeutical benefits were observed only in a minority of patients, whatever the vaccine modality (peptides, proteins, recombinant viruses, dendritic cells). Vaccination with antigenic peptide MAGE-A3168-176 induced low level anti-vaccine CTL responses, which were correlated with tumor regressions. The very low frequencies of detectable anti-vaccine CTL in most of the regressor patients led us to examine the potential role of additional effectors of the tumor rejections. A detailed analysis was carried out with melanoma patient EB81, who showed tumor regression after vaccination with MAGE-A3168-176 and has remained disease-free for six years after vaccination. Anti-vaccine CTL are rare in the blood and in tumors, whereas anti-tumor CTL, directed against other tumor antigens than those of the vaccine, were found at 1,000 to 10,000 fold higher frequencies. These anti-tumor T cells were already present at high frequency before vaccination. Two groups of anti-tumor CTL clones are present in the metastases. Some anti-tumor CTL clones were already present before vaccination, indicating that a spontaneous anti-tumor CTL response occured and became inefficient at halting tumor progression. Other CTL clones appeared after vaccination and were found to be highly enriched in regressing metastases relative to the blood. This work completes the analysis of anti-tumor CTL by identifying the antigens recognized by CTL clone 101, which appeared after vaccination, and CTL clone 10, which was already present before vaccination. CTL 101 was isolated from T lymphocytes from a metastatic lymph node. The antigenic peptide, presented by HLA-A2 molecules, is encoded by a mutated sequence in the gene coding for ClpP, a mitochondrial protease. Lysis of the tumor cells by the CTL was strongly increased by IFN-? treatment, because of a better processing of the antigenic peptide by the immunoproteasome. This could have contributed to tumor regression because a higher expression level of IFN-? was found in the regressing metastases as compared to a metastasis resected before vaccination. CTL clone 101 is also the only anti-tumor CTL clone directed against an antigenic peptide that was apparently not targeted by the spontaneous anti-tumor T cell responses that occured before vaccination. CTL clone 10, which was present in the blood and in a metastasis already before vaccination, recognized an antigenic peptide presented by HLA-A2 molecules and encoded by a mutated sequence in the gene coding for Triad3A, an E3 ubiquitin-protein ligase. Analysis of DNA extracted from tumor cells of a metastasis suggests that about half of these cells do not carry the mutated Triad3 allele. This might contribute to explain the coexistence of tumor cells and CTL 10 within the metastasis.Plusieurs études cliniques de vaccination avec des antigènes codés par les gènes MAGE ont été conduites chez des patients atteints de mélanome. Ces vaccinations apportent un bénéfice thérapeutique chez 5 à 10 % des malades, et ce quelle que soit la modalité vaccinale (peptides, protéines, virus recombinants, cellules dendritiques). La vaccination avec le peptide MAGE-A3168-176 administré sous différentes formes, induit de faibles réponses lymphocytaires T CD8+, corrélées aux régressions tumorales. Ces faibles réponses T anti-vaccinales nous ont incité à chercher d'autres effecteurs du rejet tumoral. Une analyse détaillée a été réalisée chez une patiente, EB81, qui a présenté une régression de ses métastases après vaccination avec l'antigène MAGE-A3168-176, et qui est toujours sans signe de maladie six ans plus tard. Alors que chez cette patiente, les lymphocytes T anti-vaccinaux sont rares dans le sang et dans les tumeurs, on trouve d'autres CTL anti-tumoraux, c'est à dire qui reconnaissent des antigènes différents de ceux du vaccin, et qui sont 1 000 à 10 000 fois plus fréquents que les lymphocytes T anti-vaccinaux. Ces CTL anti-tumoraux sont déjà présents en grand nombre avant la vaccination. Si on examine les métastases de cette patiente pour leur contenu en CTL anti-tumoraux, on trouve deux catégories de clones CTL. Certains sont déjà présents dans les métastases avant vaccination, suite à une réponse anti-tumorale spontanée, qui est apparemment inefficace pour arrêter la progression tumorale. D'autres CTL n'apparaissent qu'après la vaccination et se retrouvent enrichis dans les métastases en régression. Ce travail de doctorat a consisté à poursuivre l'analyse des CTL anti-tumoraux chez cette patiente, en identifiant le peptide antigénique reconnu par un clone représentatif de chacune de ces deux catégories : le CTL 101, qui apparaît après la vaccination, et le CTL 10, qui est déjà présent avant celle-ci. Le CTL 101 a été isolé à partir de lymphocytes infiltrants un ganglion métastatique. Le peptide antigénique, présenté par HLA-A2, est codé par une séquence mutée du gène CLPP, qui code une protéase mitochondriale. Le CTL 101 ne lyse que faiblement la lignée tumorale autologue sauf lorsque celle-ci est pré-traitée avec de l'IFN-?. Cet effet est dû à un meilleur apprêtage du peptide antigénique par l'immunoprotéasome et cela a pu contribuer à la régression tumorale parce que l'IFN-? est plus exprimé dans les métastases en régression que dans la tumeur prélevée avant vaccination. Par ailleurs, le CTL 101 est le seul clone CTL anti-tumoral qui est dirigé contre un antigène non ciblé par la réponse T anti-tumorale spontanée. Il n'y a donc peut-être pas eu d'immunosélection préalable de cellules tumorales résistantes à ce CTL, ce qui a pu lui conférer un rôle crucial dans la régression. Le clone CTL 10, présent dans le sang et dans la métastase déjà avant vaccination, reconnaît un peptide présenté par HLA-A2 et codé par une séquence mutée du gène Triad3, codant une ubiquitine ligase E3. Une analyse de lADN extrait de cellules tumorales suggèrent qu'in vivo la moitié de ces cellules ne possèdent pas le gène Triad3 muté, ce qui pourrait expliquer leur coexistence avec le CTL 10Thèse de doctorat en sciences biomédicales (immunologie des tumeurs) (SBIM 3) -- UCL, 200

Outcomes of bariatric surgery in patients with inflammatory bowel disease from a French nationwide database

International audienceBackground: The outcomes of bariatric surgery (BS) in patients with chronic inflammatory bowel disease (IBD) remain rarely described. We aimed to evaluate the 90-day morbidity and mortality rates, and the risk of IBD complications 2 years after BS. Method: Patients from the French Programme de Médicalisation des Systèmes d’Information (PMSI) database who underwent a primary BS between 2016 and 2018 were included. We identified patients with a previous diagnosis of IBD. Postoperative 90-day (POD90) morbidity and mortality rates were compared between the two groups. The evolution of IBD was followed 2 years after BS. Results: Between 2016 and 2018, 138 980 patients underwent primary BS, including 587 patients with IBD: 326 (55.5 per cent) with Crohn’s disease (CD) and 261 (44.5 per cent) with ulcerative colitis (UC). The preferred surgical technique was sleeve gastrectomy, especially in the IBD group (81.1 per cent), followed by gastric bypass (14.6 per cent). Patients with IBD had more comorbidities (Charlson Comorbidity Index of 1 or more, hypertension, and diabetes; P < 0.001) than those without IBD. The POD90 mortality rate did not differ between the two groups (0.049 per cent in the IBD group versus 0 per cent in the non-IBD group), but more unscheduled rehospitalizations at POD90 were observed in patients with IBD (6.0 per cent versus 3.7 per cent; P = 0.004). Two years after BS, 86 patients (14.6 per cent) in the IBD group had at least one unplanned readmission for the management of their IBD; 15 patients stayed for 3 or more days. After multivariable analysis, patients with CD had an independent elevated risk of IBD-related unplanned readmissions 2 years after BS versus UC (adjusted odds ratio 1.90, 95 per cent c.i. 1.22 to 2.97; P = 0.005). Conclusion: In a highly selected cohort of patients with well-controlled IBD, BS did not result in added mortality or morbidity. A point of vigilance must be underlined regarding BS in patients with CD

Proportions of interferon-γ-producing ascites lymphocytes in response to mycobacterial antigens: A help for early diagnosis of peritoneal tuberculosis in a low TB incidence country

International audienceBACKGROUND:Peritoneal tuberculosis (TB) remains difficult to diagnose because of its non-specific clinical features and the lack of efficient microbiological tests. As delayed diagnosis is associated with high mortality rates, new diagnostic tools are needed.METHODS AND FINDINGS:We investigated for 24 patients prospectively enrolled with a possible diagnosis of peritoneal TB, the diagnostic value of the analysis of IFN-γ production by peritoneal fluid lymphocytes in response to a short in vitro stimulation with mycobacterial antigens. The patients were classified in two groups: non-TB and confirmed or highly probable TB. Diagnosis of TB was based on microbiological and histopathological criteria and/or a favorable response to anti-TB treatment. The IFN-γ production by peritoneal CD4+ T lymphocytes was analyzed by flow cytometry after an overnight in vitro stimulation with three different mycobacterial antigens, purified protein derivative (PPD), heparin-binding haemagglutinin (HBHA) or early-secreted-antigen-target-6 (ESAT-6). The percentages of PPD-, HBHA- or ESAT-6-induced IFN-γ-producing peritoneal fluid CD4+ T lymphocytes were higher in the TB group than in the non-TB group (p = 0.0007, p = 0.0004, and p = 0.0002 respectively). Based on cut-off values determined by ROC curve analysis of the results from TB and highly probable TB compared to those of non-TB patients, the sensitivity of these three tests was 100% with a specificity of 92%.CONCLUSIONS:The analysis of mycobacterial-induced IFN-γ production by peritoneal lymphocytes is a promising tool to reliably and rapidly diagnose peritoneal TB. Further studies should be performed on larger cohorts of patients in high-TB-incidence countries to confirm the clinical value of this new diagnostic approach for peritoneal TB

Age-Stratified T Cell Responses in Children Infected with Mycobacterium tuberculosis

Tuberculosis (TB) in young children differs from adult TB in that the risk of rapid progression to active TB (aTB) is higher in children than in adults. The reasons for this increased risk are not fully understood. Early differentiation remains difficult between children at risk to develop aTB from those who will remain healthy and develop a latent TB infection (LTBI). Biomarkers to differentiate aTB from LTBI in children, especially in very young children, are urgently needed. To identify M. tuberculosis-specific functional T cell subsets related to clinical manifestations in children, we enrolled 87 children exposed to M. tuberculosis. After standard clinical assessment, the children were classified as aTB, LTBI, or uninfected. Their CD4+ T cell cytokine profiles (IFN-γ, TNF-α, IL-2, IL-17) were analyzed at the single-cell level by flow cytometry after stimulation with three mycobacterial antigens, purified protein derivative (PPD), early-secreted-antigenic target-6 (ESAT-6), or heparin-binding hemagglutinin (HBHA). This approach identified age-related discriminative markers between aTB and LTBI. Whereas among the 3- to 15-year-old children, an excellent discrimination between aTB and LTBI was provided by comparing the ratio between the proportions of ESAT-6-induced IFN-γsingle+ and ESAT-6-induced TNF-αsingle+CD4+ T lymphocytes, this was not the case for children younger than 3 years. By contrast, in this group ( < 3years), the analysis of HBHA-induced IL-17single+CD4+ T lymphocytes allowed us to identify children with LTBI by the high proportion of this cellular lymphocyte subset, whereas this was not the case for children with aTB. The analysis at the single-cell level of T cell immune responses induced by mycobacterial antigens are, thus, different in infected children younger or older than 3 years of age. HBHA-induced IL-17 production by CD4+ T lymphocytes was associated with protection only in children under 3 years who are at high risk for rapid progression to aTB. This suggests that the HBHA-induced IL-17 production by CD4+ T lymphocytes is a potential new correlate of protection against M. tuberculosis in humans, and that the distinction between children with LTBI and those with aTB is possible based on age-related diagnostic markers.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

HBHA- and rESAT-6-IGRA kinetics of LTBI subjects representative of group A, B and C.

<p>IFN-γ secretion was measured by ELISA after four days of PBMC stimulation with PPD (black circle), HBHA (violet circle) or rESAT-6 (green circle) at the indicated time points. The limits of positivity for PPD, HBHA and rESAT-6 are represented as dotted lines with the corresponding color. When available, the negative or positive results of the QFT-G-IT are shown on the bottom of the figure as “−” or “+”, respectively. Subject shown on panel A belongs to group A (n°1) characterized by stable HBHA- and rESAT-6-IGRA responses. Subject shown on panel B belongs to group B (n°14) characterized by unstable HBHA- and rESAT-6-IGRA responses. Subject shown on panel C belongs to group C (n°23) presenting disappearing HBHA- and rESAT-6-IGRA responses.</p

Definition of the heat map color and of the score values for HBHA- and rESAT-6-IGRA.

<p>The first column shows the intensity of the colors used in the heat map to represent the intensity of the IFN-γ responses, from dark blue for insignificant IFN-γ responses to dark red for very high responses. The last column defines the IFN-γ scores used to calculate the HBHA/rESAT-6 ratios. These scores were attributed to the different ranges of IFN-γ concentrations illustrated in the heat map. Values corresponding to the grey zone just below or above the cut-off values (dotted line), were attributed the same score of 1.</p