Background Traditionally, the transcriptomic and proteomic characterisation of CD4+ T cells at the single-cell level has been performed by two largely exclusive types of technologies: single-cell RNA-sequencing (scRNA-seq) and antibody-based cytometry. Here we present a multi-omics approach allowing the simultaneous targeted quantification of mRNA and protein expression in single-cells and investigate its performance to dissect the heterogeneity of human immune cell populations. Methods We have quantified the single-cell expression of 397 genes at the mRNA level and up to 68 proteins using oligo-conjugated antibodies (AbSeq) in 43,656 primary CD4+ T cells isolated from blood and 31,907 CD45+ cells isolated from blood and matched duodenal biopsies. We explored the sensitivity of this targeted scRNA-seq approach to dissect the heterogeneity of human immune cell populations and identify trajectories of functional T-cell differentiation. Results We provide a high-resolution map of human primary CD4+ T cells, and identify precise trajectories of Th1, Th17 and regulatory T-cell (Treg) differentiation in blood and tissue. The sensitivity provided by this multi-omics approach identified the expression of the B7 molecules CD80 and CD86 on the surface of CD4+ Tregs, and we further demonstrated that B7 expression has the potential to identify recently activated T cells in circulation. Moreover, we identified a rare subset of CCR9+ T cells in blood with tissue-homing properties and expression of several immune checkpoint molecules, suggestive of a regulatory function. Conclusions This transcriptomic and proteomic hybrid technology provides a cost-effective solution to dissect the heterogeneity of immune cell populations, including more precise and detailed descriptions of the differentiation and activation of circulating and tissue-resident cells in response to therapies and in stratification of patients.