Published: January 7th, 2019
Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.
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.
T-follicular helper cells (Tfh) are a CD4+ T-cell subset that was initially characterized in lymphoid tissues1. These cells express PD-1 and CXCR5 surface receptors, secrete IL-21 and IL-4 and show nuclear expression of the transcription factor, BCL-62,3. As their name suggests, they are found in germinal centers and are essential for high affinity antibody production1.
Dysregulated Tfh responses have been implicated in disease pathogenesis, most notably autoimmune disease, where they promote the expansion of autoreactive B ....
Blood samples were obtained from normal subjects (NS) (n = 12) as well as patients with marginal zone lymphoma (MZL) (n = 7) and other types of B-cell non-Hodgkin's lymphoma (BNHL) (6 FL patients, 2 lymphoplasmacytic lymphoma patients and 1 low-grade B-cell non-Hodgkin's lymphoma not otherwise specified patient). Patients were recruited from the hematology clinics at Leicester Royal Infirmary after having given informed, written consent, with ethical approval in place for all studies. Ethical approval was obtaine.......
High CD4+ purity was achieved using the CD4+ isolation protocol, which was reliable across all blood samples tested by us (mean: 96.6%, SD: 2.38, n=31) (Figure 3).
Identification of cTfh (CD4+ CXCR5+ cells) in a representative normal subject is presented (Figure 4A). The proportion of total cTfh cells within C.......
This protocol represents a simple and efficient way to analyze peripheral blood cTfh cells, enabling the detection of all relevant subsets identified in the literature thus far. Blood samples can be easily and efficiently obtained as part of standard out-patient clinics and serial samples can be collected in parallel with clinical data. In turn, this enables prospective studies evaluating cTfh subsets as biomarkers for disease progression or response to treatment. These studies would be particularly warranted in disease .......
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