Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry.

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin's lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Circulating Tfh1 (cTfh1) cell numbers and PD1 expression are elevated in low-grade B-cell non-Hodgkin's lymphoma and cTfh gene expression is perturbed in marginal zone lymphoma.

CD4+ T-cell subsets are found in the tumour microenvironment (TME) of low-grade B-cell non-Hodgkin's lymphomas such as marginal zone lymphoma (MZL) or follicular lymphoma (FL). Both numbers and architecture of activating follicular helper T-cells (Tfh) and suppressive Treg in the TME of FL are associated with clinical outcomes. There has been almost no previous work on CD4+ T-cells in MZL. It is now recognised that circulating CD4+CXCR5+ T-cells are the memory compartment of Tfh cells. We determined differences in number of circulating Tfh (cTfh) cells and cTfh subsets between normal subjects and patients with FL or MZL. Lymphoma patients showed increased numbers of cTfh1 and reduced cTfh17 cells due to decreased expression of the subset-defining marker CCR6 in patients. PD1, a surface marker associated with Tfh cells, showed increased expression on cTfh subsets in patients. Focusing on MZL we determined expression of 96 T-cell associated genes by microfluidic qRT-PCR. Analysis of differentially expressed genes showed significant differences between normal subjects and patients both for bulk cTfh (CCL4) and the cTfh1 subset (JAK3). While our findings require confirmation in larger studies we suggest that analysis of number and gene expression of circulating T-cells might be a source of clinically useful information as is the case for T-cells within lymphoma lymph nodes.