Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8(+) memory T cells.

Protection against malaria can be achieved by induction of a strong CD8(+) T-cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long-lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8(+) T-cell response and sterile protection, which is short-lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite-specific antibodies that showed protective capacity. The numbers of CSP-specific CD8(+) T cells were not substantially enhanced by sporozoite infections; however, CSP-specific memory CD8(+) T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized-only mice. CD4(+) T cells contributed to protection as well; but CD8(+) memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8(+) effector-memory T cells with increased cytotoxic activity as well as CD4(+) memory T cells and neutralizing antibodies.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 ��g naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

Selective bystander proliferation of memory CD4+ and CD8+ T cells upon NK T or T cell activation.

Ag-experienced or memory T cells have increased reactivity to recall Ag, and can be distinguished from naive T cells by altered expression of surface markers such as CD44. Memory T cells have a high turnover rate, and CD8(+) memory T cells proliferate upon viral infection, in the presence of IFN-alphabeta and/or IL-15. In this study, we extend these findings by showing that activated NKT cells and superantigen-activated T cells induce extensive bystander proliferation of both CD8(+) and CD4(+) memory T cells. Moreover, proliferation of memory T cells can be induced by an IFN-alphabeta-independent, but IFN-gamma- or IL-12-dependent pathway. In these conditions of bystander activation, proliferating memory (CD44(high)) T cells do not derive from activation of naive (CD44(low)) T cells, but rather from bona fide memory CD44(high) T cells. Together, these data demonstrate that distinct pathways can induce bystander proliferation of memory T cells.

NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8⁺ T cells.

Memory T cells exert antigen-independent effector functions, but how these responses are regulated is unclear. We discovered an in vivo link between flagellin-induced NLRC4 inflammasome activation in splenic dendritic cells (DCs) and host protective interferon-γ (IFN-γ) secretion by noncognate memory CD8(+) T cells, which could be activated by Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that CD8α(+) DCs were particularly efficient at sensing bacterial flagellin through NLRC4 inflammasomes. Although this activation released interleukin 18 (IL-18) and IL-1β, only IL-18 was required for IFN-γ production by memory CD8(+) T cells. Conversely, only the release of IL-1β, but not IL-18, depended on priming signals mediated by Toll-like receptors. These findings provide a comprehensive mechanistic framework for the regulation of noncognate memory T cell responses during bacterial immunity.

B cells Can Modulate the CD8 Memory T Cell after DNA Vaccination Against Experimental Tuberculosis

Abstract Background Although B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown. Methods In this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge. Results In vitro and in vivo studies showed that B cells efficiently present antigens after naked plasmid pcDNA3 encoding M. leprae 65-kDa heat shock protein (pcDNA3-Hsp65) internalization and protect B knock-out (BKO) mice against Mycobacterium tuberculosis infection. pcDNA3-Hsp65-transfected B cells adoptively transferred into BKO mice rescued the memory phenotypes and reduced the number of CFU compared to wild-type mice. Conclusions These data not only suggest that B cells play an important role in the induction of CD8 T cells but also that they improve bacterial clearance in DNA vaccine model.

Essential role of the Wnt pathway effector Tcf-1 for the establishment of functional CD8 T cell memory.

Immune protection from intracellular pathogens depends on the generation of terminally differentiated effector and of multipotent memory precursor CD8 T cells, which rapidly regenerate effector and memory cells during recurrent infection. The identification of factors and pathways involved in CD8 T cell differentiation is of obvious importance to improve vaccination strategies. Here, we show that mice lacking T cell factor 1 (Tcf-1), a nuclear effector of the canonical Wingless/Integration 1 (Wnt) signaling pathway, mount normal effector and effector memory CD8 T cell responses to infection with lymphocytic choriomeningitis virus (LCMV). However, Tcf-1-deficient CD8 T cells are selectively impaired in their ability to expand upon secondary challenge and to protect from recurrent virus infection. Tcf-1-deficient mice essentially lack CD8 memory precursor T cells, which is evident already at the peak of the primary response, suggesting that Tcf-1 programs CD8 memory cell fate. The function of Tcf-1 to establish CD8 T cell memory is dependent on the catenin-binding domain in Tcf-1 and requires the Tcf-1 coactivators and Wnt signaling intermediates beta-catenin and gamma-catenin. These findings demonstrate that the canonical Wnt signaling pathway plays an essential role for CD8 central memory T cell differentiation under physiological conditions in vivo. They raise the possibility that modulation of Wnt signaling may be exploited to improve the generation of CD8 memory T cells during vaccination or for therapies designed to promote sustained cytotoxic CD8 T cell responses against tumors.

Generation and function of mouse central memory and effector memory T cells

1. SUMMARY Based on functional and homing properties, two subsets of memory T lymphocytes have been defined both in humans and in mice. Central memory T cells (TCM cells) express the lymph node homing receptors CD62L and CCR7, have poor effector function and proliferate efficiently upon antigenic stimulation. Effector memory T cells (TEM cells) do not express CCR7, are mostly CD62L negative and therefore are excluded from lymph nodes, but are able to migrate to sites of inflammation where they exert immediate effector function by producing inflammatory cytokines and cytotoxic mediators. In the present work we have addressed two questions that emerged since the definition of TCM and TEM cells. Firstly, what are the priming conditions for generation of TCM and TEM and, secondly, what is the migratory capacity of TCM and TEM cells in inflammatory conditions. By using naive TCR-transgenic OT-I CD8+ T cells and OT-II CD4+ T cells and ovalbumin pulsed-mature dendritic cells (DCs) we set up an in vitro system in which the strength of T cell stimulation is controlled by varying the ratio of T cells and DCs and the duration of DC-T cell interaction. Using this system we found that precursors of TCM and TEM cells are generated at different strength of stimulation and that T cells capable of persisting in vivo in the absence of antigen and of mounting recall responses is optimally induced by intermediate stimulatory strength. In addition, we found that lymph nodes draining sites of mature DC or adjuvant inoculation recruit CD8+ CD62L- CCR7- effector and TEM cells. CD8+ T cell recruitment in reactive lymph nodes requires CXCR3 expression on T cells and occurs through high endothelial venules (HEVs) in concert with HEV lurninal expression of the CXCR3 ligand CXCL9. In reactive lymph nodes, recruited T cells establish stable interactions with and kill antigen-bearing DCs, limiting the ability of these DCs to activate CD4+ and CD8+ T cells. Taken togther these data define conditions for the generation of TCM and TEM cells and define an inflammatory pathway of effector T cell migration in lymph nodes. The inducible recruitment of blood-borne effector and TEM CD8+ cells to lymph nodes may represent a mechanism for terminating primary and limiting secondary immune responses.

Bystander-Activated Memory CD8��T Cells Control Early Pathogen Load in an Innate-like, NKG2D-Dependent Manner.

During an infection the antigen-nonspecific memory CD8 T��cell compartment is not simply an inert pool of cells, but becomes activated and cytotoxic. It is unknown how these cells contribute to the clearance of an infection. We measured the strength of T��cell receptor (TCR) signals that bystander-activated, cytotoxic CD8 T��cells (BA-CTLs) receive in��vivo and found evidence of limited TCR signaling. Given this marginal contribution of the TCR, we asked how BA-CTLs identify infected target cells. We show that target cells express NKG2D ligands following bacterial infection and demonstrate that BA-CTLs directly eliminate these target cells in an innate-like, NKG2D-dependent manner. Selective inhibition of BA-CTL-mediated killing led to a significant defect in pathogen clearance. Together, these data suggest an innate role for memory CD8 T��cells in the early immune response before the onset of a de novo generated, antigen-specific CD8 T��cell response.

Profiling of signal transduction in human memory T cells

R��sum�� pour un large public: La vaccination a eu un impact ��norme sur la sant�� mondiale. Mais, quel est le principe d'un vaccin? Il est bas�� sur la 'm��moire immunologique', qui est une particularit�� exclusive des syst��mes immunitaires des organismes ��volu��s. Suite �� une infection par un pathog��ne, des cellules sp��cialis��es de notre syst��me immunitaire (les lymphocytes) le reconnaissent et initient une r��action immunitaire qui a pour but son ��limination. Pendant cette r��action se d��veloppent aussi des cellules, appel��es cellules lymphocytaires m��moire, qui persistent pour longue dur��e et qui ont la capacit�� de stimuler une r��action immunitaire tr��s efficace imm��diatement apr��s une seconde exposition �� ce m��me pathog��ne. Ce sont ces cellules m��moires (lymphocytes B et T) qui sont �� la base de la 'm��moire immunologique' et qui sont stimul��es lors de la vaccination. Chez l'homme, deux populations distinctes des lymphocytes T m��moires ont ��t�� identifi��es: les cellules centrales (CM) et effectrices (EM) m��moires. Ces populations sont fonctionnellement h��t��rog��nes et exercent des r��les distincts et essentiels dans l'immunit�� protectrice. Typiquement, les cellules effectrices m��moires sont capables de tuer imm��diatement le pathog��ne tandis que les cellules centrales m��moires sont responsables d'initier une r��ponse immunitaire compl��te. Pourtant, les m��canismes biochimiques qui contr��lent les fonctions de ces cellules ont ��t�� jusqu'�� pr��sent peu ��tudi��s �� cause de la faible fr��quence de ces cellules et de la quantit�� limit��e de tissus humains disponibles pour les analyses. La compr��hension de ces m��canismes est cruciale pour la r��alisation de vaccins efficaces et pour le d��veloppement de nouveaux m��dicaments capables de moduler la r��ponse immunitaire lymphocytaire. Dans cette th��se, nous avons d'abord d��velopp�� et am��lior�� une technologie appel��e 'prot��ine array en phase inverse' qui poss��de un niveau de sensibilit�� beaucoup plus ��lev�� par rapport aux technologies classiquement utilis��es dans l'��tude des prot��ines. Gr��ce �� cette technique, nous avons pu comparer la composition prot��ique du syst��me de transmission des signaux d'activation des cellules CM et EM humaines. L'analyse de 8 �� 13 sujets sains a montr�� que ces populations des cellules m��moires poss��dent un syst��me de signalisation prot��ique diff��rent. En effet, les cellules EM poss��dent, par rapport aux cellules CM, des niveaux r��duits d'une prot��ine r��gulatrice (appel��e c-Cbl) que nous avons d��montr�� comme ��tant responsable des fonctions sp��cifiques de ces cellules. En effet, en augmentant artificiellement l'expression de cette prot��ine r��gulatrice dans les cellules EM jusqu'au niveau de celui des cellules CM, nous avons induit dans les cellules EM des capacit��s fonctionnelles caract��ristiques des cellules CM. En conclusion, notre ��tude a identifi��, pour la premi��re fois chez l'homme, un m��canisme biochimique qui contr��le les fonctions des populations des cellules m��moires. R��sum�� en Fran��ais: Les cellules m��moires persistent inertes dans l'organisme et produisent des r��actions immunitaires rapides et robustes contre les pathog��nes pr��c��demment rencontr��s. Deux populations distinctes des cellules m��moires ont ��t�� identifi��es chez l'homme: les cellules centrales (CM) et effectrices (EM) m��moires. Ces populations sont fonctionnellement h��t��rog��nes et exercent des r��les distincts et critiques dans l'immunit�� protectrice. Les m��canismes biochimiques qui contr��lent leurs fonctions ont ��t�� jusqu'�� pr��sent peu ��tudi��s, bien que leur compr��hension soit cruciale pour le d��veloppement des vaccins et des nouveaux traitements/m��dicaments. Les limites majeures �� ces ��tudes sont la faible fr��quence de ces populations et la quantit�� limit��e de tissus humains disponibles. Dans cette th��se nous avons d'abord d��velopp�� et am��lior�� la technologie de 'prot��ine array en phase inverse' afin d'analyser les mol��cules de signalisation des cellules m��moires CD4 et CD8 humaines isol��es ex vivo. L'excellente sensibilit��, la reproductibilit�� et la lin��arit�� de la d��tection, ont permis de quantifier des variations d'expression prot��iques sup��rieures �� 20% dans un lysat ��quivalent �� 20 cellules. Ensuite, gr��ce �� l'analyse de 8 �� 13 sujets sains, nous avons prouv�� que les cellules m��moires CD8 ont une composition homog��ne de leur syst��me de signalisation tandis que les cellules CD4 EM expriment significativement de plus grandes quantit��s de SLP-76 et des niveaux r��duits de c-Cbl, Syk, Fyn et LAT par rapport aux cellules CM. En outre, l'expression r��duite du r��gulateur n��gatif c-Cbl est corr��l��e avec l'expression des SLP-76, PI3K et LAT uniquement dans les cellules EM. L'��valuation des propri��t��s fonctionnelles des cellules m��moires a permis de d��montrer que l'expression r��duite du c-Cbl dans les cellules EM est associ�� �� une diminution de leur seuil d'activation. En effet, gr��ce a la technique de transduction cytosolique, nous avons augment�� la quantit�� de c-Cbl des cellules EM �� un niveau comparable �� celui des cellules CM et constat�� une r��duction de la capacit�� des cellules EM �� prolif��rer et s��cr��ter des cytokines. Ce m��canisme de r��gulation d��pend principalement de l'activit�� d'ubiquitine ligase de c-Cbl comme d��montr�� par l'impact r��duit du mutant enzymatiquement d��ficient de c-Cbl sur les fonctions de cellules EM. En conclusion, cette th��se identifie c-Cbl comme un r��gulateur critique des r��ponses fonctionnelles des populations de cellules T m��moires et fournit, pour la premi��re fois chez l'homme, un m��canisme contr��lant l'h��t��rog��n��it�� fonctionnelle des ces cellules. De plus, elle valide l'utilisation combin��e des 'RPP arrays' et de la transduction cytosolique comme outil puissant d'analyse quantitative et fonctionnel des prot��ines de signalisation. Summary : Memory cells persist in a quiescent state in the body and mediate rapid and vigorous immune responses toward pathogens previously encountered. Two subsets of memory cells, namely central (CM) and effector (EM) memory cells, have been identified in humans. These subsets display high functional heterogeneity and assert critical and distinct roles in the control of protective immunity. The biochemical mechanisms controlling their functional properties remain so far poorly investigated, although their clarification is crucial for design of effective T-cell vaccine and drug development. Major limitations to these studies lie in the low frequency of memory T cell subsets and the limited amount of human specimen available. In this thesis we first implemented the innovative reverse phase protein array approach to profile 15 signalling components in human CD8 and CD4 memory T cells isolated ex vivo. The high degree of sensitivity, reproducibility and linearity achieved, allowed an excellent quantification of variations in protein expression higher than 20% in as few as 20-cell equivalent per spot. Based on the analysis of 8 to 13 healthy subjects, we showed that CD8 memory cells have a homogeneous composition of their signaling machinery while CD4 EM cells express statistically significant increased amounts of SLP-76 and reduced levels of c- Cbl, Syk, Fyn and LAT as compared to CM cells. Moreover, in EM but not CM cells, reduced expression of negative regulator c-Cbl correlated with the expression of SLP-76, PI3K and LAT. Subsequently, we demonstrated that the higher functional properties and the lower functional threshold of EM cells is associated with reduced expression of c-Cbl. Indeed, by increasing c-Cbl content of EM cells to the same level of CM cells using cytosolic transduction, we impaired their proliferation and cytokine production. This regulatory mechanism was primarily dependent on c-Cbl E3 ubiquitin ligase activity as evidenced by the weaker impact of enzymatically deficient c-Cbl C381A mutant on EM cell functions. Together, these results identify c-Cbl as a critical regulator of the functional responses of memory T cell subsets and provides, for the first time in humans, a mechanism controlling the functional heterogeneity of memory CD4 cells. Moreover it validates the combined use of RPP arrays and cytosolic transduction approaches as a powerful tool to quantitatively analyze signalling proteins and functionally assess their roles.

CD4+ T cell help improves CD8+ T cell memory by retained CD27 expression

CD4+ T cell help during the priming of CD8+ T lymphocytes imprints the capacity for optimal secondary expansion upon re-encounter with antigen. Helped memory CD8+ T cells rapidly expand in response to a secondary antigen exposure, even in the absence of T cell help and, are most efficient in protection against a re-infection. In contrast, helpless memory CTL can mediate effector function, but secondary expansion is reduced. How CD4+ T cells instruct CD8+ memory T cells during priming to undergo efficient secondary expansion has not been resolved in detail. Here, we show that memory CTL after infection with lymphocytic choriomeningitis virus are CD27(high) whereas memory CTL primed in the absence of CD4+ T cell have a reduced expression of CD27. Helpless memory CTL produced low amounts of IL-2 and did not efficiently expand after restimulation with peptide in vitro. Blocking experiments with monoclonal antibodies and the use of CD27(-/-) memory CTL revealed that CD27 ligation during restimulation increased autocrine IL-2 production and secondary expansion. Therefore, regulating CD27 expression on memory CTL is a novel mechanism how CD4+ T cells control CTL memory.

Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells

The skin of an adult human contains about 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice, but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human-engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All nonrecirculating resident memory T cells (TRM) expressed CD69, but most were CD4(+), CD103(-), and located in the dermis, in contrast to studies in mice. Both CD4(+) and CD8(+) CD103(+) TRM were enriched in the epidermis, had potent effector functions, and had a limited proliferative capacity compared to CD103(-) TRM. TRM of both types had more potent effector functions than recirculating T cells. We observed two distinct populations of recirculating T cells, CCR7(+)/L-selectin(+) central memory T cells (TCM) and CCR7(+)/L-selectin(-) T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions, and TMM were depleted more slowly from skin after alemtuzumab, suggesting that TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities.

Measuring the diaspora for virus-specific CD8+ T cells

The CD8+ T cell diaspora has been analyzed after secondary challenge with an influenza A virus that replicates only in the respiratory tract. Numbers of DbNP366- and DbPA224-specific CD8+ T cells were measured by tetramer staining at the end of the recall response, then followed sequentially in the lung, lymph nodes, spleen, blood, and other organs. The extent of clonal expansion did not reflect the sizes of the preexisting memory T cell pools. Although the high-frequency CD8+ tetramer+ populations in the pneumonic lung and mediastinal lymph nodes fell rapidly from peak values, the ���whole mouse��� virus-specific CD8+ T cell counts decreased only 2-fold over the 4 weeks after infection, then subsided at a fairly steady rate to reach a plateau at about 2 months. The largest numbers were found throughout in the spleen, then the bone marrow. The CD8+DbNP366+ and CD8+DbPA224+ sets remained significantly enlarged for at least 4 months, declining at equivalent rates while retaining the nucleoprotein > acid polymerase immunodominance hierarchy characteristic of the earlier antigen-driven phase. Lowest levels of the CD69 ���activation marker��� were detected consistently on virus-specific CD8+ T cells in the blood, then the spleen. Those in the bone marrow and liver were intermediate, and CD69hi T cells were very prominent in the regional lymph nodes and the nasal-associated lymphoid tissue. Any population of ���resting��� CD8+ memory T cells is thus phenotypically heterogeneous, widely dispersed, and subject to broad homeostatic and local environmental effects irrespective of epitope specificity or magnitude.

The role of CD8+CCR4+ T-cells in axial spondyloarthritis

Axial spondyloarthritis (AxSpA) is an inflammatory disease affecting the axial skeleton. The infiltrate of T-cells in the structural lesions has been found to contribute to bone remodeling, but consensus relating the functional contribution of different T-cell subsets to pathogenesis has not been reached yet. Aim of the project was to characterize circulating T-cells and their homing markers from axSpA patients in order to identify cellular populations that could migrate to inflamed tissues and be implicated in axSpA. We found an altered proportion of circulating na��ve and memory T-cells in axSpA patients, and a skew in favor of CD8+ T-cells expressing the chemokine receptor CCR4. Since CCL17 and CCL22, the two ligands for CCR4, are found to be elevated in the sera of axSpA patients, we investigated in details the role of CD8+CCR4+ T cells in axSpA. Our data showed that circulating CD8+CCR4+ T-cells display an effector memory phenotype and express homing markers for tissues that are target of the disease. Noteworthy, CD8+CCR4+ T cells from axSpA patients were activated, expressed markers of proliferation and acquired a cytotoxic phenotype, as demonstrated by the increased production of granzyme and perforin. CD8+CCR4+ T cells from axSpA patients upregulate the transcription of genes involved in bone mineralization and downregulate genes involved in osteoclast differentiation, indicating their possible involvement in bone remodeling. Furthermore, CD8+CCR4+ T cells stimulated with PMA and ionomycin were able to produce and release TNF and IL-8, two cytokines involved in osteoclastogenesis, indicating that CD8+CCR4+ T-cells after stimulation would be able to promote osteoclasts differentiation and neutrophils recruitment. Taken together our data suggest that CD8+CCR4+ T cells might exert a pathogenic role in axSpA, by releasing mediators of tissue damage, bone remodeling and recruitment of other pro inflammatory cells.