Characterization of T cell clones from chagasic patients: predominance of CD8 surface phenotype in clones from patients with pathology

Human Chagas' disease, caused by the protozoan Trypanosoma cruzi, is associated with pathological processes whose mechanisms are not known. To address this question, T cell lines were developed from chronic chagasic patients peripheral blood mononuclear cells (PBMC) and cloned. These T cell clones (TCC) were analyzed phenotypically with monoclonal antibodies by the use of a fluorescence microscope. The surface phenotype of the TCC from the asymptomatic patient were predominantly CD4 positive (86%). On the contrary, the surface phenotype CD8 was predominant in the TCC from the patients suffering from cardiomegaly with right bundle branch block (83%), bradycardia with megacolon (75 %) and bradycardia (75%). Future studies will be developed in order to identify the antigens eliciting these T cell subpopulations.

Enrichment for murine keratinocyte stem cells based on cell surface phenotype

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [3H]thymidine ([3H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [3H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of α6 integrin and low to undetectable expression of the transferrin receptor (CD71) termed α6briCD71dim cells, are enriched for epithelial stem cells because they represent a minor (≈8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing ≈70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed α6briCD71bri, representing the majority (≈60%) of basal keratinocytes that are actively cycling, and importantly contain ≈70% of [3H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71dim cells in the hair follicle bulge region, a well documented location for KSCs.

Phenotype and function of protective T cell immune responses in HIV.

PURPOSE OF REVIEW: Definition of T cell immune correlates in HIV infection remains a lofty goal towards our understanding of the HIV-specific immune response. This review will focus upon recent developments and controversies in our understanding of protective T cell responses against HIV. RECENT FINDINGS: It has become clear that multiple functions and phenotypic markers of T cells must be assessed to accurately characterize the complexity of CD4 and CD8 T cell responses. While evidence indicates that a hallmark of protective immune responses in HIV infection is the presence of 'polyfunctional' T cell responses, a disconnect remains between the function and phenotype of effective HIV-specific T cells. Moreover, there may be inherent differences in the ability of specific human leukocyte antigen class I families to promote CD8 T cell effector versus polyfunctional responses. It remains to be determined how polyfunctional responses arise in HIV infection, which functions are important for control, and whether surface phenotype markers provide an indication of protective capacity. SUMMARY: Polyfunctional and phenotypic assessment of T cell responses have clearly advanced our understanding of HIV specific immune responses. Critical questions remain, however, especially whether polyfunctional T cell responses control, or are controlled by, HIV replication.

Unique molecular surface features of in vivo tolerized T cells

Differential expression of surface markers can frequently be used to distinguish functional subsets of T cells, yet a surface phenotype unique to T cells induced into an anergic state has not been described. Here, we report that CD4 T cells rendered anergic in vivo by superantigen can be identified by loss of the 6C10 T cell marker. Inoculation of Vβ8.1 T cell antigen receptor (TCR) transgenic mice with a Vβ8.1-reactive minor lymphocyte-stimulating superantigen (Mls-1a) induces tolerance to Mls-1a by clonal anergy. CD4 lymph node T cells from Mls-1a inoculated transgenic mice enriched for the 6C10− phenotype neither proliferate nor produce interleukin-2 upon TCR engagement, whereas 6C10+ CD4 T cells retain responsiveness. Analysis of T cell memory markers demonstrate that 6C10− T cells remain 3G11hi but express heterogeneous levels of CD45RB, CD62L, CD44, and the CD69 early activation marker, suggesting that T cells at various degrees of activation can be functionally anergic. These studies demonstrate that anergic T cells can be purified based on 6C10 expression permitting examination of issues concerning biochemical and biological features specific to T cell anergy.

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha(5) chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha(5) chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (a5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha(3)beta(1), and alpha(6)beta(4) integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.

Functional analysis of the murine T lymphocyte immune response to a protozoan parasite, Leishmania tropica

The results presented in this review summarize a seirs of experiments designed to characterize the murine T cell imune response to the protozoan parasite Leishmania tropica. Enriched T cell populations and T cell clones specific for L. tropica antigens were derived from lymph nodes of primed mice and maintained in continous culture in vitro. These T lymphocytes were shown (A) to express the Lyt 1+ 3- cell surface phenotype, (B) to proliferate specifically in vitro in response to parasite antigens, together with a source of irradiated syngeneic macrophages, (C) to transfer antigen-specific delayed-type hypersensitivity (DTH) responses to normal syngeneic mice, (D) to induce specific activation of parasitized macrophages in vitro resulting in the destruction of intracellular parasites, (E) to provide specific helper activity for antibody responses in vitro in a hapten-carrier system. Protection studies using these defiened T cell populations should allow the characterization of parasite antigen(s) implicated in the induction of cellular immune responses beneficial for the host.

Fine specificity of a BALB/c T cell clone directed against beef apo cytochrome c.

A BALB/c cloned T cell line directed against beef apo cytochrome c was shown to exhibit the Lyt-1+2- cell surface phenotype. The fine specificity of antigen recognition exhibited by the T cell clone was assessed by using a variety of peptide preparations obtained from cytochrome c of different sources. The peptide segment recognized by this T cell clone, in conjunction with I-A region gene products, appeared similar to that bound by a monoclonal antibody specific for beef apo cytochrome c derived from the same strain of mice.

NLRP3 Suppresses NK Cell-Mediated Responses to Carcinogen-Induced Tumors and Metastases.

The NLRP3 inflammasome acts as a danger signal sensor that triggers and coordinates the inflammatory response upon infectious insults or tissue injury and damage. However, the role of the NLRP3 inflammasome in natural killer (NK) cell-mediated control of tumor immunity is poorly understood. Here, we show in a model of chemical-induced carcinogenesis and a series of experimental and spontaneous metastases models that mice lacking NLRP3 display significantly reduced tumor burden than control wild-type (WT) mice. The suppression of spontaneous and experimental tumor metastases and methylcholanthrene (MCA)-induced sarcomas in mice deficient for NLRP3 was NK cell and IFN-γ-dependent. Focusing on the amenable B16F10 experimental lung metastases model, we determined that expression of NLRP3 in bone marrow-derived cells was necessary for optimal tumor metastasis. Tumor-driven expansion of CD11b(+)Gr-1(intermediate) (Gr-1(int)) myeloid cells within the lung tumor microenvironment of NLRP3(-/-) mice was coincident with increased lung infiltrating activated NK cells and an enhanced antimetastatic response. The CD11b(+)Gr-1(int) myeloid cells displayed a unique cell surface phenotype and were characterized by their elevated production of CCL5 and CXCL9 chemokines. Adoptive transfer of this population into WT mice enhanced NK cell numbers in, and suppression of, B16F10 lung metastases. Together, these data suggested that NLRP3 is an important suppressor of NK cell-mediated control of carcinogenesis and metastases and identify CD11b(+)Gr-1(int) myeloid cells that promote NK cell antimetastatic function. Cancer Res; 72(22); 5721-32. ©2012 AACR.

Human thymus exports naive CD8 T cells that can home to nonlymphoid tissues.

Functionally naive CD8 T cells in peripheral blood from adult humans can be fully described by their CD45RA(bright)CCR7(+)CD62L(+) cell surface phenotype. Cord blood lymphocytes, from healthy newborns, are homogenously functionally naive. Accordingly, the majority of cord blood CD8 T cells express the same pattern of cell surface molecules. Unexpectedly, however, a significant fraction of cord blood CD8 T cells express neither CCR7 nor CD62L. Yet these cells remain functionally naive as they contain high levels of TCR excision circles, have long telomeres, display highly polyclonal TCRs, and do not exhibit immediate effector functions. In addition, these CD8 T cells already represent a significant fraction of the mature naive CD8 single-positive thymocyte repertoire and may selectively express the cutaneous lymphocyte Ag. We suggest that CD8 single-positive thymocytes comprise two pools of naive precursors that exhibit distinct homing properties. Once seeded in the periphery, naive CCR7(+)CD62L(+) CD8 T cells patrol secondary lymphoid organs, whereas naive CCR7(-)CD62L(-) CD8 T cells selectively migrate to peripheral tissues such as skin.

Antigenic heterogeneity of clones and subclones from human melanoma cell lines demonstrated by a panel of monoclonal antibodies and flow microfluorometry analysis.

Cells from two melanoma cell lines, Me43 and GLL-19, were cloned in methylcellulose cultures and 20 randomly selected colonies from each line were picked up by micromanipulation, expanded in liquid cultures, and considered as clones of the original cell lines. The antigenic cell surface phenotype of these clones defined by panel of 12 monoclonal antibodies (MAb) was analyzed by flow microfluorometry (FMF) using a fluorescence-activated cell sorter (FACS II) and compared with the known stable phenotype of the parent cell line. The antibody panel consisted of eight MAb against melanoma-associated antigens, two MAb against monomorphic determinants of HLA-DR (la) and HLA-ABC, respectively, one MAb against the common acute lymphoblastic leukemia antigen (CALLA) and one MAb against carcinoembryonic antigen used as control. A remarkable heterogeneity in terms of qualitative and quantitative expression of the cell surface antigens studied was observed among and within the different clones. The single-cell origin of the clones was assessed by comparing the clonogenic cell frequency, determined by limiting dilutions in microculture plates, with the cloning efficiency observed in Petri dishes. Both techniques using methylcellulose medium gave the same percentages of growing colonies. Cells from four Me43 clones were recloned in methylcellulose and the phenotype of five randomly selected subclones from each clone was analysed using the same panel of monoclonal antibodies. Each subclone also displayed heterogeneity with individual phenotypes different from that of the original clone and from the parental Me43 cell line. The antigen expression by individual cells in situ within clones was analyzed on frozen sections from colonies using the same panel of MAb and a biotin-avidin immunoperoxidase method. The results confirmed the marked heterogeneity of antigen expression within and among colonies, as indicated by the FMF analysis.

Functional analysis of different haematopoietic stem cell subsets

RÉSUMÉ : Elucider les bases moléculaires et cellulaires du fonctionnement des cellules souches s'avère crucial dans la compréhension de l'organisation cellulaire au sein des tissus et des organes ainsi que pour le développement de nouvelles stratégies thérapeutiques en médecine régénérative et en oncologie. Les cellules souches adultes les mieux connues sont celles responsables de l'hématopoïèse, les cellules souches hématopoïétiques (CSH). Durant ces dernières années, la recherche a porté une attention particulière à l'isolation prospective de CSH dérivées de la moelle osseuse de souris en utilisant des marqueurs de surface cellulaire ainsi que des propriétés fonctionnelles alléguées. Par la suite, la capacité fonctionnelle des CSH a été vérifiée classiquement par leur transplantation intraveineuse dans des souris réceptrices conditionnées et par l'analyse de leur aptitude à reconstituer le système hématopoïétique à long terme. Des études récentes suggèrent que la transplantation des cellules directement dans la moelle osseuse pourrait non seulement aboutir à une prise de greffe plus rapide et plus efficace, mais pourrait même aider à l'identification de cellules qui ont certes des propriétés intrinsèques de CSH, mais qui n'ont pas la capacité de trouver leur niche au sein de la moelle osseuse et ont donc échoué dans les analyses classiques de reconstitution. Dans cette étude, nous comparons à deux niveaux la fonction de différents sous-groupes de cellules souches de la moelle osseuse, définis par leur phénotype de surface cellulaire. Premièrement, nous étudions leur capacité à reconstituer des souris létalement irradiées après injection soit intraveineuse soit intrafémorale. Deuxièmement, par analyse cytométrique de flux à 8 couleurs, nous comparons leur activité relative de « side population » (SP) par exclusion du colorant fluorescent Hoechst 33342. Nos résultats préliminaires renforcent en effet l'idée que la transplantation intrafémorale aboutit à une greffe plus rapide et plus efficace. Par contre, en utilisant cette approche, nous n'arrivons pas à identifier des cellules capables de prendre greffe spécifiquement quand elles sont injectées en intrafémorale. Finalement, bien qu'une confirmation in vivo soit encore nécessaire, nous suggérons sur la base de nos analyses cytométriques de flux, que les cellules SP Sca1t~és éie~~ CD48t~és bas sont très enrichies en CSH. Ceci permettrait l'isolation ex vivo de CSH de la moelle osseuse de souris par une stratégie à la fois nouvelle et simple. SUMMARY : Elucidating the molecular and cellular bases of stem cell function is crucial for the understanding of cellular organisation within tissues and organs as well as for the development of new therapeutic strategies in regenerative medicine and oncology. The best-known adult stem cells are those responsible for haematopoiesis, the haematopoietic stem cells (HSCs). In recent years, much effort has been put into the prospective isolation of mouse bone marrow (BM)-derived HSCs using cell-surface markers and alleged functional properties. Upon isolation, the functional capacity of putative HSCs has been classically assessed by intravenous transplantation into conditioned recipient mice and analysis of their ability to reconstitute the haematopoietic system at long-term. It has recently been suggested that transplanting the cells directly into the BM might not only result in more rapid and more effective engraftment, but even help to identify cells that have intrinsic HSC properties but lack the ability to home to their BM niche and have thus failed to succeed in classical reconstitution assays. In this study, we compare the function of different BM cell subsets, as defined by their cell surface phenotype, on two levels. Firstly, we assess their ability to reconstitute lethally irradiated mice, when injected either intravenously or intrafemorally. Secondly, using 8-colour flow cytometric analysis, we compare their relative side population (SP) activity by exclusion of the fluorescent dye Hoechst 33342. Our preliminary results indeed reinforce the idea that intrafemoral transplantation results in faster and more effective engraftment, however, using this approach, we are unable to identify cells that are capable of engrafting specifically when injected intrafemorally. Finally, although in vivo confirmation is still required, we propose, based on the results of our flow cytometric analyses, that SP Scat Very h'9h CD48Very'°W cells should be highly enriched for HSCs. This would allow for a simple new strategy for the isolation of mouse BM HSCs ex vivo.

Distinct phenotypes of antigen-selected CD8 T cells emerge at different stages of an in vivo immune response.

We have previously described a unique system for identifying Ag-selected CD8 T cells during an in vivo response in normal mice. In this system, lymphocytes isolated from DBA/2 mice injected i.p. with HLA-CW3 transfected syngeneic (H-2d) P815 cells show a remarkable expansion of CD8 cells that utilize TCR expressing the V beta 10 gene segment and additional structural features characteristic of Kd-restricted CW3-specific CTL clones. We have now taken advantage of this system to characterize the surface phenotype of CD8 cells selected by Ag in vivo. We observed several distinct phenotypes at different stages of the response. At the peak of the response, Ag-selected cells were low in CD62L and CD45RB expression but displayed high levels of CD44. In addition, there was a partial down-regulation of CD8 and TCR. Cells of this phenotype were present in lymphoid tissues for several mo after immunization. Much later in the response, Ag-selected cells expressed higher levels of CD8 and TCR. Moreover, a distinct subset of these long-term immune cells emerged that now expressed CD62L and CD45RB. Analysis of CD8 cells from different tissues also revealed certain differences, particularly in TCR and co-receptor levels from liver-derived cells compared with circulating cells at the peak of the response. Our findings suggest that the function of Ag-selected CD8 cells may be regulated over time and according to location by subtle changes in cell-surface phenotype.

Human monocytes but not dendritic cells are killed by blocking of autocrine cyclooxygenase activity

Dendritic cells (DCs), in peripheral tissues, derive mostly from blood precursors that differentiate into DCs under the influence of the local microenvironment. Monocytes constitute the main known DC precursors in blood and their infiltration into tissues is up-regulated during inflammation. During this process, the local production of mediators, like prostaglandins (PGs), influence significantly DC differentiation and function. In the present paper we show that treatment of blood adherent mononuclear cells with 10 mu M indomethacin, a dose achieved in human therapeutic settings, causes monocytes` progressive death but does not affect DCs viability or cell surface phenotype. This resistance of DCs was observed both for cells differentiated in vitro from blood monocytes and for a population with DCs characteristics already present in blood. This phenomenon could affect the local balance of antigen-presenting cells, influence the induction and pattern of immune responses developed under the treatment with non-steroidal anti-inflammatory drugs and, therefore, deserves further investigation. (C) 2009 Elsevier Inc. All rights reserved.

Stem cells in embryonic skin development

The skin is a complex stratified organ which acts not only as a permeability barrier and defense against external agents, but also has essential thermoregulatory, sensory and metabolic functions. Due to its high versatility and activity, the skin undergoes continuous self-renewal to repair damaged tissue and replace old cells. Consequently, the skin is a reservoir for adult stem cells of different embryonic origins. Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. In this review we attempt to clarify the emergence, structure, markers and embryonic development of diverse populations of stem cells from the epidermis, dermis and related appendages such as the sebaceous gland and hair follicle.