Follicular helper T (Tfh) cells constitute a specialized CD4+ T-cell subset that localizes in close proximity to B cells and are essential in the production of high-affinity, class-switched antibodies, and their dysregulations are also involved in autoimmune diseases. Modulating gene expression patterns in primary T cells is an important approach to understanding Tfh cell differentiation and function. In this chapter, we describe a protocol to evaluate Tfh cell differentiation with OT-II TCR transgenic T cells by retrovirally transducing gene of interest. This protocol adopts the recombinant retrovirus-based transduction of primary CD4+ T cells, and it also includes procedures for adoptive transfer, immunization, and flow cytometry analysis.

Our work consists of a pilot study to characterize circulating Th/c1, Th/c2, Th/c17, Treg and Tfh-like populations and IL-17 serum levels of relapsing-remitting (RR) MS patients treated with IFN-β, compared with healthy controls. In remission RRMS patients, we observe increased Th/c17 cells frequency committed to a Th1 profile and increased soluble IL-17 levels. Moreover, a shift toward Th/c2 with reduction of Tc1 cells and decrease in effector/terminal differentiated compartment of Th1 cells were also observed. Despite RRMS patients being an inactive disease phase, IL-17 and Th/c17 cells seemed to contribute to perpetuating chronic inflammation, besides the altered ratio Th1/Th2.

A 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide (LPP) immunotherapy for rhinoconjunctivitis with or without asthma over 4 physician visits is safe, well tolerated, and effective.
We sought to investigate immunologic mechanisms of LPP immunotherapy in a subset of patients who participated in a phase III, multicenter, randomized, double-blind, placebo-controlled trial (clinical.govNCT02560948).
Participants were randomized to receive LPP (n = 21) or placebo (n = 11) for 3 weeks over 4 visits. Grass pollen-induced basophil, T-cell, and B-cell responses were evaluated before treatment (visit [V] 2), at the end of treatment (V6), and after the pollen season (V8).
Combined symptom and rescue medication scores (CSMS) were lower during the peak pollen season (-35.1%, P = .03) and throughout the pollen season (-53.7%, P = .03) in the LPP-treated group compared with those in the placebo-treated group. Proportions of CD63+ and CD203cbrightCRTH2+ basophils were decreased following LPP treatment at V6 (10 ng/mL, P < .0001) and V8 (10 ng/mL, P < .001) compared to V2. No change in the placebo-treated group was observed. Blunting of seasonal increases in levels of grass pollen-specific IgE was observed in LPP-treated but not placebo-treated group. LPP immunotherapy, but not placebo, was associated with a reduction in proportions of IL-4+ TH2 (V6, P = .02), IL-4+ (V6, P = .003; V8, P = .004), and IL-21+ (V6, P = .003; V8, P = .002) follicular helper T cells. Induction of FoxP3+, follicular regulatory T, and IL-10+ regulatory B cells were observed at V6 (all P < .05) and V8 (all P < .05) in LPP-treated group. Induction of regulatory B cells was associated with allergen-neutralizing IgG4-blocking antibodies.
For the first time, we demonstrate that the immunologic mechanisms of LPP immunotherapy are underscored by immune modulation in the T- and B-cell compartments, which is necessary for its effect.

Rheumatoid arthritis (RA) is a chronic and systematic autoimmune inflammatory disease. Recently, a novel T cell subset, follicular helper CD4 T cell (Tfh cells) was found in relation to the pathogenesis and progression of RA, and increased numbers of circulating Tfh cells were found in RA patients. However, there is little evidence regarding the localization of Tfh cells in synovium tissues from RA patients, owing to the lack of an available method to characterize their localization in tissue. The aim of our present study was to characterize the Tfh cells in rheumatoid synovium tissues from RA patients by using immunohistochemistry and triple-fluorescence immunostaining methods. Our results showed that specific staining of CD4, CXCR5 and ICOS could be found on infiltrating immune cells in rheumatoid synovium tissues. The use of triple-fluorescence immunostaining and confocal laser scanning showed immunolocalization of CD4(+)CXCR5(+)ICOS(+)T cells (Tfh cells) in the rheumatoid synovium tissues, whereas these signals were absent in osteoarthritis (OA) synovium and in normal synovium tissues. Thus the data from our present preliminary study support the notion that CD4(+)CXCR5(+)ICOS(+)Tfh cells could be found in rheumatoid synovium tissues from RA patients, indicating the possibility that this T cell subset in synovium tissues may have important roles in the pathogenesis and progression of RA.