This work is focused on human T and B cell immunology in solid and hematopoietic malignancies. Human immunology has been suffering from insufficiencies to experimentally resolve the virtually unlimited richness of phenotypes and functional states within humans. To at least partially overcome technical limitations, this work presents methodologies for high-dimensional high-throughput determination of lymphocyte differentiation and specificities at the single cell level. The methodologies were applied to rectal cancer and multiple myeloma, which are prime examples for the prognostic impact of tumor-infiltrating T cells or in which the immune system (B cell compartment in multiple myeloma) is part of the malignancy. The included publications present the identification of characteristic-phenotype rectal cancer-infiltrating T cells and, with help of individually created TCR-recombinant reporter cell lines, define the spatial distribution of T cell target antigens within the colo-rectum. In multiple myeloma, the developed methodologies lead to the identification of a novel-phenotype non-plasma cell B lineage subset that is polyclonally expanded in active disease. Furthermore, the phenotypic range of malignant B lineage clones in multiple myeloma was not restricted to plasma cells but included rare normal-phenotype (memory) B cells. This work is novel and of high impact for the (human) immunology field as it i) provides the technical basis to study human lymphocyte biology at the single cell level in high-dimensional space, which is relevant beyond the presented studies on regulatory T cells, rectal cancer, and multiple myeloma. ii) determines accessibility of rectal cancer-infiltrating T cells in peripheral blood. iii) determines the spatial distribution of target antigens of rectal cancer-infiltrating T cells. iv) defines the phenotypic range of multiple myeloma at the single cell level providing a potential rationale for the therapeutic effect of CD19-targeting therapies in a presumably CD19- disease. v) determines the default differentiation pathway of the CD45RA- regulatory T cell subset during in vitro expansion, which is relevant for the design and adoptive transfer of functionally defined cell products.