Exploration of immune competencies of viral-specific CD8+T cells in MS patients using tetramers and functional CTL assays

Introduction: Epstein-Barr Virus(EBV) has been repeatedly associatedwith multiple sclerosis (MS). Wehave previously shown that there is ahigh peripheral as well as intrathecalactivation of EBV-, but not cytomegalovirus(CMV)-specific CD8+ Tcells, early in the course of MS,strengthening the link between EBVand MS. However, the trigger of thisincreased EBV-specific CD8+ T cellresponse remains obscure. It could resultfrom a higher EBV viral load. Alternatively,it could be due to an intrinsicallydeficient EBV-specificCTL response, cytotoxic granulesmediated.Thus, we performed anin-depth study of the phenotype of exvivo EBV- and CMV-specific CD8+T cells in MS patients and control patients,assessing their cytotoxic activity.Methods:We analyzed the profileof cytotoxic granules in EBV- andCMV-specific CD8+ T cells in a cohortof 13 early MS patients, 20 lateMS, 30 other neurological diseases(OND) patients and 7 healthy controlsubjects. Ex vivo analysis of EBV- orCMV-specific CD8+ T cells was performedusing HLA class I/tetramercomplexes coupled to CCR7 andCD57 markers in conjunction withperforin, granzymes A, BandKstaining.In a sub-cohort of MS patientsand controls, cytotoxic activity ofEBV- and CMV-specific CD8+ Tcells was investigated using a functionalCFSE CTL assay. Results: UsingHLA Class I tetramers for EBVand CMV, we found that the frequencyof EBV- or CMV-specificCD8+ T cells were similar in all studysubjects. Most of EBV- and CMVspecificCD8+Tcells were highly differentiated(CCR7-) and a variousproportion expressed the exhaustionmarker CD57. MS and OND patientshad increased perforin expression inEBV-specific CD8+ T cells. Most importantly,we found that MS patientswith longer disease duration tended tohave lower CTL cytotoxicity as comparedto earlyMSpatients or controls.Conclusions: Effector EBV-specificCD8+ T cells are increased in earlyMS, however their cytotoxic granuleprofile does not seem to be fully alteredand the cytotoxic activity ofthese cells is normal. However, thecytotoxic activity of CTL decreasedin late MS patients suggesting an exhaustionof EBV-specific CD8+ Tcells possibly due to EBV reactivation.This work was supported by theSwiss National Foundation PP00B3-124893, the Swiss Society for MS,and the Biaggi Foundation to RADP.

Differences in the transduction of canonical wnt signals demarcate effector and memory CD8 T cells with distinct recall proliferation capacity.

Protection against reinfection is mediated by Ag-specific memory CD8 T cells, which display stem cell-like function. Because canonical Wnt (Wingless/Int1) signals critically regulate renewal versus differentiation of adult stem cells, we evaluated Wnt signal transduction in CD8 T cells during an immune response to acute infection with lymphocytic choriomeningitis virus. Whereas naive CD8 T cells efficiently transduced Wnt signals, at the peak of the primary response to infection only a fraction of effector T cells retained signal transduction and the majority displayed strongly reduced Wnt activity. Reduced Wnt signaling was in part due to the downregulation of Tcf-1, one of the nuclear effectors of the pathway, and coincided with progress toward terminal differentiation. However, the correlation between low and high Wnt levels with short-lived and memory precursor effector cells, respectively, was incomplete. Adoptive transfer studies showed that low and high Wnt signaling did not influence cell survival but that Wnt high effectors yielded memory cells with enhanced proliferative potential and stronger protective capacity. Likewise, following adoptive transfer and rechallenge, memory cells with high Wnt levels displayed increased recall expansion, compared with memory cells with low Wnt signaling, which were preferentially effector-like memory cells, including tissue-resident memory cells. Thus, canonical Wnt signaling identifies CD8 T cells with enhanced proliferative potential in part independent of commonly used cell surface markers to discriminate effector and memory T cell subpopulations. Interventions that maintain Wnt signaling may thus improve the formation of functional CD8 T cell memory during vaccination.

Adenosine Deaminase Activities in Sera, Lymphocytes and Granulocytes in Patients with Cutaneous Leishmaniasis

Adenosine deaminase (ADA) activities in sera, lymphocytes and granulocytes in patients with cutaneous leishmaniasis were investigated and compared with control groups. Fifty patients and 50 healthy individuals were studied. The clinical diagnosis was parasitologically confirmed by culture and Giemsa stain. ADA activities were measured by colorimetric method. Serum ADA activities 37.80 ± 11.90, 18.28 ± 6.08 IU/L (p<0.0001), lymphocyte specific ADA activities 14.90 ± 7.42, 8.38 ± 7.42 U/mg protein (p = 0.04), granulocyte specific ADA activities 1.15 ± 0.73 , 1.09 ± 0.67 U/mg protein ( p>0.05) were found in patients and control groups, respectively. ADA activity increases in some infectious diseases were cell mediated immune mechanisms are dominant. In cutaneous leishmaniasis, lymphokine-mediated macrophage activity is the main effector mechanism. Increase in serum and lymphocyte ADA activities in patients with cutaneous leishmaniasis may be dependent on and reflects the increase in phagocytic activity of macrophages and maturation of T-lymphocytes.

Photoaffinity labeling of the T cell receptor on living cytotoxic T lymphocytes.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of an antigenic peptide with MHC class I molecules and the TCR on living cells. Two photoreactive derivatives of the H-2Kd (Kd) restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) were used. The first derivative contained an N-terminal photoreactive iodo, 4-azido salicyloyl (IASA) group and biotin on the TCR contact residue Lys259 [IASA-YIPSAEK(biotin)I]. As previously described, this derivative selectively bound to and labeled the Kd molecule. The second photoreactive compound, the isomeric biotin-YIPSAEK(IASA)I, also efficiently bound to the Kd molecule, but failed to label this protein. A CTL clone derived from a mouse immunized with this derivative recognized this conjugate but not the parental P. berghei circumsporozoite peptide or the [IASA-YIPSAEK-(biotin)I] derivative in an Kd-restricted manner. Incubation of the cloned CTL cells with biotin-YIPSAEK(IASA)I, but not its isomer, followed by UV irradiation resulted in photoaffinity labeling of the TCR-alpha chain that was dependent on the conjugate binding to the Kd molecule. The TCR labeling was partially inhibited by anti-LFA 1 and anti-ICAM1 mAb, but was increased by addition of beta 2m or soluble KdQ10. The exquisite labeling selectivity of the two photoprobes opens a new, direct approach to the molecular analysis of antigen presentation and recognition by living CTL.

Combination of transient lymphodepletion with busulfan and fludarabine and peptide vaccination in a phase I clinical trial for patients with advanced melanoma.

Taking advantage of homeostatic mechanisms to boost tumor-specific cellular immunity is raising increasing interest in the development of therapeutic strategies in the treatment of melanoma. Here, we have explored the potential of combining homeostatic proliferation, after transient immunosuppression, and antigenic stimulation of Melan-A/Mart-1 specific CD8 T-cells. In an effort to develop protocols that could be readily applicable to the clinic, we have designed a phase I clinical trial, involving lymphodepleting chemotherapy with Busulfan and Fludarabine, reinfusion of Melan-A specific CD8 T-cell containing peripheral blood mononuclear cells (exempt of growth factors), and Melan-A peptide vaccination. Six patients with advanced melanoma were enrolled in this outpatient regimen that demonstrated good feasibility combined with low toxicity. Consistent depletion of lymphocytes with persistent increased CD4/CD8 ratios was induced, although the proportion of circulating CD4 regulatory T-cells remained mostly unchanged. The study of the immune reconstitution period showed a steady recovery of whole T-cell numbers overtime. However, expansion of Melan-A specific CD8 T-cells, as measured in peripheral blood, was mostly inconsistent, accompanied with marginal phenotypic changes, despite vaccination with Melan-A/Mart-1 peptide. On the clinical level, 1 patient presented a partial but objective antitumor response following the beginning of the protocol, even though a direct effect of Busulfan/Fludarabine cannot be completely ruled out. Overall, these data provide further ground for the development of immunotherapeutic approaches to be both effective against melanoma and applicable in clinic.

Facteurs de radiosensibilité tardive des tissus sains [Factors of late radiosensitivity of normal tissues]

The impact of curative radiotherapy depends mainly on the total dose delivered homogenously in the targeted volume. Nevertheless, the dose delivered to the surrounding healthy tissues may reduce the therapeutic ratio of many radiation treatments. Two different side effects (acute and late) can occur during and after radiotherapy. Of particular interest are the radiation-induced sequelae due to their irreversibility and the potential impact on daily quality of life. In a same population treated in one centre with the same technique, it appears that individual radiosensitivity clearly exists. In the hypothesis that genetic is involved in this area of research, lymphocytes seem to be the tissue of choice due to easy accessibility. Recently, low percentage of CD4 and CD8 lymphocyte apoptosis were shown to be correlated with high grade of sequelae. In addition, recent data suggest that patients with severe radiation-induced late side effects possess four or more single nucleotide polymorphisms (SNP) in candidate genes (ATM, SOD2, TGFB1, XRCC1, and XRCC3) and low radiation-induced CD8 lymphocyte apoptosis in vitro. On-going studies are being analyzing the entire genome using a Genome-wide association study (GWAS) analysis.

The spleen is an important site of T cell activation during human hepatosplenic schistosomiasis

We have undertaken a comparative immunephenotypic study of spleen cells from hepatosplenic patients (HS) and uninfected individuals (NOR) using flow cytometry. Our data did not show any significant differences in the mean percentage of T-cells and B-cells between the two groups. Analysis of activated T-cells demonstrated that HS present an increased percentage of CD3+HLA-DR+ splenocytes in comparison to NOR. Analysis of T-cell subsets demonstrated a significant increase on the percentage of both activated CD4+ T-splenocytes and CD8+ cells in HS. We did not find any difference in the mean percentage of CD28+ T-cells. Analysis of the B-cell compartment did not show any difference on the percentage of B1-splenocytes. However, the spleen seems to be an important reservoir/source for B1 lymphocytes during hepatosplenic disease, since after splenectomy we found a decreased the percentage of circulating B1-lymphocytes. We observed an increase on the percentage of CD2+CD3- lymphocytes in the spleen of HS suggesting that the loss of CD3 by activated T-cells or the expansion of NK-cells might play a role in the development/maintenance of splenomegaly.

Roles of canonical Wnt/TCF-1 signaling for hematopoiesis, lymphocyte development and function

Summary : The canonical Wnt signaling pathway plays key roles in the maintenance of self-renewing tissues, like the gut or the skin. In contrast, the role of this pathway in hematopoiesis remains poorly defined. Wnt ligands transmit signals through ß-catenin which activates gene transcription upon its association with Lymphoid Cell Enhancer/T Cell Factor (LEF/TCF). Currently, v-catenin is the only alternative factor known to transduce canonical Wnt signals. The ß-/γ-catenin bindiná domain in TCF-1 is required to partly rescue thymopoiesis and NK cell development in TCF-1-deficient mice. However, T cell development and hematopoiesis w-as normal in mice deficient of ß-catenin, or of γ-catenin. Surprisingly we found that hematopoiesis and thymopoiesis was also normal in the combined absence of ß- and γ-catenin. Reporter assays showed that double-deficient lymphocytes were still able to transduce canonical wnt signals. These data provided evidence that hematopoietic cells can transduce canonical Wnt signals in the combined absence of ß- and γ-catenin. There exist numerous TCF-1 isoforrns including those that harbor the N-terminal ß-/y-catenin binding domain or that contains a C-terminal CRARF domain whose role in vivo has not been previously tested. We found that the CRARF domain influences lymphocyte development in conjunction with the N-treminal ß-/γ-catenin binding. The presence of the two domains directs thymocytes to the CD8+ T cell lineage whereas NK cell development is abolished. Roles of the canonical Wnt/TCF-1 pathway for lymphocyte function have not been defined. We demonstrate that TCF-1 deficient CDBT T cells mount a normal primary response to viral infection but these T cells fail to expand upon restimulation. The failure of CD8+ T cells to respond to IL-2 during primary infection seems to account for this phenotype. Thus, TCF-1 is essential for programming functional CD8+ T cell memory. Collectively, these data provide significant new insights into the role of Wnt/TCF-1 pathway for lymphocyte development and function and suggest a novel mechanism of Wnt signal transuction in hematopoietic cells. Résumé : La voie de signalisation canonique Wnt joue un rôle prépondérant dans le renouvellement de tissus, comme l'intestin ou la peau. Son rôle dans l'hématopoïèse est quant à lui mal défini. Le ligand Wnt transmet le signal via la ß-catenin qui active la transcription de gènes cibles quand il est associé avec Lymphoid Cell Enhancer,~T Cell Factor (LEF/TCF). Actuellement, la γ-catenin est le seul autre facteur connu pouvant se substituer à la fonction de la ß-catenin. Un variant de TCF-1 contenant le domaine liant ß-/,~-catenin est capable de restaurer le développement des lymphocytes T et NK en l'absence de TCF-1. Cependant la thymopoïèse et l'hématopoïèse sont normales dans les souris déficientes pour la ß-catenin ou la γ-catenin. De façon surprenante, nous avons trouvé que l'hématopoïèse et le développement des lymphocytes sont normaux lors de l'absence combinée de ß-/γ-catenin. De plus, la transduction des signaux de la voie de signalisation Wnt est maintenue dans des lymphocytes déficients pour ß-/γ-catenin. Ces résultats démontrent que les cellules hématopoïétiques peuvent transmettre les signaux de la voie canonique Wnt lors de l'absence combinée de la ß et la γ -catenin. Il existe de nombreuses isofonnes de TCF-1, y compris certaines qui comprennent un domaine qui lie ß-/γ-catenin du côté N-terminus ou qui contiennent un domaine CRARF du côté C-terminus. Nous montrons ici que le domaine CRARF influence le développement des lymphocytes en conjonction avec le domaine liant ß-/γ-catenin. La présence des deux domaines dirige les thymocytes vers la lignée de cellules T CD8, alors que le développement des cellules NK est aboli. Au-delà de sa fonction sur le développement des lymphocytes, le rôle de la soie de signalisation canonique Wnt/TCF-1 lors d'une infection n'a pas été défini. Nous avons montré que les cellules T CD8, déficientes pour TCF-1, développent une réponse primaire normale à une infection virale, mais qu'elles ne s'accumulent pas après restimulation. L'incapacité des cellules TCD8 à répondre à l'IL-2 durant la réponse primaire peut expliquer ce phénotype. Ainsi; TCF-1 est essentiel pour la programmation de cellules T CD8 mémoires fonctionnelles. L'ensemble de ces résultats fournit de nouveaux aperçus du rôle de la voie de signalisation Wnt/TCF-1 pour le développement et la fonction des lymphocytes et suggèrent un nouveau mécanisme de transduction du signal Wnt dans les cellules hématopoïétiques.

Effect of antiretroviral therapy on apoptosis markers and morphology in peripheral lymph nodes of HIV-infected individuals.

BACKGROUND: CD4+ T cell depletion and destruction and the involution of the lymphoid tissue are hallmarks of HIV infection. Although the underlying mechanisms are still unclear, apoptosis appears to play a central role. The objective of this study was to investigate the effect of antiretroviral therapy on the lymph node tissue, particularly with respect to morphology and apoptosis. PATIENTS AND METHODS: Between 1997 and 1999, two inguinal lymph nodes were excised from 31 previously untreated individuals who were in an early stage of HIV infection, the first one prior to treatment and the second after 16 to 20 months of treatment. Paraffin sections were investigated for lymph node architecture, distribution of cellular and viral markers, apoptosis, and expression of apoptotic key molecules which indirectly reflect apoptotic processes. RESULTS: After 16-20 months of antiretroviral therapy, a significant decrease in highly activated HIV-driven immune response was observed in the lymph node tissue as a marked reduction in follicular hyperplasia, a normalization of the follicular dendritic cell network, a significant increase in the number of CD4+ T cells, and a significant decrease in the number of CD8+ T cells. The expression of several proapoptotic (Fas, TRAIL, and active caspase 3) and antiapoptotic (Bcl-2 and IL-7Ralpha) molecules that were reconstituted in the tissues during therapy resembled their expression in lymph nodes of HIV-negative individuals. Limitations of the study are (a) the lack of untreated patients in the late stages, (b) for ethical reasons, the lack of a control group with untreated patients, and (c) for methodological reasons, the restriction of sequential measurements of apotpotic markers to one-third of the patients. CONCLUSION: Antiretroviral therapy initiated in the early stages in HIV infection may halt the irreversible destruction of the lymph node tissue and may partially normalize apoptotic processes.

Monitoring of vaccine-specific gamma interferon induction in genital mucosa of mice by real-time reverse transcription-PCR.

Monitoring of T-cell responses in genital mucosa has remained a major challenge because of the absence of lymphoid aggregates and the low abundance of T cells. Here we have adapted to genital tissue a sensitive real-time reverse transcription-PCR (TaqMan) method to measure induction of gamma interferon (IFN-gamma) mRNA transcription after 3 h of antigen-specific activation of CD8 T cells. For this purpose, we vaccinated C57BL/6 mice subcutaneously with human papillomavirus type 16 L1 virus-like particles and monitored the induction of CD8 T cells specific to the L1(165-173) H-2D(b)-restricted epitope. Comparison of the responses induced in peripheral blood mononuclear cells and lymph nodes (LN) by L1-specific IFN-gamma enzyme-linked immunospot assay and TaqMan determination of the relative increase in L1-specific IFN-gamma mRNA induction normalized to the content of CD8b mRNA showed a significant correlation, despite the difference in the readouts. Most of the cervicovaginal tissues could be analyzed by the TaqMan method if normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA was used and a significant L1-specific IFN-gamma induction was found in one-third of the immunized mice. This local response did not correlate with the immune responses measured in the periphery, with the exception of the sacral LN, an LN draining the genital mucosa, where a significant correlation was found. Our data show that the TaqMan method is sensitive enough to detect antigen-specific CD8 T-cell responses in the genital mucosa of individual mice, and this may contribute to elaborate effective vaccines against genital pathogens.

Human chronic chagasic cardiopathy: participation of parasite antigens, subsets of lymphocytes, cytokines and microvascular abnormalities

This article tries to demonstrate by new pathological findings (with the use of immunohistochemical technique and confocal laser microscopy) that chronic chagasic cardiomyopathy is a result of multiple factors involving myocarditis, immunodepression, severe fibrosis and microvessels dilatation and that all of these alterations are probably directly related with the presence of Trypanosoma cruzi parasites in the host associated with inadequate immunological response of the host.

Role of CD8+ T cells in endogenous interleukin-10 secretion associated with visceral leishmaniasis

This study examined the role and source of endogenous interleukin-10 (IL) secretion in visceral leishmaniasis (VL). The amounts of endogenous and Leishmania specific IL-10 and interferon-gamma (IFN) secreted by peripheral blood mononuclear cells (PBMC) from VL patients were compared. The correlation coefficient between endogenous IL-10 secretion and Leishmania specific IFN-gamma was -0.77, suggesting a major role for endogenous IL-10 secretion in VL. The effects of CD4+ and CD8+ T cell clones, isolated from a treated VL patient, on IL-10 secretion were assayed by mixing the clones with autologous, inactivated PBMC. The CD8+ clones mediated increased levels of IL-10 secretion in the presence of PBMC alone suggesting that CD8+ T cells may mediate endogenous IL-10 secretion.

Protective CD8+ T cell responses against the pre-erythrocytic stages of malaria parasites: an overview

CD8+ T cells have been implicated as critical effector cells in protection against the pre-erythrocytic stage of malaria in mice and humans following irradiated sporozoite immunization. Immunization experiments in animal models by several investigators have suggested different strategies for vaccination against malaria and many of the targets from liver stage malaria antigens have been shown to be immunogenic and to protect mice from the sporozoite challenge. Several prime/boost protocols with replicating vectors, such as vaccinia/influenza, with non-replicating vectors, such as recombinant particles derived from yeast transposon (Ty-particles) and modified vaccinia virus Ankara, and DNA, significantly enhanced CD8+ T cell immunogenicity and also the protective efficacy against the circumsporosoite protein of Plasmodium berghei and P. yeti. Based on these experimental results the development of a CD8+ T cell inducing vaccine has moved forward from epitope identification to planning stages of safety and immunogenicity trials of candidate vaccines.

Ex vivo detectable activation of Melan-A-specific T cells correlating with inflammatory skin reactions in melanoma patients vaccinated with peptides in IFA.

The purpose of this study was to test melanoma vaccines consisting of peptides and immunological adjuvants for optimal immunogenicity and to evaluate laboratory immune monitoring for in vivo relevance. Forty-nine HLA-A2 positive patients with Melan-A positive melanoma were repeatedly vaccinated with Melan-A peptide, with or without immune adjuvant AS02B (QS21 and MPL) or IFA. Peptide-specific CD8 T cells in PBLs were analyzed ex vivo using fluorescent HLA-A2/Melan-A multimers and IFN-gamma ELISPOT assays. The vaccines were well tolerated. In vivo expansion of Melan-A-specific CD8 T cells was observed in 13 patients (1/12 after vaccination with peptide in AS02B and 12/17 after vaccination with peptide in IFA). The T cells produced IFN-gamma and downregulated CD45RA and CD28. T-cell responses correlated with inflammatory skin reactions at vaccine injection sites (P < 0.001) and with DTH reaction to Melan-A peptide (P < 0.01). Twenty-six of 32 evaluable patients showed progressive disease, whereas 4 patients had stable disease. The two patients with the strongest Melan-A-specific T-cell responses experienced regression of metastases in skin, lymph nodes, and lung. We conclude that repeated vaccination with Melan-A peptide in IFA frequently leads to sustained responses of specific CD8 T cells that are detectable ex vivo and correlate with inflammatory skin reactions.