Carmela La Mendola

Efficient Killing of Human Colon Cancer Stem Cells by γδ T Lymphocytes

Colon cancer comprises a small population of cancer stem cells (CSC) that is responsible for tumor maintenance and resistant to cancer therapies, possibly allowing for tumor recapitulation once treatment stops. We previously demonstrated that such chemoresistance is mediated by autocrine production of IL-4 through the up-regulation of antiapoptotic proteins. Several innate and adaptive immune effector cells allow for the recognition and destruction of cancer precursors before they constitute the tumor mass. However, cellular immune-based therapies have not been experimented yet in the population of CSCs. Here, we show that the bisphosphonate zoledronate sensitizes colon CSCs to Vγ9Vδ2 T cell cytotoxicity. Proliferation and production of cytokines (TNF-α and IFN-γ) and cytotoxic and apoptotic molecules (TRAIL and granzymes) were also induced after exposure of Vγ9Vδ2 T cells to sensitized targets. Vγ9Vδ2 T cell cytotoxicity was mediated by the granule exocytosis pathway and was highly dependent on isoprenoid production by of tumor cells. Moreover, CSCs recognition and killing was mainly TCR mediated, whereas NKG2D played a role only when tumor targets expressed several NKG2D ligands. We conclude that intentional activation of Vγ9Vδ2 T cells by zoledronate may substantially increase antitumor activities and represent a novel strategy for colon cancer immunotherapy.

Differentiation, phenotype, and function of interleukin-17–producing human Vγ9Vδ2 T cells

In healthy adults, the major peripheral blood γδ T-cell subset expresses the Vγ9Vδ2 TCR and displays pleiotropic features. Here we report that coculture of naive Vγ9Vδ2 T cells with phosphoantigens and a cocktail of cytokines (IL-1-β, TGF-β, IL-6, and IL-23), leads to selective expression of the transcription factor RORγt and polarization toward IL-17 production. IL-17(+) Vγ9Vδ2 T cells express the chemokine receptor CCR6 and produce IL-17 but neither IL-22 nor IFN-γ; they have a predominant terminally differentiated (CD27(-)CD45RA(+)) phenotype and express granzyme B, TRAIL, FasL, and CD161. On antigen activation, IL-17(+) Vγ9Vδ2 T cells rapidly induce CXCL8-mediated migration and phagocytosis of neutrophils and IL-17-dependent production of β-defensin by epithelial cells, indicating that they may be involved in host immune responses against infectious microorganisms. Accordingly, an increased percentage of IL-17(+) Vγ9Vδ2 lymphocytes is detected in the peripheral blood and at the site of disease in children with bacterial meningitis, and this pattern was reversed after successful antibacterial therapy. Most notably, the phenotype of IL-17(+) Vγ9Vδ2 T cells in children with meningitis matches that of in vitro differentiated IL-17(+) Vγ9Vδ2 T cells. Our findings delineate a previously unknown subset of human IL-17(+) Vγ9Vδ2 T lymphocytes implicated in the pathophysiology of inflammatory responses during bacterial infections.

V gamma 9V delta 2 T lymphocytes efficiently recognize and kill zoledronate-sensitized, imatinib-sensitive, and imatinib-resistant chronic myelogenous leukemia cells.

Imatinib mesylate (imatinib), a competitive inhibitor of the BCR-ABL tyrosine kinase, is highly effective against chronic myelogenous leukemia (CML) cells. However, because 20–30% of patients affected by CML display either primary or secondary resistance to imatinib, intentional activation of Vγ9Vδ2 T cells by phosphoantigens or by agents that cause their accumulation within cells, such as zoledronate, may represent a promising strategy for the design of a novel and highly innovative immunotherapy capable to overcome imatinib resistance. In this study, we show that Vγ9Vδ2 T lymphocytes recognize, trogocytose, and efficiently kill imatinib-sensitive and -resistant CML cell lines pretreated with zoledronate. Vγ9Vδ2 T cell cytotoxicity was largely dependent on the granule exocytosis- and partly on TRAIL-mediated pathways, was TCR-mediated, and required isoprenoid biosynthesis by zoledronate-treated CML cells. Importantly, Vγ9Vδ2 T cells from patients with CML can be induced by zoledronate to develop antitumor activity against autologous and allogeneic zoledronate-treated leukemia cells, both in vitro and when transferred into immunodeficient mice in vivo. We conclude that intentional activation of Vγ9Vδ2 T cells by zoledronate may substantially increase their antileukemia activities and represent a novel strategy for CML immunotherapy.

CXCR5 Identifies a Subset of Vγ9Vδ2 T Cells which Secrete IL-4 and IL-10 and Help B Cells for Antibody Production

Vγ9Vδ2 T lymphocytes recognize nonpeptidic Ags and mount effector functions in cellular immune responses against microorganisms and tumors, but little is known about their role in Ab-mediated immune responses. We show here that expression of CXCR5 identifies a unique subset of Vγ9Vδ2 T cells which express the costimulatory molecules ICOS and CD40L, secrete IL-2, IL-4, and IL-10 and help B cells for Ab production. These properties portray CXCR5+Vγ9Vδ2 T cells as a distinct memory T cell subset with B cell helper function.

Phenotypical and functional analysis of memory and effector human CD8 T cells specific for mycobacterial antigens.

Mycobacterium tuberculosis infects one-third of the global population and claims two million lives every year. Because memory CD8 T cells exhibit a high heterogeneity in terms of phenotype and functional characteristic, we investigated the frequency, phenotype, and functional properties of Ag85A epitope-specific HLA-A*0201 CD8 T cells in children affected by tuberculosis (TB) before and 4 mo after chemotherapy and healthy contact children. Using Ag85A peptide/HLA-A*0201 pentamer, we found a low frequency of blood peptide-specific CD8 T cells in tuberculous children before therapy, which consistently increased after therapy to levels detected in healthy contacts. Ex vivo analysis of the expression of CD45RA and CCR7 surface markers indicated a skewed representation of Ag85A epitope-specific CD8 T cells during active TB, with a predominance of T central memory cells and a decrease of terminally differentiated T cells, which was reversed after therapy. Accordingly, pentamer-specific CD8 T cells from tuberculous patients produced low levels of IFN-γ and had low expression of perforin, which recovered after therapy. The finding of an elevated frequency of pentamer-specific CD8 T cells with T effector memory and terminally differentiated phenotypes in the cerebrospinal fluid of a child with tuberculous meningitis strongly indicates compartmentalization of such CD8 effectors at the site of disease. Our study represents the first characterization of Ag-specific memory and effector CD8 T cells during TB and may help to understand the type of immune response that vaccine candidates should stimulate to achieve protection.

Reciprocal stimulation of γδ T cells and dendritic cells during the anti-mycobacterial immune response

γδ T cells and dendritic cells (DC) are two distinct cell types of innate immunity that participate in early phases of immune response against Mycobacterium tuberculosis infection. Here we show that a close functional relationship exists between these cell populations. Using an in vitro coculture system, Vγ1 T cells from Tcrb–/– mice were found to be activated by DC infected in vitro with BCG, as indicated by the elevated CD69 expression, IFN‐γ secretion and cytotoxic activity. This activation process was due to a non‐cognate mechanism since it required neither cell to cell contact nor interaction between the TCR and a specific antigen, but was mediated by DC‐derived IL‐12. Reciprocally, Vγ1 T cells provided a key cytokine, IFN‐γ, which increased IL‐12 production by BCG‐infected DC. Moreover, exposure of BCG‐infected DC to Vγ1 T cells conditioned the former to prime a significantly stronger anti‐mycobacterial CD8 T cell response. Consequently, stimulation of γδ T cells and their non‐cognate interaction with DC could be applied as an immune adjuvant strategy to optimize vaccine‐induced CD8 T cell immunity.

Characterization of human γδ T lymphocytes infiltrating primary malignant melanomas.

T lymphocytes are often induced naturally in melanoma patients and infiltrate tumors. Given that γδ T cells mediate antigen-specific killing of tumor cells, we studied the representation and the in vitro cytokine production and cytotoxic activity of tumor infiltrating γδ T cells from 74 patients with primary melanoma. We found that γδ T cells represent the major lymphocyte population infiltrating melanoma, and both Vδ1+ and Vδ2+ cells are involved. The majority of melanoma-infiltrating γδ cells showed effector memory and terminally-differentiated phenotypes and, accordingly, polyclonal γδ T cell lines obtained from tumor-infiltrating immune cells produced IFN-γ and TNF-α and were capable of killing melanoma cell lines in vitro. The cytotoxic capability of Vδ2 cell lines was further improved by pre-treatment of tumor target cells with zoledronate. Moreover, higher rate of γδ T cells isolation and percentages of Vδ2 cells correlate with early stage of development of melanoma and absence of metastasis. Altogether, our results suggest that a natural immune response mediated by γδ T lymphocytes may contribute to the immunosurveillance of melanoma.

Aminobisphosphonate-activated gammadelta T cells in immunotherapy of cancer: doubts no more.

Background: Activated Vγ9Vδ2 T cells are able to kill most tumour cells because of recognition by T cell receptor and natural killer receptors. Objective: We discuss the possibility that the intentional activation of γδ T cells in vivo by aminobisphosphonates may represent a promising target for the design of novel and highly innovative immunotherapy in cancer patients. Methods: The antitumoural effects of γδ T cells both in vitro and in vivo have been demonstrated suggesting a new therapeutic approach for translation into the clinical setting. Results/conclusion: Vγ9Vδ2 T lymphocytes represent a particularly interesting target for immunotherapeutic protocols based on N-aminobisphosphonate administration and several Phase I-II trials are ongoing investigating the activity of zoledronic acid plus IL-2 in solid tumours.

Characterization of HLA-DR- and TCR-binding residues of an immunodominant and genetically permissive peptide of the 16-kDa protein of Mycobacterium tuberculosis

The16‐kDa protein of Mycobacterium tuberculosis represents an important antigenic target during bacillary latency and, consequently, should be considered as candidate subunit vaccine component. In this study, we have used CD4 T cell clones that recognize the peptide p91–110, an immunodominant and genetically permissive epitope, in the context of five different HLA‐DR molecules and truncated and substituted variants of this peptide, to identify the minimal binding sequence (HLA‐DR‐binding core) and the minimal stimulatory sequence (TCR‐binding core), as well as the residuesthat contact HLA‐DR molecules and the TCR. We have found a common 9‐mer sequence, spanning amino acids 93–101, as the binding core for HLA‐DR1, ‐DR11, ‐DR13 and ‐DR7, but a longer (13‐mer) sequencespanning amino acids 92–104 was required for binding to the HLA‐DR15 molecules. F93 was required for binding to all the tested HLA‐DR molecules, hence allowing us to identify it as the N‐terminal primary anchor residue (P1). Additionally, the binding requirements for other residues varied considerably between the tested alleles: A94 for HLA‐DR15, V99 for HLA‐DR1, ‐DR15, ‐DR11 and ‐DR7, R100 for HLA‐DR11 and ‐DR13, and L104 for HLA‐DR15. Concerning the residues of p91–110 peptide required for binding to the TCR, the pepscan analysis results would support the contention that P(–1) E92, P6 F98 would be important TCR contact sites because their substitutions led to full loss of T cell activation. Moreover, P8 R100 is found to be critical residue in binding to HLA‐DR11‐ and ‐DR13‐restricted T cell clones, without influencing binding to the relevant HLA‐DR molecule. Our results could be useful to design peptides with altered HLA anchor residues or TCR interaction sites to achieve remarkable increase in activity and to study their vaccine potential.

Reversible effect of MR and ELF magnetic fields (0.5 T and 0.5 mT) on human lymphocyte activation patterns

Purpose: The aim of this study was to investigate the effects of magnetic fields (MF) of different intensity generated by a magnetic resonance (MR) unit (0.5 Tesla) and a double cylindrical coil (0.5 mTesla) on human CD4+ T cell lines. Materials and methods: CD4+ T cells were exposed for two hours under isothermal conditions (37 ± 0.5°C) to the above mentioned MF; a control group was provided for each exposed sample. After exposure, the samples were analysed in the laboratory for the following endpoints: Release of cytokines, expression of surface markers, cell proliferation and levels of cytosolic free-calcium. Results: Exposure to MF for 2 h and subsequent in vitro stimulation in the presence of the appropriate mitogen, caused a decrease of interferon-γ production, a decrease of cell proliferation, a decrease of expression of CD25 and a decrease of cytosolic free calcium concentration in exposed CD4+ T cell lines. Data obtained, were statistically significant when evaluated after 24 h of in vitro culture, but were not significant, for both types of MF, when the experimental groups were analysed after prolonged in vitro culture. Conclusion: These results indicate that static magnetic fields (SMF) can give rise to transient biological effects on T lymphocytes and the present system is a sensitive model for understanding the effects of MF on the immune system.