Tumour cell generation of inducible regulatory T-cells in multiple myeloma is contact-dependent and antigen-presenting cell-independent.

Regulatory T-cells (T(Reg) cells) are increased in patients with multiple myeloma (MM). We investigated whether MM cells could generate and/or expand T(Reg) cells as a method of immuno-surveillance avoidance. In an in vitro model, CD4(+)CD25(-)FoxP3(-) T-cells co-cultured with malignant plasma cells (primary MM cells and cell lines) induced a significant generation of CD4(+)CD25(+)FoxP3(+) inducible T(Reg) cells (tT(Reg) cells; p<0.0001), in a contact-dependent manner. tT(Reg) cells were polyclonal, demonstrated a suppressive phenotype and phenotypically, demonstrated increased FoxP3 (p = 0.0001), increased GITR (p<0.0001), increased PD1 (p = 0.003) and decreased CD62L (p = 0.007) expression compared with naturally occurring T(Reg) cells. FACS-sorted tT(Reg) cells differentiated into FoxP(+)IL-17(+) and FoxP3(-)IL-17(+) CD4(+) cells upon TCR-mediated stimulation. Blocking experiments with anti-ICOS-L MoAb resulted in a significant inhibition of tT(Reg) cell generation whereas both IL-10 & TGFß blockade did not. MM tumour cells can directly generate functional T(Reg) cells in a contact-dependent manner, mediated by ICOS/ICOS-L. These features suggest that tumour generation of T(Reg) cells may contribute to evasion of immune surveillance by the host.

Reduced immune effector cell NKG2D expression and increased levels of soluble NKG2D ligands in multiple myeloma may not be causally linked.

BACKGROUND: There is limited understanding of the dysregulation of the innate immune system in multiple myeloma (MM). We analysed the expression of the activating receptor NKG2D on NK cells and T cells of MM patients and investigated the impact of soluble versus membrane-bound NKG2D ligands on the expression of NKG2D. DESIGN: NKG2D expression on NK cells and CD8+ alphabeta T cells from patients with MM or monoclonal gammopathy of uncertain significance and healthy controls was examined flow-cytometrically. Sera from patients and controls were analysed for soluble NKG2D ligands (sNKG2D ligands). RESULTS: Significantly fewer NK cells and CD8+ alphabeta T cells from patients expressed NKG2D compared to healthy controls (NK cells: median 54% interquartile range (IQR) 32-68 versus 71% IQR 44-82%, P = 0.017, CD8+ alphabeta T cells: median 63% IQR 52-81 versus 77% IQR 71-90%, P = 0.018). The sNKG2D ligand sMICA was increased in patients [median 175 (IQR 87-295) pg/ml] versus controls [median 80 (IQR 32-129) pg/ml, P < 0.001], but levels of sMICA did not correlate with NKG2D expression on effector cells. To elucidate the mechanism of NKG2D down-regulation, we incubated lymphocytes from healthy donors in the presence of sNKG2D ligands or in co-culture with MM cell lines. sNKG2D ligands in clinically relevant concentrations did not down-regulate NKG2D expression, but co-culture of effector cells with myeloma cells with high surface expression of NKG2D ligands reduced NKG2D expression significantly. CONCLUSIONS: These results indicate that MM is associated with a significant reduction in NKG2D expression which may be contact-mediated rather than caused by soluble NKG2D ligands.

CD4(+)CD25(+)FoxP3(+) regulatory T cells are increased whilst CD3(+)CD4(-)CD8(-)alphabetaTCR(+) Double Negative T cells are decreased in the peripheral blood of patients with multiple myeloma which correlates with disease burden.

Increased levels of naturally occurring regulatory T cells (T(Reg) cells) have been found in a variety of solid tumours and haematological malignancies. In multiple myeloma (MM), evidence suggests that T(Reg) cells are increased though controversy exists with regards to their function and no relationship to disease stage and treatment has been demonstrated. Here, we demonstrate significantly elevated levels of functional CD4(+)CD25(+)FoxP3(+) T(Reg) cells in a large cohort of patients with MM as well as monoclonal gammopathy of uncertain significance (MGUS) in comparison to age-matched, healthy controls. The frequency of Double Negative T(Reg) cells was also evaluated, demonstrating that these cells were reduced in patients with MM. Furthermore, a characteristic profile of immunomodulatory cytokines in the peripheral blood and bone marrow of patients with MM and MGUS was demonstrated, compared with healthy controls. This data adds further evidence to the understanding of the role of T(Reg) cell subsets in tumour immunology and the fundamentals of the host/tumour immune conflict.

T cell receptor-induced phosphoinositide-3-kinase p110delta activity is required for T cell localization to antigenic tissue in mice.

The establishment of T cell-mediated inflammation requires the migration of primed T lymphocytes from the blood stream and their retention in antigenic sites. While naive T lymphocyte recirculation in the lymph and blood is constitutively regulated and occurs in the absence of inflammation, the recruitment of primed T cells to nonlymphoid tissue and their retention at the site are enhanced by various inflammatory signals, including TCR engagement by antigen-displaying endothelium and resident antigen-presenting cells. In this study, we investigated whether signals downstream of TCR ligation mediated by the phosphoinositide-3-kinase (PI3K) subunit p110delta contributed to the regulation of these events. T lymphocytes from mice expressing catalytically inactive p110delta displayed normal constitutive trafficking and migratory responses to nonspecific stimuli. However, these cells lost susceptibility to TCR-induced migration and failed to localize efficiently to antigenic tissue. Importantly, we showed that antigen-induced T cell trafficking and subsequent inflammation was abrogated by selective pharmacological inhibition of PI3K p110delta activity. These observations suggest that pharmacological targeting of p110delta activity is a viable strategy for the therapy of T cell-mediated pathology.

Physiologic and aberrant regulation of memory T-cell trafficking by the costimulatory molecule CD28.

Productive T-cell immunity requires both the activation and the migration of specific T cells to the antigenic tissue. The costimulatory molecule CD28 plays an essential role in the initiation of T-cell-mediated immunity. We investigated the possibility that CD28 may also regulate migration of primed T cells to target tissue. In vitro, CD28-mediated signals enhanced T-cell transendothelial migration, integrin clustering, and integrin-mediated migration. In vivo, T cells bearing a mutation in the CD28 cytoplasmic domain, which abrogates PI3K activation, displayed normal clonal expansion but defective localization to antigenic sites following antigenic rechallenge. Importantly, antibody-mediated CD28 stimulation led to unregulated memory T-cell migration to extra-lymphoid tissue, which occurred independently of T-cell receptor (TCR)-derived signals and homing-receptor expression. Finally, we provide evidence that CD28- and CTLA-4-mediated signals exert opposite effects on T-cell trafficking in vivo. These findings highlight a novel physiologic function of CD28 that has crucial implications for the therapeutic manipulation of this and other costimulatory molecules.

Antigen presentation by the endothelium: a green light for antigen-specific T cell trafficking?

The functional consequences of recognition of antigen displayed by the endothelium during T cell extravasation in the development of an immune response have been a matter of debate for a long time. Most investigations have focused on the induction of proliferative responses and cytokine production by T cells. In parallel, endothelial cells have been shown to express costimulatory molecules with positive and negative regulatory effects on T cell responses. Recent studies have provided an alternative view of the antigen presenting cell function of endothelial cells, suggesting that cognate recognition of the endothelium by trafficking T cells is a key event in selecting the migration of antigen-specific lymphocytes into the site of inflammation.