Tumour Cell Generation of Inducible Regulatory T-Cells in Multiple Myeloma Is Contact-Dependent and Antigen-Presenting Cell-Independent

Regulatory T-cells (TReg cells) are increased in patients with multiple myeloma (MM). We investigated whether MM cells could generate and/or expand TReg 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 TReg cells (tTReg cells; p<0.0001), in a contact-dependent manner. tTReg 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 TReg cells. FACS-sorted tTReg 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 tTReg cell generation whereas both IL-10 & TGFβ blockade did not. MM tumour cells can directly generate functional TReg cells in a contact-dependent manner, mediated by ICOS/ICOS-L. These features suggest that tumour generation of TReg cells may contribute to evasion of immune surveillance by the host

Subcoracoid acromioclavicular joint dislocation (Rockwood type VI) sustained in motorcycle crash: A case report

We present the case of a young male who sustained a rare acromioclavicular joint (ACJ) injury during a road traffic accident. A left-sided ACJ injury was identified on plain radiographs fourteen days following a motorcycling accident during which significant distracting injuries were sustained. Owing to persistent shoulder pain during awake tertiary surveillance, repeat shoulder plain radiographs were obtained, along with re-examination of the patient's index computed tomography (CT) shoulder imaging, indicating a grade VI left-sided acromioclavicular subluxation.The patient underwent operative management of the above injury at three weeks, with initial examination under anesthetic revealing a stiff shoulder joint significantly limited external rotation requiring extensive release of fibrosis. The left ACJ was reduced under anesthesia, being temporarily secured with Kirschner wire insertion. A Synthes locking distal tibial “L” plate was contoured and applied across the AC joint, and secured with locking screws. Intensive post-operative physiotherapy resulted in an significantly improved post-operative range of motion in the patient's left shoulder.Acromioclavicular joint injuries, commonly shortened to ACJ injuries, are most regularly traumatic in etiology, ranging in severity from mild sprain to complete joint disruption.ACJ injuries are classified according to the position of the clavicle with respect to the acromion and coracoid. The above case highlights the requirement for comprehensive tertiary surveillance of trauma patients both pre and postextubation, in order to identify such injuries that may require prompt surgical management in order to restore range of motion and function in affected joints

T cell receptor–induced phosphoinositide-3-kinase p110δ 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 p110δ contributed to the regulation of these events. T lymphocytes from mice expressing catalytically inactive p110δ 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 p110δ activity. These observations suggest that pharmacological targeting of p110δ activity is a viable strategy for the therapy of T cell–mediated pathology

Functional characteristics of tumour-induced regulatory T-cells.

<p>A. Suppression of anti-CD3/anti-CD28-induced autologous T-cell proliferation by tumour-generated and naturally occurring T_Reg cells (n = 3), as determined by tritiated thymine incorporation. Results expressed as counts per minute (cpm)± SEM representing assays performed in triplicate. <b>Key:</b> Unstim – resting CD4⁺CD25^- T cells, Stim – CD3/CD28 stimulated CD4⁺CD25^- T-cells, 4∶1 etc – ratio of stimulated autologous T-cells to T_Reg cells. B. The generation of IL-10 in co-cultures of CD25^-CD4⁺ sorted T-cells and HMCL, compared with HMCl alone and culture medium (n = 6, p = 0.0004). Results represent all experiments, expressed as mean±SEM and analyzed using student t-test. C. IL-10 production by tT_Reg cells after 7 days of co-cultures of CD25^-CD4⁺ sorted T-cells and HMCL. Results represent all experiments, expressed as mean±SEM (n = 3) and analyzed using student t-test. D. IL-10 production by tT_Reg cells after 7 days of co-cultures of CD25^-CD4⁺ sorted T-cells and HMCL. Results represent all experiments, expressed as mean±SEM (n = 3) and analyzed using student t-test. E. Representative flow cytometry plots demonstrating the generation of IFNγ⁺<i>FoxP3</i>⁺CD25⁺CD4⁺ T-cells from CD4⁺CD25^- T-cells in a 7 day co-culture assay with mitomycin C-treated U266B cells. F. The proportion of IFNγ-producing <i>FoxP3</i>⁺CD25⁺CD4⁺ T-cells detectable in the peripheral blood of age-matched controls (n = 15), patients with MM (n = 15) and tTReg cells generated <i>in vitro</i> after 7 days of co-cultures of CD25^-CD4⁺ sorted T-cells and HMCL (n = 3). Histograms represent IFNγ production by cells gated on <i>FoxP3</i>/CD25/CD4 positive staing. Results expressed as mean±SEM.</p

Natural and tumour-induced regulatory T-cell plasticity.

<p>A. The generation of IL-17 in co-culture supernatants of CD25^-CD4⁺ sorted T-cells with HMCL, compared with HMCl alone and culture medium (n = 3). Results represent all experiments, expressed as mean±SEM. B. Representative dot-plots of IL-17 producing cells generated from re-stimulation of sorted tumour-generated and naturally occurring T_Reg cells after 5 days of re-stimulation. C. The proportion of IL-17 producing cells generated from re-stimulation of sorted tumour-generated and naturally occurring T_Reg cells, expressing <i>FoxP3</i> after 5 days of re-stimulation, expressed as a percentage of CD4⁺ T-cells. Results expressed as mean±SEM.</p

<i>In vitro</i> mechanisms of tumour regulatory T-cell induction.

<p>A. The generation of <i>FoxP3</i>⁺CD25⁺CD4⁺ tT_Reg cells from CD4⁺CD25^- T-cells, expressed as a percentage of CD4⁺T-cells, in a co-culture assay with mitomycin C-treated U266 cells with and without transwell inserts (n = 7). Results represent all experiments, represented as mean±SEM and analysed using a student t-Test. B. Surface expression of HLA-DR, ICOSL (CD275) and TGFβ by human myeloma cell lines. C. Inhibition of tT_Reg cells generation from CD4⁺CD25^- T-cells by co-culture with HMCL (n = 3) through blockade of TGFβ and IL-10 using monoclonal antibodies and Latency-associated Peptide (LAP). Results represent all experiments, expressed as mean (±SEM). D. Surface expression of ICOS by T_Reg cells, presented as both percentage expression of CD4⁺CD25⁺<i>FoxP3</i>⁺ cells and representative dot plots. Results represent all experiments, expressed as mean ±SEM (n = 3) and analysed using a student t-test. E. Inhibition of tT_Reg cells generation from CD4⁺CD25^- T-cells by co-culture with HMCL through blockade of anti-ICOS-L (αICOS 1, 10, 100 µM) monoclonal antibody (n = 6), expressed as percentage of CD4⁺ T-cells and percent inhibition of tT_Reg cell generation. Results represent all experiments, illustrated as median with maximum and minimum values and analysed using a student t-test.</p

Regulatory T-cell induction by Myeloma tumour cells.

<p>A. The expansion of <i>FoxP3</i>⁺CD25⁺CD4⁺ natural T_Reg cells, expressed as a percentage of CD4⁺T-cells. T_Reg cells were enumerated in the PB of healthy donors, after 7 days in CM and 7 days co-cultured with mitomycin C-treated U266B cells (n = 4). Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test. B. Expansion of nT_Reg cells when co-culture with HMCL results from cell division, illustrated by a representative flow cytometry plot of CFSE dilution. C. The generation of <i>FoxP3</i>⁺CD25⁺CD4⁺ T-cells, expressed as a percentage of CD4⁺T-cells, in a co-culture assay with mitomycin C-treated U266B cells (n = 6) with varying starting populations: PB MNC, PBL CD25 depleted and CD4⁺CD25^- T-cells. Results demonstrate that increased generation of tumour-induced regulatory T-cells (tT_Reg cells) is seen with increasing purity of the seeded population. Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test. D. Representative flow cytometry plots demonstrating the generation of <i>FoxP3</i>⁺CD25⁺CD4⁺ T-cells from CD4⁺CD25^- T-cells through cell division of de novo generated <i>FoxP3</i>⁺ T-cells in a 7 day co-culture assay with mitomycin C-treated U266B cells. E. The generation of <i>FoxP3</i>⁺CD25⁺CD4⁺ T-cells, expressed as a percentage of CD4⁺T-cells, in a co-culture assay of CD4⁺CD25⁻ T-cells (n = 10) with mitomycin C-treated MM cell lines (U266B, JJN3, JIM3 & KMS11), an erythro-leukaemia cell line (K562) and non-heamatopoietic cell lines (Mel888 & HeLa). Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test (**p<0.001, *p<0.01). F. The generation of <i>FoxP3</i>⁺CD25⁺CD4⁺ T-cells, expressed as a percentage of CD4⁺T-cells, in a co-culture assay with fresh BM-derived myeloma plasma cells from patient samples (n = 7). Results demonstrate that increased generation of tumour-induced regulatory T-cells (tT_Reg cells) is seen with primary myeloma cells. Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test.</p