Phenotypically and functionally distinct subsets of natural killer cells in human PBMCs

Human natural killer (NK) cells are one major component of lymphocytes that mediate early protection against viruses and tumor cells, and play an important role in immune regulatory functions. In this study, we demonstrated that human NK cells could be divided into four subsets, CD56hi CD16(-), CD56lo CD16(-), CD56+CD16+ and CD56(-)CD16+, based on the expression of cell surface CD56 and CD16 molecules. Phenotypic analysis of NK cell subsets indicated that the expression of activation markers, adhesion molecules, memory cell markers, inhibitory and activating receptors, and intracellular proteins (granzyme B and perforin) were heterogeneous. Following interleukin (IL)-2 stimulation, interferon-gamma was preferentially produced by CD56+CD16(-) NK cells and this subset showed more proliferative capacity. The cytolytic activity of both CD56+CD16(-) and CD56+/-CD16+ subsets could be augmented in response to IL-2. The data provided a new definition for NK cell subsets demonstrating their phenotypic and functional diversity and possible stage of NK cell differentiation in peripheral blood.     

Characterization of lymphocyte subsets in patients with common variable immunodeficiency reveals subsets of naive human B cells marked by CD24 expression

Increased proportions of naive B cell subset and B cells defined as CD27(neg)CD21(neg)CD38(neg) are frequently found in patients with common variable immunodeficiency (CVID) syndrome. Current methods of polychromatic flow cytometry and PCR-based detection of κ deletion excision circles allow for fine definitions and replication history mapping of infrequent B cell subsets. We have analyzed B cells from 48 patients with CVID and 49 healthy controls to examine phenotype, frequency, and proliferation history of naive B cell subsets. Consistent with previous studies, we have described two groups of patients with normal (CVID-21norm) or increased (CVID-21lo) proportions of CD27(neg)CD21(neg)CD38(neg) B cells. Upon further analyses, we found two discrete subpopulations of this subset based on the expression of CD24. The B cell subsets showed a markedly increased proliferation in CVID-21lo patients as compared with healthy controls, suggesting developmental arrest rather than increased bone marrow output. Furthermore, when we analyzed CD21(pos) naive B cells, we found two different subpopulations based on IgM and CD24 expression. They correspond to follicular (FO) I and FO II cells previously described in mice. FO I subset is significantly underrepresented in CVID-21lo patients. A comparison of the replication history of naive B cell subsets in CVID patients and healthy controls implies refined naive B cell developmental scheme, in which human transitional B cells develop into FO II and FO I. We propose that the CD27(neg)CD21(neg)CD38(neg) B cells increased in some of the CVID patients originate from the two FO subsets after loss of CD21 expression.     

Attenuation of T-lymphocyte demargination and adhesion molecule expression in response to moderate exercise in physically fit individuals.

The effects of physical fitness on leukocyte demargination and cellular adhesion molecule (CAM) responses to moderate exercise were examined. We assessed leukocyte subsets and CAM expression before, immediately after, and 10 min after a 20-min treadmill exercise at 65-70% peak oxygen consumption in fit vs. nonfit individuals. Physical fitness was determined by peak oxygen consumption during a treadmill test. Catecholamine levels were determined by radioenzymatic assay, and enumeration of cells and detection of CAM expression were assessed by flow cytometry. As expected, exercise led to significant increases in numbers of leukocyte subsets, regardless of fitness level (P < 0.01). Values returned to near resting levels 10 min after exercise. More importantly, physically fit individuals showed attenuated responses to the moderate-exercise challenge in numbers of CD3(+), CD4(+), CD8(+), memory (CD45RO(+)) CD4, and naive (CD45RA(+)62L(+)) CD4 and CD8 lymphocytes. Postexercise human leukocyte antigen-DR absent memory CD4(+) cell numbers were also lower in fit subjects. Increases in CD62L-expressing CD4(+) and CD8(+) lymphocytes and CD11a- expressing lymphocytes after exercise were also attenuated in fit individuals compared with nonfit individuals (P < 0.05). Catecholamine levels increased to a similar extent (P < 0.01) in both fitness groups. The findings suggest that physical fitness attenuates demargination of selected lymphocyte subsets in response to moderate exercise. Although the differences in plasma catecholamine responses were not significant between the groups, a possible mediating role of the sympathetic system remains to be further investigated. Being physically fit may offset exaggerated immune cell responses to stress.     

Transient expression of human interleukin-2 and interferon-γ genes is regulated by interaction between distinct cell subsets

The level of transient expression of human IL-2 and IFN-γ genes, we show, is regulated by dynamic interaction between two functionally distinct cell populations. One is able to express these genes, while the other, bearing one of several specific surface markers, actively inhibits their expression. Defined cell subsets were isolated from PBMC and tonsil cells using immunomagnetic beads coated with monoclonal antibodies directed against surface markers. Depletion of CD8, CD11a (Leu15), or Leu8 subsets led to a pronounced superinduction of IL-2 and IFN-γ gene expression when the remaining cell population was stimulated with mitogen (PHA) or antigen (SEB). Thus, a 10-fold increase in production of IFN-γ was observed after removal of CD11a (Leu15) cells constituting only a small percentage of the total cell population. By contrast, depletion of cells expressing CD19, a B cell marker, did not yield any superinduction. Conversely, CD8, CD11a (Leu15), or Leu8 cell subsets, but not CD19 cells, each inhibited the induction of IL-2 and IFN-γ gene expression almost completely in depleted or total cell populations from which they were derived. Gene expression occurring within one cell subset could be effectively inhibited by cells from a second subset. Introduction of inhibitory cells (Leu8) into a population that actively expressed IL-2 and IFN-γ mRNA resulted in an immediate cessation of gene expression. This suppression involves a soluble mediator, since the culture medium in which such cells were activated exerted a similarly effective inhibition.     

The role of CD4+ cells in sustaining lymphocyte proliferation during lymphocytic choriomeningitis virus infection.

The murine immune response to lymphocytic choriomeningitis virus (LCMV) infection involves the activation of CD8+, class I MHC-restricted and virus-specific CTL. At times coinciding with CTL activation, high levels of IL-2 gene expression and production occur, the IL-2R is expressed, and T cell blastogenesis and proliferation are induced. We have previously found that, although both CD4+ and CD8+ T cell subsets transcribe IL-2, the CD4+ subset appears to be the major producer of IL-2 whereas the CD8+ subset appears to be the major proliferating population when the subsets are separated after activation in vivo. The studies presented here were undertaken to examine the contribution made by the CD4+ subset to lymphocyte proliferation in vivo. Responses to LCMV infection were examined in intact mice and in mice depleted of CD4+ or CD8+ subsets by antibody treatments in vivo. Protocols were such that in vivo treatments with anti-CD4 or anti-CD8 depleted the respective subset by greater than 90%. In situ hybridizations demonstrated that the IL-2 gene was expressed in non-B lymphocytes isolated from either CD4+ cell-depleted or CD8+ cell-depleted mice on day 7 post-infection with LCMV. When placed in culture, however, cells from CD8+ cell-depleted mice produced significantly higher levels of detectable IL-2 than did cells isolated from CD4+ cell-depleted mice on day 7 post-infection. IL-2 was apparently produced in vivo in mice depleted of either CD4+ or CD8+ cells, as expression of the gene for the p55 chain of the IL-2R, IL-2 responsiveness, and lymphocyte proliferation were observed with cells isolated from both sets of mice. Lymphocyte proliferation was shown to be sustained in mice depleted of CD4+ cells in vivo by three criteria: 1) non-B lymphocytes isolated from infected mice depleted of CD4+ cells underwent more DNA synthesis than did those isolated from uninfected mice or from infected mice depleted of CD8+ cells; 2) leukocyte yields were expanded during infection of CD4+ cell-depleted mice; and 3) CD8+ cell numbers were increased during infection of CD4+ cell-depleted mice. The majority of non-B lymphocytes having the characteristics of blast lymphocytes was recovered in the CD8+ populations isolated from infected CD4+ cell-depleted mice. These findings suggest that the requirement for the CD4+ subset to sustain CD8+ lymphocyte proliferation in vivo is limited, and that CD4+ and CD8+ cell types can function independently in many aspects of their responses to viral infections.     

The normalization of guinea pig leukocyte fractions and lymphocyte subsets in blood and lymphoid tissues using a flow cytometric procedure.

Many hematological and immunological parameters remain unclear in the study of the guinea pig. In this study, we established the mean values of blood counts, the percentage of leukocyte fractions and lymphocyte subsets in blood and various lymphoid tissues of the guinea pig with a flow cytometric procedure using MIL4/SSC. The mean counts of WBC and RBC in the blood were lower, and MCV and MCH were higher than those of other rodents, resembling those of humans. Furthermore, the mean percentages of blood lymphocytes were smaller and that of granulocyte was larger than those of other rodents, resembling those of humans. We further established a flow cytometric procedure for lymphocyte subsets and clarified the mean percentages of T- and B-cells, CD4(+)-, CD8(+)- and MHC Class II(+)- T-cells, and CD4(-)CD8 (-) T-cells. The latter were morphologically larger in cell size and cytoplasm than CD4(+)- plus CD8(+) T-cells, and this subset had a significantly higher percentage in newborn animals. Furthermore, the appearance of the MHC Class II(+) T-cell subset was suggested to be a marker of hyper-activation of T-cells in BCG-immunized animals. Thus, both the novel flow cytometric procedure for leukocyte fractions and lymphocyte subsets, and the established normal values will be useful tools in studying guinea pigs as models of various diseases and biological phenomena.     

Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-1) and three other molecules (UCHL1, CDw29, and Pgp-1) and have enhanced IFN-gamma production

Studies of cell-surface molecules involved in human T cell interaction reveal that differential expression of each of three adhesion molecules (LFA-3, CD2, and LFA-1) subdivides human peripheral blood T cells into major subpopulations. Systematic analysis of the relationship between expression of these and other markers of T cell subsets demonstrates a single major subset of human peripheral blood T lymphocytes distinguished by enhanced expression of LFA-3, CD2, LFA-1, and three other markers (CDw29 [4B4], UCHL1, and Pgp-1). Large differences in relative expression are observed for UCHL1 (29-fold) and LFA-3 (greater than 8-fold), and smaller differences (2- to 4-fold) are seen for CDw29, CD2, LFA-1, and Pgp-1. Bimodal distribution of LFA-3 is found on both CD4+ cells and on CD8+ cells as well as on B lymphocytes (CD19+). Neonatal T cells (CD3+) are comprised almost exclusively of the subset expressing low LFA-3, CD2, LFA-1, CDw29, and UCHL1. Activation of cord peripheral blood mononuclear leukocytes with PHA leads to uniform enhanced expression of each of these molecules on CD3+ cells. Functional analyses of these T cell subsets were performed after sorting of adult T cells based on differential LFA-3 expression. Only the LFA-3+ subset proliferated in response to the Ag tetanus toxoid, even though the LFA-3- subset proliferated more strongly to PHA. Furthermore, the LFA-3+ subset made greater than fivefold more IFN-gamma than the LFA-3- subset in response to PHA, despite the fact that both subsets made equivalent amounts of IL-2. This phenotypic and functional analysis of resting and activated newborn and adult T cells indicates that human memory T cells express enhanced levels of LFA-3, CD2, LFA-1, UCHL1, CDw29, and Pgp-1; we speculate that the increase in expression of T cell adhesion molecules LFA-3, CD2, and LFA-1 on memory cells is functionally important in their enhanced responsiveness.     

Memory functions and death proneness in three CD4+CD45RO+ human T cell subsets

We propose a classification of human CD4(+)CD45RO(+) memory T cells into three new subsets based on cell surface expression levels of CD43. The first subset consists of cells whose CD43 expression is relatively high; this subset also contains the highest proportion of recall Ag-reactive precursors, and its constituent cells respond far more strongly than cells in either of the other subsets to immobilized CD3 Ab in addition to secreting substantially more IFN-gamma and IL-4. Cells of the second subset express similar levels of CD43 to naive cells, and they also respond weakly to TCR-mediated stimuli as judged by either their ability to proliferate or capacity for cytokine production. The third subsets consists of cells whose CD43 expression levels are clearly down-regulated; its cells appear to be anergic to TCR-mediated stimuli, and when examined ex vivo many of them appear to be undergoing either spontaneous apoptosis via a caspase-independent pathway or Fas-mediated apoptosis via a caspase-dependent pathway, even in the resting state. An analysis of telomere lengths revealed that the typical telomere of a cell in the second subset was significantly longer than the typical telomere in the first or third subset. Taken together, these results appear to indicate that CD4(+)CD45RO(+) T cells fall into three functionally differing subsets, one being a subset of cells with fully matured memory phenotype, a second being a less mature subset of cells that retain longer telomeres and whose memory functionality is marginal, and a third consisting of anergic cells that give every appearance of being death-prone and/or in the process of dying.     

A circulating bovine gamma delta T cell subset, which is found in large numbers in the spleen, accumulates inefficiently in an artificial site of inflammation: correlation with lack of expression of E-selectin ligands and L-selectin

Tissue-specific localization of TCR-defined subsets of gamma delta T cells has been widely reported; however, the mechanisms responsible for this phenomenon are poorly understood. We describe a bovine gamma delta T cell TCR-associated subset that preferentially localizes in the spleen. This subset was characterized by coexpression of CD8, and was found to lack surface expression of E-selectin ligands, GR Ag ligands, as well as low expression of L-selectin. The CD8-positive gamma delta T cell subset did not accumulate at sites of inflammation as efficiently as CD8-negative gamma delta T cells that, in contrast, express E-selectin and GR ligands and high levels of L-selectin. This is the first demonstration of a gamma delta T cell subset, which exhibits a defined tissue tropism, having a unique adhesion molecule expression profile. These results demonstrate that in some cases tissue-specific accumulation of gamma delta T cell subsets can be predicted by expression, or lack of expression, of defined homing molecules.     

IL-12 controls cytotoxicity of a novel subset of self-antigen-specific human CD28+ cytolytic T cells

Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28(-)) T cells strongly expressing granzyme/perforin, and two EM28(+) subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28(-)-derived clones lysed target cells with high efficacy. In contrast, EM28(+)-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28(+) conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.     

Leukocyte Populations in Human Preterm and Term Breast Milk Identified by Multicolour Flow Cytometry

Background

Extremely preterm infants are highly susceptible to bacterial infections but breast milk provides some protection. It is unknown if leukocyte numbers and subsets in milk differ between term and preterm breast milk. This study serially characterised leukocyte populations in breast milk of mothers of preterm and term infants using multicolour flow cytometry methods for extended differential leukocyte counts in blood.

Methods

Sixty mothers of extremely preterm (<28 weeks gestational age), very preterm (28–31 wk), and moderately preterm (32–36 wk), as well as term (37–41 wk) infants were recruited. Colostrum (d2–5), transitional (d8–12) and mature milk (d26–30) samples were collected, cells isolated, and leukocyte subsets analysed using flow cytometry.

Results

The major CD45+ leukocyte populations circulating in blood were also detectable in breast milk but at different frequencies. Progression of lactation was associated with decreasing CD45+ leukocyte concentration, as well as increases in the relative frequencies of neutrophils and immature granulocytes, and decreases in the relative frequencies of eosinophils, myeloid and B cell precursors, and CD16- monocytes. No differences were observed between preterm and term breast milk in leukocyte concentration, though minor differences between preterm groups in some leukocyte frequencies were observed.

Conclusions

Flow cytometry is a useful tool to identify and quantify leukocyte subsets in breast milk. The stage of lactation is associated with major changes in milk leukocyte composition in this population. Fresh preterm breast milk is not deficient in leukocytes, but shorter gestation may be associated with minor differences in leukocyte subset frequencies in preterm compared to term breast milk.     

CD27 dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity

Lineage differentiation and the formation of heterogeneous mature subsets are crucial for immune cells to maintain a breadth of responsiveness to pathogens while controlling reactivity to self. In this study, we report that CD27 is a key marker of the NK cell lineage, dissecting the mature Mac-1high NK cell pool into two functionally distinct subsets. The CD27low NK cell subset possesses a higher threshold to stimulation and appears to be tightly regulated by the expression of NK cell inhibitory receptors. Comparatively, the CD27high NK cell subset displays a greater effector function, exhibits a distinct tissue distribution and responsiveness to chemokines, and interacts productively with dendritic cells. Importantly, we have verified that CD27high and CD27low subsets with distinct cell surface phenotypes also exist in human peripheral blood. These findings clearly reclassify mature NK cells into two distinct subsets and begin to discern their specific role in immune responses.     

Identification of novel dendritic cell subset markers in human blood

Human dendritic cells (DC) are key regulators of innate and adaptive immunity that can be divided in at least three major subpopulations: plasmacytoid DC (pDC), myeloid type 1 DC (mDC1) and myeloid type 2 DC (mDC2) exhibiting different functions. However, research, diagnostic and cell therapeutic studies on human DC subsets are limited because only few DC subset markers have been identified so far. Especially mDC2 representing the rarest blood DC subset are difficult to be separated from mDC1 and pDC due to a paucity of mDC2 markers. We have combined multiparameter flow cytometry analysis of human blood DC subsets with systematic expression analysis of 332 surface antigens in magnetic bead-enriched blood DC samples. The initial analysis revealed eight novel putative DC subset markers CD26, CD85a, CD109, CD172a, CD200, CD200R, CD275 and CD301 that were subsequently tested in bulk peripheral blood mononuclear cell (PBMC) samples from healthy blood donors. Secondary analysis of PBMC samples confirmed three novel DC subset markers CD26 (dipeptidyl peptidase IV), CD85a (Leukocyte immunoglobulin-like receptor B3) and CD275 (inducible costimulator ligand). CD85a is specifically expressed in mDC1 and CD26 and CD275 represent novel mDC2 markers. These markers will facilitate human DC subset discrimination and additionally provide insight into potentially novel DC subset-specific functions.     

Single-cell Immune Landscape of Human Recurrent Miscarriage

Successful pregnancy in placental mammals substantially depends on the establishment of maternal immune tolerance to the semi-allogenic fetus. Disorders in this process are tightly associated with adverse pregnancy outcomes including recurrent miscarriage(RM). However, an indepth understanding of the systematic and decidual immune environment in RM remains largely lacking. In this study, we utilized single-cell RNA-sequencing(sc RNA-seq) to comparably analyze the cellular and molecular signatures of decidual and peripheral leukocytes in normal and unexplained RM pregnancies at the early stage of gestation. Integrative analysis identifies 22 distinct cell clusters in total, and a dramatic difference in leukocyte subsets and molecular properties in RM cases is revealed. Specifically, the cytotoxic properties of CD8+effector T cells, nature killer(NK), and mucosal-associated invariant T(MAIT) cells in peripheral blood indicates apparently enhanced pro-inflammatory status, and the population proportions and ligand–receptor interactions of the decidual leukocyte subsets demonstrate preferential immune activation in RM patients.The molecular features, spatial distribution, and the developmental trajectories of five decidual NK(d NK) subsets have been elaborately illustrated. In RM patients, a d NK subset that supports embryonic growth is diminished in proportion, while the ratio of another d NK subset with cyto-toxic and immune-active signature is significantly increased. Notably, a unique pro-inflammatory CD56+CD16+d NK subset substantially accumulates in RM decidua. These findings reveal a comprehensive cellular and molecular atlas of decidual and peripheral leukocytes in human early pregnancy and provide an in-depth insight into the immune pathogenesis for early pregnancy loss.     

CD127 and CD25 expression defines CD4+ T cell subsets that are differentially depleted during HIV infection

Decreased CD4(+) T cell counts are the best marker of disease progression during HIV infection. However, CD4(+) T cells are heterogeneous in phenotype and function, and it is unknown how preferential depletion of specific CD4(+) T cell subsets influences disease severity. CD4(+) T cells can be classified into three subsets by the expression of receptors for two T cell-tropic cytokines, IL-2 (CD25) and IL-7 (CD127). The CD127(+)CD25(low/-) subset includes IL-2-producing naive and central memory T cells; the CD127(-)CD25(-) subset includes mainly effector T cells expressing perforin and IFN-gamma; and the CD127(low)CD25(high) subset includes FoxP3-expressing regulatory T cells. Herein we investigated how the proportions of these T cell subsets are changed during HIV infection. When compared with healthy controls, HIV-infected patients show a relative increase in CD4(+)CD127(-)CD25(-) T cells that is related to an absolute decline of CD4(+)CD127(+)CD25(low/-) T cells. Interestingly, this expansion of CD4(+)CD127(-) T cells was not observed in naturally SIV-infected sooty mangabeys. The relative expansion of CD4(+)CD127(-)CD25(-) T cells correlated directly with the levels of total CD4(+) T cell depletion and immune activation. CD4(+)CD127(-)CD25(-) T cells were not selectively resistant to HIV infection as levels of cell-associated virus were similar in all non-naive CD4(+) T cell subsets. These data indicate that, during HIV infection, specific changes in the fraction of CD4(+) T cells expressing CD25 and/or CD127 are associated with disease progression. Further studies will determine whether monitoring the three subsets of CD4(+) T cells defined based on the expression of CD25 and CD127 should be used in the clinical management of HIV-infected individuals.