Phenotypic and functional characterisation of regulatory T cells in head and neck squamous cell carcinoma

Head and neck cancer is the sixth most common cancer worldwide. Despite advances in therapy, the five year survival rate remains poor. The presence of regulatory T cells (Tregs) may be a mechanism by which head and neck squamous cell carcinoma (HNSCC) evades the immune system. Using flow cytometry to identify distinct Treg populations on the basis of phenotype and a CFSE proliferation assay, the frequency and suppressive activity of Treg populations from the peripheral circulation and tumour microenvironment of newly-presenting HNSCC patients was assessed. No difference in the circulating Treg prevalence was observed between HNSCC patients (n=39) and healthy controls (n=14), or between patients with HNSCC from different subsites. However, patients with advanced stage tumours and those with nodal involvement had significantly elevated levels of peripheral CD4+CD25highCD127low/- Tregs compared with patients who had early stage tumours (p=0.03) and those without nodal involvement (p=0.03) respectively. Circulating CD4+CD25highCD127low/- Tregs from the entire HNSCC patient cohort and patients whose tumours had metastasised to the lymph nodes suppressed the proliferation of effector T cells significantly more compared with those from healthy controls (p=0.04) or patients with no nodal involvement (p=0.04). Additionally, peripheral CD4+CD25interCD127low/- Tregs consistently induced greater suppressive activity than CD4+CD25highCD127low/- Tregs. The tumour microenvironment had an elevated frequency of Tregs (p≤0.002) and a lower frequency of effector T cells (p≤0.03) compared with the patient's peripheral circulation (n=15). No difference was observed in the level of suppression between tumour and peripheral Tregs. Using immunohistochemistry, patients with oropharyngeal tumours showed a significantly greater infiltration of Foxp3+ cells in the tumour and stroma compared with laryngeal tumours. Furthermore, as determined by ELISA, the dispersed tumour specimens secreted detectable levels of TGF-β and IL-10, but secretions from HNSCC (dissociated tumour samples and cell lines) did not influence the suppressive activity of Tregs. Clarifying the role of CD127low/- Tregs in HNSCC and the influence the tumour may have on the regulatory population will provide the opportunity, through future work, to establish whether Tregs can be used as a prognostic determinant or manipulated by immunotherapy

Frequency over function : raised levels of CD127low/- regulatory T cells in the tumour microenvironment compared with the periphery of head and neck cancer patients

Objective: Regulatory T cells (Tregs) are known to infiltrate the tumour microenvironment of many cancers, including head and neck malignancies, and are thought to contribute to the host's impaired anti-tumour immune response. However, their immunosuppressive function remains poorly understood within the tumour microenvironment and this study aimed to address this. Methods: The frequency and suppressive capacity of two CD4?CD127low/- Treg populations, separated on the basis of different levels of CD25 expression (CD25inter and CD25high), from the tumour/node microenvironment and peripheral circulation of newly-presenting head and neck squamous cell carcinoma patients (n=19), were assessed using multicolour flow cytometry. Results: The proportion of Tregs (CD4?CD25high/?interCD127low/-) in the tumour/node microenvironment was significantly elevated compared with the peripheral circulation (p<0.001) and similar percentages were present in both the primary tumour and metastatic lymph node. The percentage of suppression induced by Tregs isolated from tumour associated nodes on the proliferation of nodal effector T cells was similar to that of peripheral Tregs on peripheral effector T cells. However, when the suppressive activity of both nodal and peripheral Tregs was compared on the same peripheral effectors, peripheral Tregs suppressed proliferation to a greater extent. Conclusion: This work shows that the recruitment and percentages of tumour infiltrating Tregs are key factors in modulating the immune environment of head and neck tumours

Laptop loans in UCD Library

This article describes the introduction of a new laptop loan service in University College Dublin’s Health Sciences Library.Not applicabl

Increased frequency and suppressive activity of CD127low/- regulatory T cells in the peripheral circulation of patients with head and neck squamous cell carcinoma are associated with advanced stage and nodal involvement

The presence of regulatory T (Treg) cells is thought to be an important mechanism by which head and neck squamous cell carcinoma (HNSCC) successfully evades the immune system. Using multicolour flow cytometry, the frequency and functional capacity of two CD4+CD127low/- Treg cell populations, separated on the basis of different levels of CD25 expression (CD25inter and CD25high), from the peripheral circulation of newly presenting HNSCC patients were assessed with regard to clinicopathological features and healthy controls. The frequency of circulating Treg cells was similar between HNSCC patients and healthy controls, and for patients with HNSCC developing from different subsites (laryngeal compared with oropharyngeal). However, patients with advanced stage tumours and those with nodal involvement had significantly elevated levels of CD4+CD25highCD127low/- Treg cells compared with patients who had early stage tumours (P=0·03) and those without nodal involvement (P=0·03), respectively. CD4+CD25highCD127low/- Treg cells from the entire HNSCC patient cohort and from patients whose tumours had metastasized to the lymph nodes were also shown to suppress the proliferation of effector T cells significantly more, compared with those from healthy controls (P=0·04) or patients with no nodal involvement (P=0·04). Additionally, CD4+CD25interCD127low/- Treg cells consistently induced greater suppressive activity than CD4+CD25highCD127low/- Treg cells on the proliferation of the effector T-cell populations (CD4+CD25-CD127-/+ and CD4+CD25+CD127+). Peripheral Treg cells, identified by the CD127low/- phenotype, have been shown to be influenced by a patient's tumour stage and/or nodal status in HNSCC; suggesting a role in tumour progression that could be manipulated by future immunotherapy

Ibrutinib Therapy Releases Leukemic Surface IgM from Antigen Drive in Chronic Lymphocytic Leukemia Patients

Purpose: In chronic lymphocytic leukemia (CLL), disease progression associates with surface IgM (sIgM) levels and signaling capacity. These are variably downmodulated in vivo and recover in vitro, suggesting a reversible influence of tissue-located antigen. Therapeutic targeting of sIgM function via ibrutinib, an inhibitor of Bruton tyrosine kinase (BTK), causes inhibition and tumor cell redistribution into the blood, with significant clinical benefit. Circulating CLL cells persist in an inhibited state, offering a tool to investigate the effects of drug on BTK-inhibited sIgM.Experimental Design: We investigated the consequences of ibrutinib therapy on levels and function of sIgM in circulating leukemic cells of patients with CLL.Results: At week 1, there was a significant increase of sIgM expression (64% increase from pretherapy) on CLL cells either recently released from tissue or persisting in blood. In contrast, surface IgD (sIgD) and a range of other receptors did not change. SIgM levels remained higher than pretherapy in the following 3 months despite gradual cell size reduction and ongoing autophagy and apoptotic activity. Conversely, IgD and other receptors did not increase and gradually declined. Recovered sIgM was fully N-glycosylated, another feature of escape from antigen, and expression did not increase further during culture in vitro The sIgM was fully capable of mediating phosphorylation of SYK, which lies upstream of BTK in the B-cell receptor pathway.Conclusions: This specific IgM increase in patients underpins the key role of tissue-based engagement with antigen in CLL, confirms the inhibitory action of ibrutinib, and reveals dynamic adaptability of CLL cells to precision monotherapy

Mutations in SARS-CoV-2 spike protein impair epitope-specific CD4⁺ T cell recognition

CD4+ T cells are essential for protection against viruses, including SARS-CoV-2. The sensitivity of CD4+ T cells to mutations in SARS-CoV-2 variants of concern (VOCs) is poorly understood. Here, we isolated 159 SARS-CoV-2-specific CD4+ T cell clones from healthcare workers previously infected with wild-type SARS-CoV-2 (D614G) and defined 21 epitopes in spike, membrane and nucleoprotein. Lack of CD4+ T cell cross-reactivity between SARS-CoV-2 and endemic beta-coronaviruses suggested these responses arose from naïve rather than pre-existing cross-reactive coronavirus-specific T cells. Of the 17 epitopes located in the spike protein, 10 were mutated in VOCs and CD4+ T cell clone recognition of 7 of them was impaired, including 3 of the 4 epitopes mutated in omicron. Our results indicated that broad targeting of epitopes by CD4+ T cells likely limits evasion by current VOCs. However, continued genomic surveillance is vital to identify new mutations able to evade CD4+ T cell immunity.</p

Surface IgM expression and function are associated with clinical behavior, genetic abnormalities, and DNA methylation in CLL

15Chronic lymphocytic leukemia (CLL) with unmutated (U-CLL) or mutated (M-CLL) immunoglobulin gene heavy-chain variable region (IGHV) displays different states of anergy, indicated by reduced surface immunoglobulin M (sIgM) levels and signaling, consequent to chronic (super)antigen exposure. The subsets also differ in the incidence of high-risk genetic aberrations and in DNA methylation profile, preserved from the maturational status of the original cell. We focused on sIgM expression and function, measured as intracellular Ca(2+) mobilization following stimulation, and probed correlations with clinical outcome. The relationship with genetic features and maturation status defined by DNA methylation of an 18-gene panel signature was then investigated. sIgM levels/signaling were higher and less variable in U-CLL than in M-CLL and correlated with disease progression between and within U-CLL and M-CLL. In U-CLL, increased levels/signaling associated with +12, del(17p) or NOTCH1 mutations. In M-CLL, there were fewer genetic lesions, although the methylation maturation status, generally higher than in U-CLL, varied and was increased in cases with lower sIgM levels/signaling. These features revealed heterogeneity in M-CLL and U-CLL with clear clinical correlations. Multivariate analyses with phenotype, genetic lesions, or DNA methylation maturation status identified high sIgM levels as a new potential independent factor for disease progression. Multiple influences on sIgM include the cell of origin, the clonal history of antigen encounter in vivo, and genetic damage. This simple marker compiles these different factors into an indicator worthy of further investigations for prediction of clinical behavior, particularly within the heterogeneous M-CLL subset.nonenoneD'Avola, Annalisa; Drennan, Samantha; Tracy, Ian; Henderson, Isla; Chiecchio, Laura; Larrayoz, Marta; Rose-Zerilli, Matthew; Strefford, Jonathan; Plass, Christoph; Johnson, Peter W; Steele, Andrew J; Packham, Graham; Stevenson, Freda K; Oakes, Christopher C; Forconi, FrancescoD'Avola, Annalisa; Drennan, Samantha; Tracy, Ian; Henderson, Isla; Chiecchio, Laura; Larrayoz, Marta; Rose Zerilli, Matthew; Strefford, Jonathan; Plass, Christoph; Johnson, Peter W; Steele, Andrew J; Packham, Graham; Stevenson, Freda K; Oakes, Christopher C; Forconi, Francesc

High surface IgM levels associate with shorter response to ibrutinib and BTK bypass in CLL patients

Chronic lymphocytic leukemia (CLL) cells have variably low surface IgM (sIgM) levels/signaling capacity, influenced by chronic antigen engagement at tissue sites. Within these low levels, CLL with relatively high sIgM (CLLhigh) progress more rapidly than CLL with low sIgM (CLLlow). During ibrutinib therapy, surviving CLL cells redistribute into the peripheral blood and can recover sIgM expression. Return of CLL cells to tissue may eventually recur, where cells with high sIgM could promote tumor growth. We analyzed time to new treatment (TTNT) following ibrutinib in 70 CLL patients (median follow-up of 66 months) and correlated it with pre-treatment sIgM levels and signaling characteristics. Pre-treatment sIgM levels correlated with signaling capacity, as measured by intracellular Ca2+ mobilization (iCa2+), in vitro (r=0.70; p&lt;0.0001). High sIgM levels/signaling strongly correlated with short TTNT (p&lt;0.05), and 36% CLLhigh versus 8% CLLlow progressed to require a new treatment. In vitro, capacity of ibrutinib to inhibit sIgM-mediated signaling inversely correlated with pre-therapy sIgM levels (r=-0.68, p=0.01) or iCa2+ (r=-0.71, p=0.009). In patients, sIgM-mediated iCa2+ and ERK phosphorylation levels were reduced by ibrutinib therapy, but not abolished. The residual signaling capacity downstream of BTK was associated with high expression of sIgM, while it was minimal when sIgM expression was low (p&lt;0.05). These results suggested that high sIgM levels facilitated CLL cell resistance to ibrutinib in patients. The CLL cells, surviving in the periphery with high sIgM expression, include a dangerous fraction, able to migrate to tissue and receive proliferative stimuli, which may require targeting by combined approaches