Efficient ex vivo analysis of CD4+ T-cell responses using combinatorial HLA class II tetramer staining

Abstract

MHC tetramers are an essential tool for characterizing antigen-specific CD4+ T cells. However, their ex vivo analysis is limited by the large sample requirements. Here we demonstrate a combinatorial staining approach that allows simultaneous characterization of multiple specificities to address this challenge. As proof of principle, we analyse CD4+ T-cell responses to the seasonal influenza vaccine, establishing a frequency hierarchy and examining differences in memory and activation status, lineage commitment and cytokine expression. We also observe cross-reactivity between an established epitope and recent variant and provide a means for probing T-cell receptor cross-reactivity. Using cord blood samples, we correlate the adult frequency hierarchy with the naive precursor frequencies. Last, we use our combinatorial staining approach to demonstrate that rheumatoid arthritis patients on therapy can mount effective responses to influenza vaccination. Together, these results demonstrate the utility of combinatorial tetramer staining and suggest that this approach may have broad applicability in human health and disease.

CD4 T cells in protection from influenza virus: Viral antigen specificity and functional potential

“…If we extrapolate the results established in mouse models of primary infection to humans, who are each likely to express at least 8–10 different HLA-class II molecules due to co-expression of different alleles and isotypes (HLA-DR, DQ and DP), as well as heterozygosity in HLA genotype (57), we anticipate that the CD4 T cell response to the initial influenza infection would encompass more than 100 different epitope specificities. This has not been tested experimentally because of the limited sampling that can take place from the peripheral blood of young children, typically 2–10 ml (58), and the relatively low frequency of CD4 T cells that are specific for single peptides, even in adults who have presumably encountered influenza multiple times (59–64). …”

“…There is a growing appreciation that vaccine strategies that more fully engage the cellular response can enhance protection. A number of recent studies have sought to quantify and characterize the repertoire of circulating memory CD4 T cells with specificity towards influenza antigens (59, 60, 62, 63, 83–87). Many studies by other groups have concluded that the most prominent influenza-reactive CD4 T cells are those specific for internal virion proteins, particularly M1 and NP (84–86), as well as HA (63).…”

Autoreactive T cells in type 1 diabetes

“…The ability to detect and phenotype autoreactive T cells in circulation, where they are present at extremely low frequencies, has greatly improved. For example, HLA-II tetramers or HLA-I multimers/monomers allow measurement of autoreactive T cells in the circulation ex vivo, without in vitro amplification that might alter phenotypic features (8)(9)(10). This Review integrates experimental, human pathology, and clinical research studies and identifies key outstanding questions related to autoreactive T cells in T1D.…”

Recent developments and highlights in immune monitoring of allergen immunotherapy

“…113 Furthermore, allergen-specific Tregs have been shown to expand following AIT for HDM and grass pollen allergies thereby increasing the ratio of allergen-specific Tregs over effector T cells. 32,33,114,115 To facilitate translation towards routine measurements of T-cell biomarkers, immunophenotyping approaches have been developed that only use membrane markers and allow for direct ex vivo T-cell evaluation without in vitro cell culture. The CD4+CD25+CD127phenotype specifically distinguishes human Treg, 116,117 and these cells can be quantified in allergic patients 31 with increases following SLIT for grass pollen 11 and AIT in HDM-allergic individuals.…”

“…42,123 Many abnormalities in Treg and Th cell number and frequency have been observed within the total pool of circulating T cells, independent of antigen specificity. 30,31,42,121 With the use of MHC-peptide reagents to detect antigen specificity, 115 the sensitivity of these changes can be significantly enhanced to detect early effects of AIT. 32,113 As the application of MHC tetramers, especially for MHC class II, is limited by the diversity of alleles in the human population, there is a need for alternative markers for allergen-specific cells.…”