Specific features of endothelial cells in primary tumors

RESUMO: As células endoteliais definem e delineiam todo o sistema vascular...Nesta tese procurámos explorar o papel que o ambiente tumoral exerce sobre as células endoteliais. ... Avaliamos também a capacidade anti-angiogénica de alguns derivados do estrogénio... Em suma os nossos resultados mostram a importância de um controlo rigoroso da regulação transcricional...

A study of the endocervical columnar cells II - effects of physiological changes and of oathological lesions of the uterus on the specific numerical composition of the endocervical epithelium

In the second part of this paper we nalysed the correlation between the clinical pathological alterations and the sum of the types of columnar cells of 300 histological sections of cervix. Fifty histological sections of normal cervix of sexually mature women were selected and considered as normal in pattern. The specific counts of the columnar cells which line the endocervical mucosa and those of the glands of 50 normal cervices were compared with other similar counts made in 50 histological sections of cervices of old women and emphasized the differences. Comparisons were made also between 50 normal cervices and 50 sections of cervices with chronic inflammation, 50 cervices with epidermoid metaplasia and 50 cervices with myoma of the corpus. Counts were made from 50 cervices of patients who on the occasion of the surgical operation were in the proliferative phase of the menstrual cycle; these were compared with the counts of 50 cervices of uteri in the luteal phase. Finally, the numerical frequency of the following data encountered in the 300 cervices was recorded: 1. aspects of the ectocervical epithelium; 2. number of Nabothian cysts; 3. number of cervical glands; 5. number of deliveries and 6. aspect of the material within the cervical canal.

T Cells Bearing a Chimeric Antigen Receptor against Prostate-Specific Membrane Antigen Mediate Vascular Disruption and Result in Tumor Regression.

Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of protumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here, we describe the ability of chimeric antigen receptor (CAR)-bearing T cells to recognize human prostate-specific membrane antigen (hPSMA) on endothelial targets in vitro as well as in vivo. CAR T cells were generated using the anti-PSMA scFv, J591, and the intracellular signaling domains: CD3ζ, CD28, and/or CD137/4-1BB. We found that all anti-hPSMA CAR T cells recognized and eliminated PSMA(+) endothelial targets in vitro, regardless of the signaling domain. T cells bearing the third-generation anti-hPSMA CAR, P28BBζ, were able to recognize and kill primary human endothelial cells isolated from gynecologic cancers. In addition, the P28BBζ CAR T cells mediated regression of hPSMA-expressing vascular neoplasms in mice. Finally, in murine models of ovarian cancers populated by murine vessels expressing hPSMA, the P28BBζ CAR T cells were able to ablate PSMA(+) vessels, cause secondary depletion of tumor cells, and reduce tumor burden. Taken together, these results provide a strong rationale for the use of CAR T cells as agents of tumor vascular disruption, specifically those targeting PSMA. Cancer Immunol Res; 3(1); 68-84. ©2014 AACR.

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression.

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.

Phenotypic heterogeneity of antigen-specific CD4 cells under different conditions of antigen persistence and antigen load

RESUME Nous n'avons pas de connaissance précise des facteurs à l'origine de l'hétérogénéité phénotypique des cellules T CD4 mémoires. Une troisième population phénotypique des cellules T CD4 mémoires, caractérisée par les marqueurs CD45RA+CCR7- a été identifiée dans cette étude. Cette population présente un état de différentiation avancée, comme en témoigne son histoire de réplication, ainsi que sa capacité de prolifération homéostatique. Les réponses des cellules T CD4 mémoires à différentes conditions de persistance et charge antigénique ont trois patterns phénotypiques différents, caractérisés par les marqueurs CD45RA et CCR7. La réponse CD4 mono -phénotypique CD45RA-CCR7+ ou CD45RA- CCR7- est associée à des conditions d'élimination de l'antigène (telle la réponse CD4 tétanos spécifique) ou à des conditions de persistance antigénique et de virémie élevée (telle la réponse HIV chronique ou la primo-infection CMV) respectivement. D'autre part, les réponses T CD4 multi -phénotypiques CD45RA-CCR7+ sont associées à des conditions d'exposition antigénique prolongée et de faible virémie (telles les infections CMV, EBV et HSV ou les infections HIV chez les long term non progressons). La réponse mono -phénotypique CD45RA- CCR7+ est propre aux cellules T CD4 secrétant de IL2, définies également comme centrales mémoires, la réponse CD45RA- CCR7- aux cellules T CD4 secrétant de l'IFNγ et finalement la réponse mufti-phénotypique aux cellules T CD4 secrétant à la fois de l'IL2 et de l' IFNγ. En conclusion, ces résultats témoignent d'une régulation de l'hétérogénéité phénotypique par l'exposition et la charge antigénique. ABSTRACT The factors responsible for the phenotypic heterogeneity of memory CD4 T cells are unclear. In the present study, we have identified a third population of memory CD4 T cells characterized as CD45RA+CCRT that, based on its replication history and the homeostatic proliferative capacity, was at an advanced stage of differentiation. Three different phenotypic patterns of memory CD4 T cell responses were delineated under different conditions of antigen (Ag) persistence and load using CD45RA and CCR7 as markers of memory T cells. Mono-phenotypic CD45RA'CCR7+ or CD45RA'CCR7' CD4 T cell responses were associated with conditions of Ag clearance (tetanus toxoid-specific CD4 T cell response) or Ag persistence and high load (chronic HIV-1 and primary CMV infections), respectively. Multi-phenotypic CD45RA CCR7+, CD45RA'CCRT and CD45RA+CCRT CD4 T cell responses were associated with protracted Ag exposure and low load (chronic CMV, EBV and HSV infections and HIV-1 infection in long-term nonprogressors). The mono-phenotypic CD45RA'CCR7+ response was typical of central memory (TCM) IL-2-secreting CD4 T cells, the mono-phenotypic CD45RA CCRT response of effector memory (TEM) IFN-γ -secreting CD4 T cells and the multi-phenotypic response of both IL-2- and IFN-γ -secreting cells. The present results indicate that the heterogeneity of different Ag-specific CD4 T cell responses is regulated by Ag exposure and Ag load.

Effect of vitamin D on Epstein-Barr (EBV)-specific CD8+T cells in patients with early multiple sclerosis (MS)

Background: Infection with EBV and a lack in vitamin D may be important environmental triggers of MS. 1,25-(OH)2D3 mediates a shift of antigen presenting cells (APC) and CD4+ T cells to a less inflammatory profile. Although CD8+ T cells do express the vitamin D receptor, a direct effect of 1,25(OH)2D3 on these cells has not been demonstrated until now. Since CD8+ T cells are important immune mediators of the inflammatory response in MS, we examined whether vitamin D directly affects the CD8+ T cell response, and more specifically if it modulates the EBV-specific CD8+ T cell response. Material and Methods: To explore whether the vitamin D status may influence the pattern of the EBV-specific CD8+ T cell response, PBMC of 10 patients with early MS and 10 healthy controls (HC) were stimulated with a pool of immunodominant 8-10 mer peptide epitopes known to elicit CD8+ T cell responses. PBMC were stimulated with this EBV CD8 peptide pool, medium (negative control) or anti- CD3/anti-CD28 beads (positive control). The following assays were performed: ELISPOT to assess the secretion of IFN-gamma by T cells in general; cytometric beads array (CBA) and ELISA to determine whichcytokines were released by EBV-specific CD8+ T cells after six days of culture; and intracellular cytokine staining assay to determine by which subtype of T cells secreted given cytokines. To examine whether vitamin D could directly modulate CD8+ T cell immune responses, we depleted CD4+ T cells using negative selection. Results: We found that pre-treatment of vitamin D had an antiinflammatory action on both EBV-specific CD8+ T cells and on CD3/ CD28-stimulated T cells: secretion of pro-inflammatory cytokines (IFNgamma and TNF-alpha) was decreased, whereas secretion of antiinflammatory cytokines (IL-5 and TGF-beta) was increased. At baseline, CD8+ T cells of early MS patients showed a higher secretion of TNFalpha and lower secretion of IL-5. Addition of vitamin D did not restore the same levels of both cytokines as compared to HC. Vitamin D-pretreated CD8+T cells exhibited a decreased secretion of IFN-gamma and TNF-alpha, even after depletion of CD4+ T cells from culture. Conclusion: Vitamin D has a direct anti-inflammatory effect on CD8+ T cells independently from CD4+ T cells. CD8+ T cells of patients with earlyMS are less responsive to the inflammatory effect of vitamin D than HC, pointing toward an intrinsic dysregulation of CD8+ T cells. The modulation of EBV-specific CD8+T cells by vitaminDsuggests that there may be interplay between these twomajor environmental factors of MS. This study was supported by a grant from the Swiss National Foundation (PP00P3-124893), and by an unrestricted research grant from Bayer to RDP.

Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response.

Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4(+) T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-kappaB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.

Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein.

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.

PSMA-Specific CAR-Engineered T Cells Eradicate Disseminated Prostate Cancer in Preclinical Models.

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting.

Rapid in vitro detection of HIV-1-specific antibody secretion by cells-culture with virus antigens

The present report describes an alternative method for in vitro detection of HIV-1 -specific antibody secretion in 24h of culture employing as stimulant of peripheral blood mononuclear cells the disrupted inactivated whole virus adsorbed onto microwells in a commercial ELISA kit plates. The results obtained from this technique have showed high sensitivity and specificity since it was capable of detecting HIV-1 infection early after birth. There were neither false-positivity nor false-negativity when blood samples obtained from HIV-1 seronegative asymptomatic individuals, and HIV-1 seropositive adult patients were analized. This rapid, low cost, simple, highly sensitive and specific assay can be extremely useful for early diagnosis of pediatric HIV infection.

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes.

To demonstrate that antibody-guided targeting of antigenic MHC class I-peptide tetramer on tumor cells can render them susceptible to lysis by relevant cytotoxic T lymphocytes (CTL), biotinylated HLA-A*0201/Flu matrix peptide complexes were tetramerized on streptavidin molecules previously coupled to Fab' fragments from monoclonal antibodies (mAb) specific for cell surface markers such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. Flow cytometry analysis showed that coating of the HLA-A2-peptide complexes on the four HLA-A2-negative human cancer lines tested (including a CEA-positive colon carcinoma, an ErbB-2(+) breast carcinoma and two CD20(+) B lymphomas) was entirely dependent upon the specificity of the conjugated antibody fragments. More importantly, HLA-A2-restricted Flu matrix peptide-specific CTL were then found to lyse specifically and efficiently the MHC-coated target cells. These results open the way to the development of new immunotherapy strategies based on antibody targeting of MHC class I-peptide complexes.