Direct presentation of a melanocyte-associated antigen in peripheral lymph nodes induces cytotoxic CD8+ T cells.

Encounter of self-antigens in the periphery by mature T cells induces tolerance in the steady-state. Hence, it is not understood why the same peripheral antigens are also promiscuously expressed in the thymus to mediate central tolerance. Here, we analyzed CD8(+) T-cell tolerance to such an antigen constituted by ovalbumin under the control of the tyrosinase promoter. As expected, endogenous CD8(+) T-cell responses were altered in the periphery of transgenic mice, resulting from promiscuous expression of the self-antigen in mature medullary epithelial cells and deletion of high-affinity T cells in the thymus. In adoptive T-cell transfer experiments, we observed constitutive presentation of the self-antigen in peripheral lymph nodes. Notably, this self-antigen presentation induced persisting cytotoxic cells from high-affinity CD8(+) T-cell precursors. Lymph node resident melanoblasts expressing tyrosinase directly presented the self-antigen to CD8(+) T cells, independently of bone marrow-derived antigen-presenting cells. This peripheral priming was independent of the subcellular localization of the self-antigen, indicating that this mechanism may apply to other melanocyte-associated antigens. Hence, central tolerance by promiscuous expression of peripheral antigens is a mandatory, rather than a superfluous, mechanism to counteract the peripheral priming, at least for self-antigens that can be directly presented in lymph nodes. The peripheral priming by lymph node melanoblasts identified here may constitute an advantage for immunotherapies based on adoptive T-cell transfer.

Four functionally distinct populations of human effector-memory CD8+ T lymphocytes.

In humans, the pathways of memory and effector T cell differentiation remain poorly defined. We have dissected the functional properties of ex vivo effector-memory (EM) CD45RA-CCR7- T lymphocytes present within the circulating CD8+ T cell pool of healthy individuals. Our studies show that EM T cells are heterogeneous and are subdivided based on differential CD27 and CD28 expression into four subsets. EM(1) (CD27+CD28+) and EM(4) (CD27-CD28+) T cells express low levels of effector mediators such as granzyme B and perforin and high levels of CD127/IL-7Ralpha. EM(1) cells also have a relatively short replicative history and display strong ex vivo telomerase activity. Therefore, these cells are closely related to central-memory (CD45RA-CCR7+) cells. In contrast, EM(2) (CD27+CD28-) and EM(3) (CD27-CD28-) cells express mediators characteristic of effector cells, whereby EM(3) cells display stronger ex vivo cytolytic activity and have experienced larger numbers of cell divisions, thus resembling differentiated effector (CD45RA+CCR7-) cells. These data indicate that progressive up-regulation of cytolytic activity and stepwise loss of CCR7, CD28, and CD27 both characterize CD8+ T cell differentiation. Finally, memory CD8+ T cells not only include central-memory cells but also EM(1) cells, which differ in CCR7 expression and may therefore confer memory functions in lymphoid and peripheral tissues, respectively.

Human effector CD8+ T lymphocytes express TLR3 as a functional coreceptor.

TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-gamma secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-gamma production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.

The hope for an HIV vaccine based on induction of CD8+ T lymphocytes: a review

The only long-term and cost-effective solution to the human immunodeficiency virus (HIV) epidemic in the developing world is a vaccine that prevents individuals from becoming infected or, once infected, from passing the virus on to others. There is currently little hope for an AIDS vaccine. Conventional attempts to induce protective antibody and CD8+ lymphocyte responses against HIV and simian immunodeficiency virus (SIV) have failed. The enormous diversity of the virus has only recently been appreciated by vaccinologists, and our assays to determine CD8+ lymphocyte antiviral efficacy are inadequate. The central hypothesis of a CTL-based vaccine is that particularly effective CD8+ lymphocytes directed against at least five epitopes that are derived from regions under functional and structural constraints will control replication of pathogenic SIV. This would be somewhat analogous to control of virus replication by triple drug therapy or neutralizing antibodies.

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-α blockade

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-α) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-α levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-α, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-α+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-α treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-α-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-α treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.

Mechanisms regulating the proliferative potential of human CD8+ T lymphocytes overexpressing telomerase.

In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division, eventually leading to a state of cellular senescence. Ectopic expression of telomerase results in the extension of their replicative life spans without inducing changes associated with transformation. However, it is yet unknown whether somatic cells that overexpress telomerase are physiologically indistinguishable from normal cells. Using CD8+ T lymphocyte clones overexpressing telomerase, we investigated the molecular mechanisms that regulate T cell proliferation. In this study, we show that early passage T cell clones transduced or not with human telomerase reverse transcriptase displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in human telomerase reverse transcriptase-transduced cells with extended life spans. These cells, despite maintaining high expression levels of genes involved in the cell cycle progression, also showed increased expression in several genes found in common with normal aging T lymphocytes. Strikingly, late passage T cells overexpressing telomerase accumulated the cyclin-dependent inhibitors p16Ink4a and p21Cip1 that have largely been associated with in vitro growth arrest. We conclude that alternative growth arrest mechanisms such as those mediated by p16Ink4a and p21Cip1 still remained intact and regulated the growth potential of cells independently of their telomere status.

Study of the mechanisms regulating the proliferative potential of human CD8+T lymphocytes over-expressing telomerase reverse transcriptase (hTERT)

R��sum�� La plupart des cellules issues du sang ont une dur��e de vie limit��e. Dans les cellules somatiques humaines, y incluant les lymphocytes T, la taille des t��lom��res diminue progressivement �� chaque division cellulaire, pouvant aboutir �� des instabilit��s chromosomiques. L'expression ectopique du g��ne de la transcriptase r��verse de la t��lom��rase (hTERT) dans les cellules restaure l'activit�� de la t��lom��rase, et permet un rallongement de leur vie r��plicative. Malgr�� l'absence de signes caract��ristiques de transformation, nous ne savons pas encore si les cellules somatiques qui surexpriment hTERT sont physiologiquement indiscernables des cellules normales. Certaines ��tudes r��centes proposent que la t��lom��rase joue plusieurs r��les additionnels dans d'autres ph��nom��nes biologiques tels que la r��paration de l'ADN, la survie et la croissance des cellules. Dans notre ��tude, nous avons utilis�� des clones issus de lymphocytes T cytotoxiques surexprimant la t��lom��rase afin d'��tudier les m��canismes mol��culaires qui r��glent leur prolif��ration et leur s��nescence. Nous avons montr�� que les ��jeunes �� cellules T exprimant ou non hTERT r��v��lent des taux de croissance identiques suite �� des r��ponses de stimulation induites par des mitog��nes. De plus, aucun changement global dans leur expression des g��nes n'a pu ��tre mis en ��vidence. Curieusement, nous avons observ�� des r��ponses r��duites dans la prolif��ration des cellules transduites avec la t��lom��rase qui pr��sentaient une ��longation des t��lom��res et une dur��e de vie prolong��e. Ces cellules, malgr�� le maintien d'un niveau ��lev�� de l'expression de g��nes impliqu��s dans la progression du cycle cellulaire, ont ��galement montr�� une expression accrue de plusieurs g��nes trouv��s en commun avec nos lymphocytes T vieillissants n'exprimant pas de t��lom��rase. En particulier, les cellules ayant une dur��e de vie prolong��e gr��ce �� l'expression de la t��lom��rase accumulaient ��galement certains inhibiteurs du cycle cellulaire tels que p16Ink4a et p21Cip1, associ��s �� l'arr��t de la croissance cellulaire. En r��sum��, nos r��sultats indiquent la pr��sence fonctionnelle de m��canismes alternatifs pouvant contr��ler la croissance r��plicative de ces cellules; ils sont donc encourageants dans l'optique d'une utilisation �� moindre risque de lymphocytes T ��immortalis��s �� �� des fins th��rapeutiques pour traiter les tumeurs malignes ou les infections. Summary Most mature blood cells have a finite life span. In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division eventually leading to chromosomal instability. Ectopic expression of the human telomerase reverse transcriptase (hTERT) gene in cells restores telomerase activity and results in the extension of their replicative life span. Despite lack of transformation characteristics, it is yet unknown whether somatic cells that over-express telomerase are biologically and physiologically indistinguishable from normal cells. Recent data suggest that telomerase might mediate additional functions in DNA repair, cell survival and cell growth. Using CD8+ T lymphocyte clones over-expressing telomerase we investigated the molecular mechanisms that regulate T cell proliferation and senescence. Here we show that early-passage T cell clones transduced or not with hTERT displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in hTERT-transduced cells with elongated telomeres and extended life span. These cells, despite maintaining high expression level of genes involved in cell cycle division and progression, also showed increased expression of several genes associated with normal aging T lymphocytes. In particular, late passage T cells over-expressing telomerase accumulated the cyclin-dependent inhibitors p16INK4a and p21CIP1 that have largely been associated with in vitro growth arrest. Whether tumor-reactive CD8+ T cells that ectopically express telomerase could now be used for adoptive transfer therapy in cancer patients remains unclear at this point. Nevertheless, our results regarding the safe and effective use of hTERT-transduced lymphocytes are encouraging, since they indicate that alternative growth arrest mechanisms such as p 16 and p21 are still functional in these cells and regulate to some extend their growth potential.

Clinical proteomics : a focus on transformed human T and B lymphocytes

R��sum�� large public La prot��omique clinique est une discipline qui vise l'��tude des prot��ines dans un but diagnostique ou th��rapeutique. Nous avons utilis�� cette approche pour ��tudier les lymphocytes T ��tueurs �� ou cytotoxiques qui font partie des globules blancs du syst��me sanguin et agissent dans la lutte contre les infections et les tumeurs. Ces cellules sont impliqu��es dans l'immunoth��rapie cellulaire qui se fonde sur la capacit�� naturelle des ces lymphocytes �� rep��rer les cellules tumorales et �� les d��truire. L'introduction du g��ne de la t��lom��rase dans les lymphocytes T r��sulte en une prolongation de leur long��vit��, ce qui en ferait des candidats int��ressants pour l'immunoth��rapie cellulaire. Il subsiste cependant des doutes quant aux cons��quences de l'utilisation de ces lymphocytes ��immortalis��s ��. Pour r��pondre �� cette question, nous avons compar�� le profile prot��ique de lymphocytes T cytotoxiques ��jeunes �� et vieux �� avec celui des lymphocytes ��immortalis��s ��. Nous avons trouv�� que ces derniers pr��sentent une double face et partagent �� la fois les caract��ristiques de la jeunesse et de la vieillesse. Dans une seconde ��tude de prot��omique clinique, nous nous sommes pench��s sur les lymphocytes B ��immortalis��s �� cette fois-ci non pas avec la t��lom��rase, mais avec le virus d'Epstein-Barr. Ces derniers sont utilis��s comme mod��le dans l'��tude de la leucodystrophie, une maladie g��n��tique rare qui affecte le cerveau. Notre but est d'identifier des marqueurs biologiques potentiels qui pourraient aider le diagnostic et le traitement de cette maladie neurod��g��n��rative. Nous avons pour ce faire compar�� les profiles prot��iques des lymphocytes B ��immortalis��s �� provenant d'individus sains et malades. Malheureusement, notre analyse n'a pas r��v��l�� de diff��rences notoires entre ces deux classes de lymphocytes. Ceci nous permet toutefois de conclure que la maladie n'affecte pas la synth��se des prot��ines de mani��re pr��pond��rante dans ces cellules sanguines. En r��sum��, le travail pr��sent�� dans cette th��se montre �� la fois le potentiel et les limites de l'analyse des prot��ines lymphocytaires, dans diff��rentes situations biologiques. R��sum�� La prot��omique clinique ouvre la porte vers de multiples horizons relatifs au traitement de diverses maladies. Ce domaine particulier alliant la prot��omique �� la m��decine, implique l'intervention de la biologie mol��culaire et cellulaire. Dans notre ��tude, nous nous sommes d'abord int��ress��s aux lymphocytes T CD8+ cytotoxiques dans le contexte de l'immunoth��rapie adoptive. Le fondement de cette th��rapie repose sur la capacit�� naturelle de ces lymphocytes �� reconna��tre les cellules tumorales et �� les d��truire chez les patients atteints de cancer. L'introduction du g��ne de la transcriptase r��verse de la t��lom��rase (hTERT) dans les lymphocytes T humains permet de rallonger leur dur��e de vie, sans toutefois induire d'alt��rations li��es �� la transformation. Cependant, des incertitudes subsistent quant �� la ressemblance physiologique et biochimique entre ces cellules surexprirnant la t��lom��rase et les cellules normales. Afin de r��pondre �� cette question, nous avons compar�� l'expression des prot��ines de lymphocytes humains T CD8+ ��jeunes �� et ��vieux ��avec celle de lymphocytes transduits avec hTERT. Nous avons trouv�� que les lymphocytes T surexprimant la t��lom��rase ont un profile prot��ique interm��diaire, avec certaines expressions prot��iques similaires aux jeunes cellules T et d'autres se rapprochant des cellules vieilles. Dans la seconde partie de notre ��tude, nous nous sommes int��ress��s aux lymphocytes B transform��s avec le virus d'Epstein-Barr provenant de patients atteints d'une maladie g��n��tique rare du cerveau, la leucodystrophie. Dans cette maladie, des mutations dans le facteur de transcription eIF2B, impliqu�� dans la synth��se prot��ique, ont ��t�� trouv��es. Afin d'analyser les cons��quences de ces mutations et de trouver des biomarqueurs sp��cifiques �� cette maladie, nous avons effectu�� une analyse prot��omique des lymphoblastes provenant de malades et d'individus sains. Nous avons trouv�� que les mutations dans le complexe ubiquitaire eIF2B n'affectent pas de mani��re significative l'expression des prot��ines des lymphoblastes mut��s. En conclusion, notre travail illustre le potentiel et les limitations des technologies prot��omiques utilis��es pour diss��quer l'implication des prot��ines dans diff��rentes situations biologiques. Summary Clinical proteomics opens the door to multiple applications related to the treatment of diseases. This particular field is at the crossroad of proteomics and medicine and involves tools from cellular and molecular biology. We focused first our investigations on cytotoxic T cells in the context of adoptive immunotherapy, which is an interesting and evolving field. The basis of this therapy relies on the natural capacity of cytotoxic CD8+ T lymphocytes in recognizing tumor cells and destroying them in cancer patients. As their number is reduced, the idea would be to use transformed T lymphocytes with extended life span. Overexpression of telomerase into human T lymphocytes results in the extension of their replicative life span, but it still remains unclear whether these cells are physiologically indistinguishable from normal ones. To address this question, we compared the proteome of young and aged CD8+ T lymphocytes with that of T cells transduced with hTERT and found that the latter cells displayed an intermediate protein pattern, sharing similar protein expression with young, but also with aged T cells. We were then interested in studying Epstein-Barr virus transformed B lymphocytes in the context of a rare human brain genetic disorder called leukodystrophy. In this disease, mutations in the ubiquitous factor eIF2B involved in protein synthesis and its regulation have been reported. In order to analyze the functional consequences of the mutations and to find out specific biomarkers of eIF2B-related disorders, proteomic and peptidomic studies were carried out on lymphoblasts from eIF2Bmutated patients versus healthy patients. Following two-dimensional gel electrophoresis and mass fingerprints, mutations in the eIF2B complex did not appear to significantly affect the proteome of the mutated lymphoblasts extracts. To conclude, our work emphasizes the potentials and the limitations of the proteomic technologies used to analyze the role of lymphocyte proteins in different biological situations.

A key regulatory role of the transcription factor NFATc2 in bronchial adenocarcinoma via CD8+ T lymphocytes.

The Ca(2+)-regulated calcineurin/nuclear factor of activated T cells (NFAT) cascade controls alternative pathways of T-cell activation and peripheral tolerance. Here, we describe reduction of NFATc2 mRNA expression in the lungs of patients with bronchial adenocarcinoma. In a murine model of bronchoalveolar adenocarcinoma, mice lacking NFATc2 developed more and larger solid tumors than wild-type littermates. The extent of central tumor necrosis was decreased in the tumors in NFATc2((-/-)) mice, and this finding was associated with reduced tumor necrosis factor-alpha and interleukin-2 (IL-2) production by CD8(+) T cells. Adoptive transfer of CD8(+) T cells of NFATc2((-/-)) mice induced transforming growth factor-beta(1) in the airways of recipient mice, thus supporting CD4(+)CD25(+)Foxp-3(+)glucocorticoid-induced tumor necrosis factor receptor (GITR)(+) regulatory T (T(reg)) cell survival. Finally, engagement of GITR in NFATc2((-/-)) mice induced IFN-gamma levels in the airways, reversed the suppression by T(reg) cells, and costimulated effector CD4(+)CD25(+) (IL-2Ralpha) and memory CD4(+)CD127(+) (IL-7Ralpha) T cells, resulting in abrogation of carcinoma progression. Agonistic signaling through GITR, in the absence of NFATc2, thus emerges as a novel possible strategy for the treatment of human bronchial adenocarcinoma in the absence of NFATc2 by enhancing IL-2Ralpha(+) effector and IL-7Ralpha(+) memory-expressing T cells.

Proteomic and transcriptomic analysis of human CD8(+) T lymphocytes over-expressing telomerase.

Human T lymphocytes have a finite life span resulting from progressive telomere shortening that occurs at each cell division, eventually leading to chromosomal instability. It has been shown that ectopic expression of the human telomerase reverse transcriptase (hTERT) gene into various human cells results in the extension of their replicative life span, without inducing changes associated with transformation. However, it is still unclear whether cells that over-express telomerase are physiologically and biochemically indistinguishable from normal cells. To address this question, we compared the proteome of young and aged human CD8(+) T lymphocytes with that of T cells transduced with hTERT. Interestingly, we found no global changes in the protein pattern in young T cells, irrespective of telomerase expression. In contrast, several relevant proteins with differential expression patterns were observed in hTERT-transduced T cells with extended life span upon long-term culture. Altogether, our data revealed that T lymphocytes over-expressing telomerase displayed an intermediate protein pattern, sharing a similar protein expression not only with young T cells, but also with aged T cells. Finally, the results obtained from this global proteomic approach are in agreement with the overall gene transcription profiling performed on the same T-cell derived clones.

L���impact de la grossesse sur l���amplitude et la diversit�� de la reconnaissance antig��nique des lymphocytes T cytotoxiques dirig��s contre le VIH-1

La transmission m��re-enfant (TME) du VIH-1 est un des enjeux majeurs de la pand��mie. Une meilleure compr��hension de la r��ponse des lymphocytes T cytotoxiques CD8+ (LTC) VIH-sp��cifiques lors de la grossesse facilitera le design de strat��gies optimales pour diminuer la TME. Notre objectif est donc de caract��riser l���amplitude et la diversit�� de la reconnaissance antig��nique des LTC VIH-sp��cifiques avant, pendant et apr��s la grossesse chez des femmes infect��es par le VIH-1. Nos r��sultats montrent pour la premi��re fois que l���initiation et la progression de la grossesse, �� elles seules, n'ont que peu d���influence sur l���amplitude et la diversit�� de la reconnaissance antig��nique des r��ponses LTC en termes de production d���IFN������. Ces r��sultats indiquent que les femmes infect��es par le VIH conservent une immunocomp��tence durant leur grossesse, du moins dans le contexte d���un traitement antir��troviral efficace. Ceci pourrait ��ventuellement aider �� promouvoir l���immunisation comme strat��gie pour pr��venir la TME du VIH���1.

��tude de l���expression de la mol��cule d���adh��rence CD146 dans les lymphocytes T

Les tumeurs solides sont infiltr��es par des cellules immunes (TIIC) dont la nature, la fonction et la composition varient d���un patient �� l'autre. Ces cellules inflammatoires influencent l'invasion tumorale en contr��lant la croissance et le potentiel m��tastatique d���une tumeur. Ainsi, il est propos�� d���utiliser cette infiltration comme outil diagnostic et pronostic de routine. Certaines cellules sont bien connues pour jouer un r��le important dans le contr��le de la progression tumorale, comme c���est le cas des lymphocytes T cytotoxiques CD8+ alors que d���autres poss��dent un r��le contradictoire. ��tant donn�� la d��pendance des tumeurs sur l�����quilibre entre ces diff��rentes cellules, il est important d���identifier les fonctions pr��cises des cellules immunes au sein de la tumeur. De nombreuses ��tudes sont r��alis��es afin d���identifier des marqueurs descriptifs du ph��notype et la fonction des cellules immunes dans la tumeur. Ce projet de doctorat se divise en deux parties : 1- Identifier la m��thode de d��sagr��gation des tissus tumoraux alt��rant le moins la biologie des TIIC pour leur caract��risation. 2- Caract��riser l���expression de la mol��cule d���adh��rence CD146 dans les TIIC et en identifier l���origine. L���identification de marqueurs pour la caract��risation ph��notypique et fonctionnelle des TIIC a ��t�� r��alis��e, entre autres, par la d��tection de prot��ines exprim��es par la cellule. Dans la premi��re partie de ce projet, nous avons d��montr�� que les m��thodes utilis��es pour d��sagr��ger les tissus tumoraux dans le but d���isoler les TIIC induisent des changements dans la biologie de ces cellules ce qui peut fausser les conclusions qui en d��rivent. Nous avons donc compar�� l'impact de trois m��thodes de d��sagr��gation : une dissociation m��canique utilisant la M��dimachineTM et deux digestions enzymatiques utilisant une collag��nase de type I seule ou combin��e �� de la collag��nase de type IV et de la DNase I de type II. Nous nous sommes int��ress��s �� l'effet de ces m��thodes sur des param��tres tels que la viabilit�� cellulaire, l���alt��ration des prot��ines de surface et la capacit�� des cellules �� prolif��rer. Nous avons d��montr�� que ces m��thodes affectent la viabilit�� des cellules de mani��re comparable, alors que la d��tection de certaines prot��ines de surface et la capacit�� de prolif��rer est r��duite/inhib��e par les traitements enzymatiques. Nous concluons qu���une m��thode m��canique utilisant la M��dimachineTM est mieux adapt��e �� la caract��risation des TIIC afin de conserver leurs propri��t��s. Dans la deuxi��me partie de notre projet, nous avons adapt�� cette m��thode �� la caract��risation des TIIC. Nous avons port�� une attention particuli��re �� la mol��cule d���adh��rence CD146 dont l���implication dans la migration des cellules immunes �� travers l���endoth��lium vers les sites d���inflammation est de plus en plus ��tudi��e dans les maladies autoimmunes. Nous avons mis en ��vidence une augmentation des proportions de cellules immunes exprimant CD146 dans les tumeurs comparativement au sang de patients de cancers. Cette expression est induite par les cellules tumorales tout en ��tant accrue par la n��crose de celles-ci. Nous d��montrons que ces cellules sont majoritairement des lymphocytes T CD4+ pr��sentant un profil immunosuppressif. En conclusion, nos r��sultats sugg��rent que CD146 participe �� la mise en place du contexte immunitaire dans la tumeur et augmente la capacit�� de migration des lymphocytes T CD4+. L���induction par les cellules tumorales de cette mol��cule d���adh��rence dans les cellules suppressives pourrait contribuer aux m��canismes immunor��gulateurs mis en place par la tumeur. CD146 pourrait ��tre un marqueur d���int��r��t pour l���identification des cellules immunosuppressives et pour le d��veloppement de nouvelles th��rapies.

Chronic colitis associated with HIV infection can be related to intraepithelial infiltration of the colon by CD8+T lymphocytes

Gastrointestinal complications in AIDS patients with diarrhoea are common clinical manifestations, frequently diagnosed by colonoscopy as non-specific colitis. We retrospectively study colon biopsies diagnosed as chronic colitis associated with HIV (CCH). Biopsies were sorted as patients with AIDS (serum CD4 < 200 cell/mm(3)) but without any clear infectious process (n = 12) and patients without HIV infection (n = 24). There are low numbers of CD4+ T lymphocytes in lamina propria of AIDS patients, but CD8+ T populations in this area appear to be similar in all studied groups, regardless of HIV infection or laboratory evidence of a specific agent. We found the clear evidence of CD8+ T cells infiltration in colonic mucosa in HIV patients with microscopic colitis. An imbalance of lymphocyte subpopulations in the colon, both in the lamina propria and epithelium, could result in an intraepithelial CD8 infiltration, involved in the pathogenesis of CCH in AIDS patients.

Infiltrating CD8(+) T lymphocytes, natural killer cells, and expression of IL-10 and TGF-beta 1 in chemically induced neoplasms in male Wistar rats

The present study aimed to estimate the number of CD8(+) T and natural killer (NK) infiltrating cells and the expression of interleukin-10 (IL-10) and transforming growth factor beta 1 (TGF-beta1) in chemically induced neoplasms in an initiation-promotion bioassay for carcinogenesis. Male Wistar rats were treated with N-nitrosodiethylamine, N-methyl-N-nitrosourea, N-butyl-N-(4-hydroxybutyl) nitrosamine, dihydroxy-di-N-propylnitrosamine, and 1,2-dimethylhydrazine for 4 weeks. Two groups were subsequently exposed through diet to phenobarbital (0.05%) or 2-acetylaminofluorene (0.01%) for 25 weeks. An untreated group was used as a control. Immune cells and cytokines were immunohistochemically evaluated in neoplasms and in surrounding normal tissues at the liver, kidneys, lung, and small and large intestines. When compared to the respective normal tissues, an increased number of NK cells was verified infiltrating the colon, lung, and kidney neoplasms, while the number of CD8+ T cells decreased in the intestine and lung neoplasms. Expression of IL-10 was found mainly in kidney tumors. TGF-beta1 was expressed mainly in the liver and kidneys tumors. The results indicate that the differential occurrence of immune cells between neoplastic and normal tissues could be dependent upon tumor microenvironment.

Identification of preferentially targeted tumor-associated antigens in melanoma patients via mRNA stimulation of CD8+ blood lymphocytes

Until now, therapeutic vaccination of cancer patients has mainly relied on rather few T cell epitopes processed from structurally normal shared tumor antigens and presented by frequent HLA alleles. So far the design of these studies has not addressed the individuality of tumor-host interactions, which are not only determined by the antigenic tumor phenotype or the natural HLA polymorphism, but also by the individual T cell repertoire. The procedure described herein was developed to identify the preferential targets of the individual repertoire from a panel of known shared tumor-associated antigens. Lymphocytes were isolated from the peripheral blood of cancer patients or healthy donors and stimulated twice with autologous mRNA-transfected FastDC (Dauer et al., J Immunol. 170:4069, 2003). FastDC were generated from blood monocytes and separately transfected via lipofection with in vitro transcribed mRNAs encoding the panel antigens. Responder lymphocytes were tested on day 12 in a 20-hour IFN-g ELISPOT assay for recognition of 293T cells co-transfected pairwise with plasmids encoding the stimulation antigens and the respective individual���s HLA class I alleles. In a first step, stimulation parameters were optimized for the detection of anti-HCMV pp65 responses. A maximum amplification of pp65-specific CD8+ T cell responses was obtained at a rather low IL-2 concentration (25 IU/ml) and at a minimum APC-to-effector ratio of 1:10. Addition of IL-4, IL-7 or IL-15 did not substantially improve the stimulatory potential. The test was applied to the human melanoma models D05 and MZ2, in both of which multiple T cell-defined antigens had previously been identified by expression screening. Blood lymphocytes were stimulated in parallel with autologous tumor cells and with mRNA-transfected FastDC. In D05, T cell reactivities against three out of eleven epitopes induced by stimulation with tumor cells were also found after stimulation with mRNA-transfected FastDC. Two further T cell target epitopes were identified with mRNA but not with tumor cell stimulation. In MZ2, T cell responses against five distinct epitopes were detected on day 12 after stimulation with mRNA transfectants. The same responses were detectable after stimulation with tumor cells only on day 32. mRNA stimulations against 21 tumor-associated antigens in addition to HCMV pp65 were performed in four healthy individuals. In all cases, CD8+ T cells against HCMV pp65 could be expanded. Among tumor-associated antigens, only reactivity against Melan-A/MART-1 in association with HLA-A*0201 was detectable in one of the donors. The vaccination of patients with targets a priori known to be recognized by their T cell repertoire may help to improve the outcome of therapeutic vaccination.