These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

Class I and class II MHC-restricted T cells specific for proteins present in myelin have been shown to be involved in autoimmunity in the central nervous system (CNS). It is not yet known whether CD1d-restricted T cells reactive to myelin-derived lipids are present in the CNS and might be targeted to influence the course of autoimmune demyelination. Using specific glycolipid-CD1d tetramers and cloned T cells we have characterized a T cell population reactive to a myelin-derived glycolipid, sulfatide, presented by CD1d. This population is distinct from the invariant Vα14+ NK T cells, and a panel of Vα3/Vα8+ CD1d-restricted NK T cell hybridomas is unable to recognize sulfatide in the presence of CD1d+ antigen-presenting cells. Interestingly, during experimental autoimmune encephalomyelitis a model for human multiple sclerosis, sulfatide-reactive T cells but not invariant NK T cells are increased severalfold in CNS tissue. Moreover, treatment of mice with sulfatide prevents antigen-induced experimental autoimmune encephalomyelitis in wild-type but not in CD1d-deficient mice. Disease prevention correlates with the ability of sulfatide to suppress both interferon-γ and interleukin-4 production by pathogenic myelin oligodendrocyte glycoprotein-reactive T cells. Since recognition of sulfatide by CD1d-restricted T cells has now been shown both in mice and humans, study of murine myelin lipid-reactive T cells may form a basis for the development of intervention strategies in human autoimmune demyelinating diseases.

NKT cells are associated with immunological control of autoimmune disease and cancer and can recognize cell surface mCD1d without addition of exogenous antigens. Cellular antigens presented by mCD1d have not been identified, although NKT cells can recognize a synthetic glycolipid, alpha-GalCer. Here we show that after addition of a lipid extract from a tumor cell line, plate-bound mCD1d molecules stimulated an NKT cell hybridoma. This hybridoma also responded strongly to three purified phospholipids, but failed to recognize alpha-GalCer. Seven of sixteen other mCD1d restricted hybridomas also showed a response to certain purified phospholipids. These findings suggest NKT cells can recognize cellular antigens distinct from alpha-GalCer and identify phospholipids as potential self-antigens presented by mCD1d.

SummaryRather unexpectedly, major histocompatibility complex class II-deficient mice have a significant population of peripheral CD4 + T lymphocytes. We have investigated these cells at the population and clonal levels. CD4 + T lymphocytes from class II-deficient animals are thymically derived, appear early in ontogeny, exhibit the phenotype of resting memory cells, are potentially functional by several criteria, and have a diverse T cell receptor repertoire. They do not include substantially elevated numbers of NKI.1 § cells. Hybridomas derived after polyclonal stimulation of the CD4 + lymphocytes from class II-deficient animals include a subset with an unusual reactivity pattern, responding to splenocytes from many mouse strains including the strain of origin. Most members of this subset recognize the major histocompatibllity complex class Ib molecule CD1; their heterogenous reactivities and T cell receptor usage further suggest the involvement of peptides and/or highly variable posttranslational modifications.M ost CD4 + T cells depend on an interaction with the MHC class II molecules expressed on thymic stroma to complete differentiation. As a consequence, mice that lack the conventional class II molecules (II ~ mice) 1 have a drastic reduction in the number of mature CD4 + T cells in the thymus and peripheral lymphoid organs (1-3). Unexpectedly, though, a small population of CD4 + T cells was detected in the spleen and lymph nodes of these animals. This population did not appear to result from nonspecific "leakage" because the cells had atypical properties: they expressed markers reminiscent of memory cells and were preferentially localized in the B cell areas of lymphoid organs.The existence ofCD4 + T cells in II ~ mice contradicts previous dogma on positive selection, which states that CD8 + cells result from a TCtL/MHC class I molecule engagement in the thymus and react in the periphery to antigen presented by class I molecules, while CD4 + cells emerge after a TCR/class II interaction and have class II-restricted antigen reactivities (reviewed in reference 4). Since the CD4 + cells in the II ~ mice could not have been selected on 1Abbreviations used in this paper: BrdU, bromodeoxyuridine; ~2m ~ J32 microglobulin-deficient mice; Ii ~ invariant chain deficient; II ~ mice, mice that lack the conventional class II molecules; TAP, transporter associated with antigen processing. conventional class II molecules, several questions arose: When and where are these cells generated? Does their cell surface phenotype show other atypical features? Are they functional? If so, what ligands do they recognize? Here, we address these questions at the population and clonal levels. Besides determining some important general properties of the peripheral CD4 + T cell population in II ~ mice, we describe an interesting set of hybridomas derived from this population. Materials and MethodsMice. II ~ mice (1), originally on a mixed C57BL/6J 036) X 129 background, were mostly from the fifth to eighth generation backcross to B6; in...

We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. Protease- and protease inhibitor–specific microarray analyses (CLIP-CHIP) of laser-dissected leukocyte infiltrated and noninfiltrated pancreatic islets and confirmatory quantitative real time PCR and protein analyses identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. Interestingly, the peri-islet BM and underlying IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies.