Cool recent studies utilizing human PBMC

CD4_t_cellThe availability of human peripheral blood mononuclear cells (PBMC) from healthy individuals and from patients with various diseases allows for many studies on normal and abnormal functions of human immune cells.  Because human and murine immune biology differs in many ways, it is important that various methodologies for studying human immunology are established.  The two reports highlighted below demonstrate the usage of human PBMCs for mechanistic and pre-clinical human immune cell studies.

A recent report in The Journal of Immunology by Edwards et. al, demonstrated the usage of healthy human PBMC to elucidate the mechanisms involved in modulation of TH2 T cell responses by Toll-Like Receptor-7 (TLR7) agonists.  Because TLR7 stimulation perturbs TH2 responses, the potential use of rapidly metabolized 8-oxoadenine TLR7 antedrugs for treatment of allergic diseases was explored in this study in collaboration between AstraZeneca and Dainippon Sumitomo.  The TLR7 agonistic antedrug AZ12441970 was found to inhibit TH2 responses via at least two different mechanisms: TLR7-induced production of type I interferons (IFN) and induction of Notch-ligand expression on TLR7-responsive antigen presenting cells.  These led to inhibition of IL-5 production by T cells via the IFN and Notch signaling pathways, respectively.  TLR7-induction of IFNα was found to be intact in PBMCs from asthmatic patients when compared with healthy volunteers, and thus the authors proposed that this therapeutic strategy may be effective in allergic disease patients.

This study provides a demonstration of the usage of human PBMCs for elucidating signaling and immune-cell crosstalk mechanisms as well as determining the potential for the effectiveness of candidate drugs in patients with different disease states.

TLR7 Stimulation of APCs Results in Inhibition of IL-5 through Type I IFN and Notch Signaling Pathways in Human Peripheral Blood Mononuclear Cells. Edwards S, Jones C, Leishman AJ, Young BW, Matsui H, Tomizawa H, Murray CM, Biffen M. J Immunol. 2013 Mar 15;190(6):2585-92. doi: 10.4049/jimmunol.1200780.

The role of the cytokine IL-17 in cancer immunity continues to be controversial.  IL-17 and the IL-17-producing TH17 T cell subsets have been shown to have both pro-tumor as well as anti-tumor immune modulating functions in different cancer contexts.  Monocytes isolated from human PBMC can be differentiated into various myeloid cell types including dendritic cells (DC), providing a tool for studies on these human immune cell types.  In a recent Plos One article, Olsson Åkefeldt et. al, explore the role of IL-17 in survival of human monocyte-derived DCs in vitro, and the relevance of this during chemotherapy.

IL-17 was found to significantly prolong DC survival in vitro, however the cells took on expression of macrophage markers (CD14/CD68) and exhibited a pre-M2 macrophage phenotype.  The prolonged survival was associated with upregulated expression of pro-survival gene BCL-2A1.  Interestingly, IL-17 plus IFNγ treatment in vitro rendered these M2 macrophage-like DCs resistant to cell death induced by 11 of 17 tested chemotherapeutic agents.  Thus, to determine if IL-17 treatment would benefit patients by allowing DC survival during therapy, future studies should address whether this chemoresistance of IL-17 treated DCs occurs in patients undergoing chemotherapy, and to determine how IL-17 affects the anti- versus pro-tumor function of these DCs in various types of cancer.

Chemoresistance of Human Monocyte-Derived Dendritic Cells Is Regulated by IL-17A. Olsson Åkefeldt S, Maisse C, Belot A, Mazzorana M, Salvatore G, Bissay N, Jurdic P, Aricò M, Rabourdin-Combe C, Henter JI, Delprat C. PLoS One. 2013;8(2):e56865. doi: 10.1371/journal.pone.0056865. Epub 2013 Feb 18.

Studies like these are examples of the utility of using human PBMCs to elucidate mechanisms of human immune cell biology under normal and diseased conditions.

Natural IgMs: A possible therapeutic role in autoimmunity?

Antibodies play a significant role in countering foreign threats and ensuring specificity of self/non-self recognition. However, in healthy individuals, a substantial proportion of circulating IgM antibodies, secreted constitutively by B1 cells has been observed to demonstrate self-reactivity in the absence of antigen activation. These antibodies, which are present from birth without external stimulation, are known as natural auto-antibodies (NAA). Interestingly, such antibodies were found in human cord blood as well as in mice raised in germ-free conditions. One important feature of these molecules is their broad spectrum of reactivity to antigens ranging from proteins, polysaccharides and nucleotides to phospholipids. Though there are numerous reports that describe the crucial functions of NAAs in protection against infection, here I would like to discuss more about the role of these polymeric IgMs in maintaining tissue homeostasis and autoimmunity, with a brief look at the future of using this class of antibodies for therapeutic purposes.                                        

Though the immune system has strict mechanisms of ensuring removal of self reactive B cells to prevent autoimmune diseases, growing evidence suggests that auto IgMs produced by B1 cells play a major role in clearance of apoptotic cells (AC) and maintaining immune homeostasis. The innate immune system recognizes markers on cells destined to undergo apoptosis and clears them by a process called efferocytosis. Any defect in this clearance mechanism could lead to release and accumulation of autoantigenic proteins from these dying cells, resulting in the development of autoimmune diseases in predisposed individuals. A large number of the NAAs bind to oxidation associated markers like phosphorylcholine (PC) and malondialdehyde (MDA) that become exposed on apoptotic cells, but are conformationally inaccessible in the healthy ones. Recognition of such cells by auto IgMs promotes their complement mediated clearance (Fig. 1, Silverman 2012).

 

describe the imageFigure 1: A simplified overview of the clearance of apoptotic cells by natural IgMs via recruitment of complement factors (Silverman 2012)

Recent reports have demonstrated that mice engineered to be deficient in IgMs are more susceptible to autoimmune diseases resulting from the defective clearance of ACs. In other murine studies, it has been observed that IgMs against PC and MDA may lead to direct suppression of pro-inflammatory responses. In certain murine models of arthritis, infusion of anti PC natural IgMs conferred protection against inflammation. Also, in healthy humans a major portion of the natural IgMs is found to be generated against oxidation-associated epitopes on apoptotic cells. Indeed, recent studies in patients with systemic lupus erythromatosus substantiate the fact that higher levels of natural IgMs are associated with protection against autoimmunity. Moreover, several recent reports indicate that low levels of natural IgMs are associated with higher chances of atherosclerotic plaques, increased occurrence of strokes and even Alzheimer’s disease

With growing evidence of the beneficial role of natural IgMs in autoimmunity and other diseases, efforts are on to develop effective therapeutic strategies based on the properties of these unique antibodies. In recent reports, different strategies such as injection of anti-idiotypic antibodies, administration of IL-18 and immunization with PC containing antigens have been described to increase natural IgM response. Moreover, pooled human IgM from healthy donors have been used with favorable effects in experimental autoimmune disease models. In fact, a preparation of IgG containing pooled 12% IgM is currently used for treatment of sepsis. In the future, further understanding of the role of these natural antibodies and their progenitor B cells could lead the way for the development of effective IgM based therapies against autoimmunity. 

 






 

arijit bhowmickArijit Bhowmick is currently a postdoctoral researcher at the Immunology institute of the Mount Sinai Medical Center, NY. He received his PhD in structural immunology from the National Institute of Immunology, New Delhi. His current research interests encompass autoimmunity, Th17 cells and structure based inhibitor designing. 







 

 

NRAS MUTATION IN MELANOMA

Melanoma, the most dangerous type of skin cancer, is the leading cause of death from skin disease (refer to my previous post titled “Targeting B-RAF in melanoma”). Melanoma results from a neoplastic transformation of pigment-producing melanocytes, with cutaneous neoplasms of stratified epithelium comprising the majority. Traditionally melanoma has been difficult to treat and exhibited resistance to all available standard therapies. However, recent progresses in the treatment of melanoma, including the FDA approved B-RAF inhibitor vemurafenib, have shown great promise in shrinking tumor size and improving the survival of patients. Studies have shown that melanoma is a complex disease that arises through multiple etiologic pathways. Therefore, detailed understanding of the underlying molecular mechanisms associated with melanoma pathogenesis and drug resistance is warranted in achieving a sustained clinical response. Over the past decade, molecular characterization of melanoma has progressed with the identification of the influence of various important oncogenes. These include oncogenic activation of BRAF, KIT, NRAS, cyclin D, and cyclin-dependent kinase 4 and alterations in the ERBB4 gene. Among these, the most frequently occurring genetic alteration associated with melanoma progression is the activating somatic mutation of B-RAF serine/thereonine kinase (for details please refer to my post titled “Targeting B-RAF in melanoma”). In addition to B-RAF mutations, which occurs in 50% of cases of melanoma, mutations of NRAS or neuroblastoma RAS gene were also identified in 15 to 20% of melanomas.

NRAS mutation resized 600

Approximately one-third of all human malignancies have mutations in RAS oncogene. RAS is a small sized plasma membrane-associated GTP binding protein. The RAS family of proteins consists of KRAS, HRAS, and NRAS. These proteins primarily regulate growth and, as a molecular switch, they connect signals from cell surface receptors to transcription factors and cell cycle regulating proteins in the nucleus. RAS proteins exist either in GTP-bound state (active) or GDP-bound (inactive) state. In normal cells, following binding of a ligand to its cognate receptor tyrosine kinase (RTK) RAS becomes activated. Once activated, RAS recruits and stimulates a number of signaling pathways including mitogen-activated protein kinas (MAPK) pathway and the phosphoinositide 3-kinase/AKT (PI3K/AKT) pathway.

Even though mutation of KRAS gene is the most common type of RAS mutation in human malignant disease, in melanoma, a point mutation of NRAS is most frequent and was the first oncogene to be identified. The most common mutation in NRAS is observed in codon 61 which occurs as a replacement of glutamine residue by lysine or arginine. This leads to constitutive activation of the MAPK signal transduction pathway resulting in proliferation and promotion of tumor growth. In addition, the RAS oncogene also activates signaling via the Rho GTPase Rac1, which can mediate growth, survival, and motility signaling.

Several studies analyzed the role of NRAS mutation in melanoma. In a study of 100 primary and metastatic melanoma samples by Ball et al. (1994), 36% of melanomas were found to contain mutations in RAS that, in 69% of cases, were at the codon 61. It was observed in multiple studies that patients with NRAS-mutated tumors are older at diagnosis than are patients with BRAF mutations (median age 55·7 years for NRAS vs 49·8 years for BRAF) and more frequently have melanoma due to chronic sun damage. Two large (>240 samples) studies of melanomas with NRAS mutations indicate that these tumors appear to exhibit more aggressive behavior, being associated with shorter overall survival. These tumors have also exhibited higher rates of mitosis and are thicker at presentation. A meta-analysis of studies from 1989 to 2010 reported that NRAS mutations were associated with nodular histology and location on the extremities. Collectively all these studies suggest a prominent role of NRAS as an oncogene in melanoma, and recommend scope of therapeutic targeting of NRAS for the treatment of advanced and high-risk melanoma.

With the increasing knowledge about the roles of various oncogenes (especially BRAF and NRAS), substantial advances in the targeted therapy to treat melanoma was achieved, however, only in BRAF-mutated melanomas. Compared to patients with BRAF mutations, as of today no approved targeted therapies exist for patients with NRAS-mutated melanoma. Complete inhibition of NRAS oncogenic signaling has proven to be challenging in part due to existence of redundant feedbacks to activate NRAS-MEK-ERK (MAPK) pathway. Various alternative strategies have thus been proposed, including (a) targeting membrane localization of RAS protein, required for RAS activity, through inhibitors of farnesyl transferase or galectin 1; (b) targeting NRAS mRNA with interfering RNAs; and (c) targeting signaling downstream of NRAS protein through inhibitors of PI3K/Akt. In addition, in in vitro studies some NRAS-mutated cell lines exhibited sensitivity to MEK inhibition. In conclusion, even though all these approaches hold promise, none of them got translated into the clinic. Therefore, more studies are required to formulate strategies to effectively treat NRAS-mutated melanoma.

 

References:

1.Devitt B, Liu W, Salemi R, Wolfe R, Kelly J, Tzen CY, Dobrovic A, McArthur G: Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma. Pigment Cell Melanoma Res 2011, 24:666-672.

2. Ball NJ, Yohn JJ, Morelli JG, Norris DA, Golitz LE, Hoeffler JP: Ras mutations in human melanoma: a marker of malignant progression. J Invest Dermatol 1994, 102:285-290.

Artifacts and non-specific staining in flow cytometry, Part II

flow cytometryIn Part I, I talked about un-specific binding and Fc-receptor binding. Besides these cases of non-specific binding, there are also other cases of antibody/fluorochrome binding that appears non-specific but that actually represents a real specific interaction – even though it is usually one that is not welcomed. I call these ‘pseudo-artifacts’ and you will read about some really odd stuff here.

 

(1) Binding of fluorochromes to Fc-receptors

The fact that Fc-receptors (FcR) bind antibodies is obvious, but lesser known is the fact that some of the fluorochrome linked to your antibody can also bind some FcRs.

It has been reported that R-phycoerythrin (PE) can bind to mouse Fc-gamma-RII  (CD16) and Fc-gamma-RIII (CD32) (Takizawa et al.). Furthermore, FcR binding of fluorochromes apparently applies to most or maybe even all cyanine fluorochromes, either alone or in tandem conjugates (Shapiro). So far I found reports for Cy5 (Jahrsdorfer et al.), PE-Cy5 (van Vugt et al.; Steward and Steward; Jahrsdorfer et al.) and APC-Cy7 (Beavis et al.). In this case, human CD64 (Fc-gamma-RI) was suggested to be the culprit of some of the binding (van Vugt et al.; Jahrsdorfer et al.), but binding also to CD64neg leukemia cells has been reported (Steward and Steward), so the role of FcR is not solved for all cases yet.

Þ Potential solution:

(a)  PE: For the binding of PE to mouse CD16/32 the use of a ‘Fc-block’, i.e. adding blocking monoclonal antibody 2.4G2 (rat IgG2b kappa), will avoid the problem (Takizawa et al.).

(b)  Cyanine: If you work with FcR+ cells, especially monocytes, you might consider avoiding cyanine-containing fluorochromes for the staining of your cells of interest.

 

(2) Binding of fluorochromes to antigen-receptors

Phycoerythrin (PE) and allophycocyanin (APC) are large proteins of 240kD and 110kD respectively that were original derived from cyanobacteria or red algae. As it turns out these phycobiliproteins are also a specific antigen for some T and B cells. Approximately 0.1% of all mouse B cells recognize PE as antigen in a BCR-dependent manner (Pape et al.; Wu et al.). Similar, about 0.02% of all mouse B cells are APC antigen-specific (Pape et al.). Furthermore, about 0.02-0.4% of all gamma-delta-T cells (mouse and human) recognized PE as a specific antigen (Zeng et al.) as well.

Þ Potential solution: Given their low frequency, these cells only pose a problem if you study tiny subsets of B and gamma-delta-T cells. In that case, you should avoid the use of PE for your cells of interest.

(3) Binding of fluorochromes to other receptors or interaction partners

(a) Cross-reactivity of the antibody:  Epitopes might be shared between different proteins, i.e. your antibody might not only recognize your protein in question, but recognizes also a similar epitope of another protein. This is for obvious reasons more likely with polyclonal antibodies.

Þ Potential solution: Usage of monoclonal antibodies reduces the risk of such cross-reactivity. If you suspect a cross-reactivity of your antibody, using a different clone for the same epitope will likely solve this problem.

(b) Intracellular biotin: Biotin is an important component of the cell metabolism. Therefore, biotin is present in the cells and the use of a streptavidin for intracellular staining will lead to binding of the streptavidin also to the cellular biotin.

Þ Potential solution: If you need to use a biotin-conjugated antibody for your intracellular staining you could cover all intracellular biotin by incubation of your cells with unconjugated streptavidin (followed by thorough washing) before the addition of your biotin-conjugated antibody.

(c) FITC charge: FITC is a charged molecule and antibodies with many FITC molecules (i.e. high F/P ratio) result in a highly charged antibody that binds, presumably through electrostatic interactions, nonspecifically to cytoplasmic elements (Hulspas et al.). This seems to be mainly a problem with intracellular staining and not with surface stains.

Þ Potential solution: For this reason FITC is not ideal for intracellular staining and you might try your antibody conjugated to a different fluorochrome.

(d) CD205: CD205 (DEC205) is a C-type lectin that is highly expressed on dendritic cells. Recently, it has been demonstrated that PE-Cy5.5 binds with high specificity to mouse CD205 (Park et al.). No staining was observed towards human CD205 and the binding of other Cy5.5 conjugates (PerCP-Cy5.5, APC-Cy5.5 and Cy5.5) to mouse CD205 was much weaker than that of PE-Cy5.5 (Park et al.).

Þ Potential solution: Given the high specificity of the interaction, you should avoid the use of PE-Cy5.5, and to a lesser extent other Cy5.5 containing fluorochromes, when your cells of interest expresses mouse CD205.

(4) Other effects

Finally, another odd-ball has been reported for APC tandems. Apparently, living cells have some way, which depends on their metabolism, to degrade the APC-Cy7 and APC-H7 tandems, leaving you with an APC signal (Le Roy et al.). APC-Cy7 seemed to be more affected than APC-H7 and monocytes are more active at degrading this signal than lymphocytes.

Þ Potential solution: Given that this requires live cells, fixation of your cell solution after staining will solve this problem. Alternatively, as this degradation requires metabolically active cells, storing your cell solution at 4°C or on ice or adding sodium azide (NaN3) to your storing buffer will reduce the effect.

That’s all I have for now, but if you know of other such ‘pseudo-artifacts’, or if you have any corrections and comments please share them with us!

 





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References:

An amazing source for odd questions on flow cytometry is the ‘Cytometry mailing list’ hosted by the Purdue University, which can be found under: https://lists.purdue.edu/mailman/listinfo/cytometry

Beavis, A.J. & Pennline, K.J., 1996. Allo-7: a new fluorescent tandem dye for use in flow cytometry. Cytometry, 24(4), pp.390–395.

Hulspas, R. et al., 2009. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry, 76B(6), pp.355–364.

Jahrsdörfer, B., Blackwell, S.E. & Weiner, G.J., 2005. Phosphorothyoate oligodeoxynucleotides block nonspecific binding of Cy5 conjugates to monocytes,

Le Roy, C. et al., 2009. Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism. Cytometry A, 75(10), pp.882–890.

Pape, K.A. et al., 2011. Different B cell populations mediate early and late memory during an endogenous immune response. Science, 331(6021), pp.1203–1207.

Park, C.G., Rodriguez, A. & Steinman, R.M., 2012. PE-Cy5.5 conjugates bind to the cells expressing mouse DEC205/CD205. J Immunol Methods, 384(1-2), pp.184–190.

Shapiro, H.M., 2004. Practical Flow Cytometry 4th Edt,

Stewart, C.C. & Stewart, S.J., 2001. Cell preparation for the identification of leukocytes. Methods Cell Biol, 63, pp.217–251.

Takizawa, F., Kinet, J.P. & Adamczewski, M., 1993. Binding of phycoerythrin and its conjugates to murine low affinity receptors for immunoglobulin G. Journal of Immunological Methods, 162(2), pp.269–272.

van Vugt, M.J., van den Herik-Oudijk, I.E. & van de Winkle, J.G., 1996. Binding of PE-CY5 conjugates to the human high-affinity receptor for IgG (CD64). Blood, 88(6), pp.2358–2361.

Wu, C.J. et al., 1991. Murine memory B cells are multi-isotype expressors. Immunology, 72(1), pp.48–55.

Zeng, X. et al., 2012. gd T Cells Recognize a Microbial Encoded B Cell Antigen to Initiate a Rapid Antigen-Specific Interleukin-17 Response. Immunity, 37(3), pp.524–534.

 

Gerhard WingenderGerhard Wingender is currently an Instructor at the La Jolla Institute for Allergy and Immunology (La Jolla, CA). His main lab toy is flow cytometry and his research interest involve invariant Natural Killer T (iNKT) cells.

 

 





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Photo credit: PNNL – Pacific Northwest National Laboratory / Foter.com / CC BY-NC-SA

Can shutting down the IRF3 kinases be the magic pill to treat obesity?

amlexanoxCan amlexanox, a small molecule drug that has been approved for asthma, allergic rhinitis and aphthous ulcers be an effective treatment for obesity? Many are excited about this prospective presented in a recent article in Nature Medicine by Reilly et. al. as simply Googling “amlexanox” and “obesity” resulted in a plethora of news articles on this report.  Interestingly, the two highly related proteins allegedly inhibited by amlexanox that led to this result in mice are the IRF3-kinases, TBK1 (TANK-binding Kinase-1, T2K, NAK) and IKK-ε (IκB kinase-epsilon, IKK-i), whose major known functions are during pathogen infections: the activation of IRF-3 (interferon regulatory kinase-3), the major transcription factor regulating expression of interferon-β (IFNβ).

Inflammation is considered a key link between obesity and insulin resistance.  The canonical NF-ĸB signaling pathway has been shown to playing a major role in this linkage.  IKK-ε and TBK1 are two IKK-β-related kinases with an unclear role in NF-ĸB activation, and thus their role in obesity and insulin resistance was explored in this and a previous study by the same group (Chiang et. al).

In this study, the authors show that expression and kinase activity of IKK-ε and TBK1 were increased in mice fed a high-fat diet and in response to TNFα in an in vitro adipocyte inflammation model. In the previous study, IKK-ε deficient mice were partially resistant to development of obesity and insulin resistance when fed a high fat diet.  Thus, the authors sought to determine if inhibiting these kinases would have a therapeutic effect on obesity.  A screen for small molecule inhibitors to block IKK-ε and TBK1 identified amlexanox, a drug currently used to treat asthma, allergic rhinitis and aphthous ulcers.  Amlexanox treatment inhibited the in vitro kinase activity of both IKK-ε and TBK1 at a much lower concentration than it did the related canonical NF-ĸB pathway kinase IKK-β.

To determine the effect of in vivo inhibition of these kinases on obesity indexes, mice fed a high fat diet were treated with amlexanox.  Amlexanox-treated mice gained significantly less weight than non-treated mice, and treatment of mice with pre-established diet-induced obesity led to a significant, but reversible weight loss accompanied by a decrease in adipose tissue, without a lower food intake.  Additionally, ob/ob mice which are genetically disposed to overt obesity due to deficient leptin expression, also lost adipose tissue mass when treated with amlexanox.

Interestingly, compared with mice fed a normal diet, mice on a high fat diet have a decreased core body temperature. Amlexanox-treated mice had body temperatures raised to normal levels along with increased oxygen consumption, indicating that an increase in energy expenditure of these mice may contribute to their weight loss.  Other indexes of obesity and glucose intolerance also returned to normal levels following amlexanox treatments.

Despite the promising results achieved with this drug in decreasing obesity and insulin resistance, the mechanisms by which inhibition of these kinases lead to this effect remains unclear.  Thus, future studies need to clarify the effects of amlexanox on IKK-ε and TBK1 regulation of the NF-ĸB pathway and obesity-associated inflammation, as well as address effects on the IRF3-interferon pathway.  Finally, it will be important to determine if other molecules are targeted by this drug.

 

Further Reading:

An inhibitor of the protein kinases TBK1 and IKK-ɛ improves obesity-related metabolic dysfunctions in mice.  Reilly SM, Chiang SH, Decker SJ, Chang L, Uhm M, Larsen MJ, Rubin JR, Mowers J, White NM, Hochberg I, Downes M, Yu RT, Liddle C, Evans RM, Oh D, Li P, Olefsky JM, Saltiel AR. Nat Med. 2013 Mar;19(3):313-21.

The protein kinase IKKepsilon regulates energy balance in obese mice.  Chiang SH, Bazuine M, Lumeng CN, Geletka LM, Mowers J, White NM, Ma JT, Zhou J, Qi N, Westcott D, Delproposto JB, Blackwell TS, Yull FE, Saltiel AR. Cell. 2009 Sep 4;138(5):961-75.

Turning off the inflammatory, but not the metabolic, flames.  Calay ES, Hotamisligil GS. Nat Med. 2013 Mar 6;19(3):265-7.

Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription.  Bonnard M, Mirtsos C, Suzuki S, Graham K, Huang J, Ng M, Itié A, Wakeham A, Shahinian A, Henzel WJ, Elia AJ, Shillinglaw W, Mak TW, Cao Z, Yeh WC. EMBO J. 2000 Sep 15;19(18):4976-85.

Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood.  Herman M, Ciancanelli M, Ou YH, Lorenzo L, Klaudel-Dreszler M, Pauwels E, Sancho-Shimizu V, Pérez de Diego R, Abhyankar A, Israelsson E, Guo Y, Cardon A, Rozenberg F, Lebon P, Tardieu M, Heropolitanska-Pliszka E, Chaussabel D, White MA, Abel L, Zhang SY, Casanova JL. J Exp Med. 2012 Aug 27;209(9):1567-82.

TANK-binding kinase 1 (TBK1) controls cell survival through PAI-2/serpinB2 and transglutaminase 2.  Delhase M, Kim SY, Lee H, Naiki-Ito A, Chen Y, Ahn ER, Murata K, Kim SJ, Lautsch N, Kobayashi KS, Shirai T, Karin M, Nakanishi M. Proc Natl Acad Sci U S A. 2012 Jan 24;109(4):E177-86.

*Image courtesy of RXlist.com*

Markers and functions of human CD4+ follicular helper T cells

human white blood cellCXCR5 is a chemokine receptor expressed by and used to identity human CD4+ follicular helper T cells (TFH).  TFH cells, as their name implies, promote the differentiation and survival of memory and plasma B cells in the B cell follicular and germinal center regions of secondary lymphoid organs. CXCR5+ TFH-like central memory CD4+ T cells (CD4+ TCM) also circulate in peripheral blood and can be detected among human peripheral blood mononuclear cells (PBMC).  CXCR5+ cells comprise 20-25% of CD4+ TCM cells in human PBMC.  However, what are the identifying markers and functional differences of CXCR5+ vs. CXCR5 CD4+ T cells from human PBMC and the prototypical CXCR5+ TFH cells in secondary lymphoid organs?

I have previously discussed markers that can be used for identification of human CD4+ TH1, TH2, and TH17 T cell helper subsets as well as CD4+ FoxP3+ regulatory T cells.  CXCR5 can be upregulated transiently on activated T cells, however a subset of PBMC T cells constitutively express CXCR5, indicating this is a uniquely functioning subset identifiable by this marker using flow cytometry or other methods.  PBMC CXCR5+ CD4+ TCM cells exhibit many but not all features of TFH cells present in secondary lymphoid organs, and thus may be the circulating memory counterpart of TFH cells.

CXCL13, the chemokine ligand for CXCR5, is highly expressed in B cell follicles and likely plays an important role in recruitment of CXCR5+ TFH cells to B cell zones.  Expression of ICOS by TFH cells has been demonstrated to be essential for their function in B cell help.  Additionally, TFH cells are higher expressers of CXCL13, as well as IL-21, IL-10, Bcl-6, and PD-1 than other helper T cell subsets.

Studies by Chevalier et al, and Morita et. al. compared the functional properties of CXCR5 and CXCR5+ CD4+ TCM cells from human PBMC.  PBMC CXCR5+ CD4+ cells are resting central memory cells in phenotype, being CD45RA, CCR7+ and CD62L+, but not expressing activated TFH markers such as ICOS and CD69.  Upon stimulation, CXCR5+ CD4+ TCM promote significantly higher B cell plasmablast differentiation and Ig secretion than CXCR5 CD4+ TCM cells, attributable to enhanced expression of ICOS and IL-10.  However, Bcl-6 expression was not found to be different between these PBMC subsets, and conclusions for expression levels and role of IL-21 were contradictory between these studies.

The question of whether PBMC CXCR5+ CD4+ TCM are distinct from TH1, TH2, and TH17 T cells was addressed by these studies as well. While Chevalier et al. found that CXCR5+ TCM cells were more non-polarized and secreted comparatively lower levels of cytokines associated with TH1, TH2, and TH17 T cells, Morita et. al, identified TH1, TH2, and TH17 T cells within CXCR5+ compartment, albeit at somewhat different frequencies than CXCR5 cells. Thus PBMC CXCR5+ CD4+ TCM are a heterogenous subset with features of both TFH cells and the various TH1, TH2, and TH17 subsets.  Further interrogation of the functions of these populations are needed.

PBMC CXCR5+ CD4+ cells have been identified as a highly relevant population to study in the context of vaccination and human disease.  Patients with systemic lupus erythematosus (SLE) have higher percentages of circulating CD4+CXCR5+ ICOS+ cells.  Patients with autoimmune juvenile dermatomyositis (JDM) were found to have an altered CXCR5+ compartment where the overall frequency of CXCR5+ cells was not different, but the ratio of TH2 and TH17 to TH1 cells within the CXCR5+ population was enhanced and associated with disease activity.

In the vaccine setting, emergence of a population of circulating ICOS+CXCR3+CXCR5+CD4+ T cells was found in individuals 7 days after influenza vaccination, and correlated with increased antibody titers and B cell plasmablasts. These cells could also induce plasma cell differentiation in vitro and  thus are important in the development of vaccine elicited protective antibody responses.

In conclusion, PBMC CXCR5+ CD4+ T cells are an important cellular subset to study in the context of human disease.  These cells are likely the circulating memory component of TFH cells, and alterations in frequencies and functions are associated with various human diseases and protective antibody responses following vaccination.

 

Further Reading:

Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion.  Morita R, Schmitt N, Bentebibel SE, Ranganathan R, Bourdery L, Zurawski G, Foucat E, Dullaers M, Oh S, Sabzghabaei N, Lavecchio EM, Punaro M, Pascual V, Banchereau J, Ueno H. Immunity. 2011 Jan 28;34(1):108-21.

CXCR5 expressing human central memory CD4 T cells and their relevance for humoral immune responses.  Chevalier N, Jarrossay D, Ho E, Avery DT, Ma CS, Yu D, Sallusto F, Tangye SG, Mackay CR. J Immunol. 2011 May 15;186(10):5556-68.

Expansion of circulating T cells resembling follicular helper T cells is a fixed phenotype that identifies a subset of severe systemic lupus erythematosus. N. Simpson, P.A. Gatenby, A. Wilson, S. Malik, D.A. Fulcher, S.G. Tangye, H. Manku, T.J. Vyse, G. Roncador, G.A. Huttley et al.  Arthritis Rheum., 62 (2010), pp. 234–244.

Induction of ICOS+CXCR3+CXCR5+ TH Cells Correlates with Antibody Responses to Influenza Vaccination.  Bentebibel S. E. et al.  Sci Transl Med. 13 March 2013: Vol. 5, Issue 176, p. 176ra32.

Photo credit: wellcome images / Foter.com / CC BY-NC-ND

Artifacts and non-specific staining in flow cytometry, Part I

If you add your antibody, lets say anti-CD3-epsilon antibody, to your cell solution you’d expect that only T cells will be labeled, right? Well, if it were so easy then it wouldn’t be biology!

 

antibodiesIn this first half of the two part blog, I will talk about the two reasons, namely unspecific binding and Fc-receptors, which most people think of when they talk about non-specific binding in flow cytometry.

Some lesser known, but intriguing and important, ‘pseudo-artifacts’ will be covered later in Part II of the blog.

 

(1) Unspecific binding

Unspecific binding is defined as any sticking of an antibody or a fluorochrome to a cell in a fashion that does not require a specifically (currently) defined interaction. This might occur due to electrostatic interactions, glycolipid interaction on the cell membrane, protein-protein interactions and DNA binding.

As such unspecific binding of a cell depends heavily on the surface area (for surface stains) and/or its volume (intracellular staining). For example a cell with twice the size (as seen in the FSC) has 4-times the surface area (SF = 4pi r2) and 8-times the volume (V = 4/3pi r3) and consequently the unspecific binding will be 4 to 8-times higher. So, if you see the whole population shifting a bit in your histogram, you might want to check the scatter of the cells. For example, activated cells start proliferating, which increase their cell size along the way.

 

Aggravating factors and potential solutions:

Antibody amount: A surplus of antibody can increase the non-specific binding, leading to a reduction in the separation of your positive cells and reducing the signal:noise ratio.

Þ Potential solution: Titrate your antibody. As a starting point, antibodies with the same fluorochrome conjugate can often be used at similar concentrations.

Extracellular matrix/cell content: All cells bind proteins including antibodies to some degree via various interactions

Þ Potential solution: Addition of protein to the wash and staining solutions will cover many of these binding sites. Most staining protocols include BSA or serum (either human or FCS) for this purpose.

Dead cells: Dead cells are notorious for non-specifically binding antibodies and appear very ‘sticky’. This is partially due to DNA, but including DNAse would only partially solve the problem.

Þ Potential solution: A live/dead differentiation should be included, if possible, in every staining. Dead cells cannot be entirely separated just by FSC/SSC characteristics, especially not after fixation. Keep in mind though, that fixation of your cells after staining with e.g. PI or 7AAD will partially permeabilize all your cells, so that PI or 7AAD can leak out of the labeled cells to other cells eventually homogenously staining all your cells. In the case of 7AAD this can be avoided by inclusion of non-fluorescent actinomycin D (Schmid et al.). However, nowadays multiple live/dead discriminating reagents are available that can be fixed, thereby stopping potential leakage and avoiding this problem altogether.

 

(2) Binding of antibodies to Fc-receptors:

Obviously Fc-receptors (FcR) bind antibodies with high specificity, but the common misconception is that this is solely species-specific. However, FcRs from one species readily bind antibodies from other species to varying degrees. For example, hamster anti-mouse CD3-epsilon (clone 145.2C11) can bind to all mouse FcRs (Wingender et al.).

Þ Potential solution:

(a)   Fab or F(ab)2 fragments: Utilizing antibodies without their Fc-end avoids the problem altogether, but most commercially available antibodies do contain their Fc part.

(b)   ‘Fc-Block’: Adding antibodies that are specific for particular FcRs that block the undesired interaction with your experimental antibody. However, the ‘Fc-block’ commonly used for mice is the blocking monoclonal antibody 2.4G2 (rat IgG2b kappa) which is specific for mouse Fc-gamma-RII  (CD16) and Fc-gamma-RIII (CD32). Therefore, other FcRs are not directly blocked by 2.4G2. However, the majority of commercial antibodies are of an IgG subtype, most of the potential unspecific Fc-binding will be blocked by 2.4G2. Similar products for staining of human cells are widely available.

(c)     Unconjugated antibody: Adding unconjugated antibody of the same species and isotype as your experimental antibody to your staining cocktail will saturate most potential FcR binding sites.

As a positive side effect, adding unconjugated antibodies, either 2.4G2 or any other isotype, to your stain will incidentally also saturate most other potential unspecific bindings, as they were outlined under (1). Therefore, adding unconjugated antibody to your surface and also your intracellular staining cocktails will reduce unspecific binding.

 

So much for part one. As always, corrections and comments are highly welcomed.

 

References:

Schmid, I. et al., 2001. Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation. J Immunol Methods, 247(1-2), pp.175–186.

Wingender, G. et al., 2006. Rapid and preferential distribution of blood-borne alphaCD3epsilonAb to the liver is followed by local stimulation of T cells and natural killer T cells. Immunology, 117(1), pp.117–126.

 





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Gerhard WingenderGerhard Wingender is currently an Instructor at the La Jolla Institute for Allergy and Immunology (La Jolla, CA). His main lab toy is flow cytometry and his research interest involve invariant Natural Killer T (iNKT) cells.

 

 





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References:

Schmid, I. et al., 2001. Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation. J Immunol Methods, 247(1-2), pp.175–186.

Wingender, G. et al., 2006. Rapid and preferential distribution of blood-borne alphaCD3epsilonAb to the liver is followed by local stimulation of T cells and natural killer T cells. Immunology, 117(1), pp.117–126.

 

Photo credit: AJC1 / Foter.com / CC BY-NC-SA

The Fascinating System of Eye-induced Immune Regulation

The immune privilege of the eye is a widely recognized but frequently oversimplified concept. The notion that the eye possessed unusual immunological characteristics was recognized in the 19th century by van Dooremaal, who observed prolonged survival of murine skin grafts transplanted into the anterior chamber (AC) of the dog eye. The term ocular ‘immune privilege’ was articulated by Medawar, who recognized that the extended survival of foreign grafts in the AC was a remarkable departure from the fate of similar grafts transplanted to sites outside of the eye. Almost 30 years later, the seminal studies of Kaplan et al. demonstrated that alloantigenic cells introduced into the AC in fact did escape from the eye and induced a deviant immune response in which serum alloantibodies were generated, while systemic cell-mediated immune responses were suppressed in an antigen-specific manner. Subsequent studies in mice confirmed this AC-associated immune deviation (ACAID) and demonstrated that it is an important contributor to the immune privilege of the eye.

ACAID

Figure 1. Organ systems involved in the induction of ACAID.
A brief description of the complex cellular-interplay, that causes ACAID (From Jerry Niederkorn’s review in Nature Immunology 7, 354 – 359;2006). Removal of the thymus, eye or spleen within 72 h of injection of antigen into the anterior chamber prevents the induction of ACAID. Chemical sympathectomy before anterior chamber injection of antigen also prevents the induction of ACAID. IL-, interleukin; BCR, B cell receptor. 

 

Several labs have since confirmed that antigens introduced into the AC elicit a deviant immune response, which is characterized by the antigen-specific suppression of classical Th1 immune responses, such as delayed-type hypersensitivity (DTH) and complement-fixing antibodies, while preserving the generation of noncomplement-fixing antibodies of the IgG1 isotype in the mouse. This complex phenomenon called ACAID involves multiple cell types that interact to create unique, antigen-specific immune suppression. Briefly, the injection of antigen into the ocular AC results in the antigen being taken up by circulating F4/80+ cells (a type of dendritic cell) that migrate to the spleen and thymus. Within 3 days of entering the thymus,  the F4/80+ cells induce the generation of CD4-CD8-NK1.1+ thymocytes that are believed to enter the circulation as recent thymic emigrants and home to the spleen, where they contribute to the generation of splenic regulatory cells. The spleen is the terminal organ in ACAID, where there is a complex interaction between the F4/80+ cells, natural killer T (NKT) cells, NK1.1 cell, gamma delta T cells and B cells, which in turn elicit the generation of CD4+ and CD8+ regulatory T cells (T regs) specific for the antigen that was introduced in the AC. These T regs are the key players in causing the antigen specific immune-suppression. That ACAID may occur in humans is suggested by the demonstration that individuals with acute retinal necrosis develop antibodies but not cell-mediated immunity to Varicella zoster.

Because the injection of antigen into the AC induces different phenotypes of Tregs and classically Tregs have been known to suppress autoimmunity, Bhowmick et al in 2011 investigated the ability of splenic regulatory T cells induced by an intracameral injection of MOG35-55 peptide to regulate MOG35-55-induced EAE (Experimental Autoimmune Encephalomyelitis), the animal model of human Multiple Sclerosis. In this animal model, MOG35-55 immunization results in an immune response against MOG35-55 peptide which is a component of the Myelin protein, hence causing an inflammatory auto-reaction against myelin and neurodegeneration, similar to the human disease – Multiple Sclerosis. Bhowmick et al found that the injection of MOG35-55 peptide into the ocular AC could suppress MOG35-55 peptide induced EAE, both as a cure (when injected after disease initiation) and as a prophylactic measure (when injected prior to disease induction). The suppression was antigen specific because when they injected an unrelated antigen e.g ovalbumin into the AC, it did not affect EAE in the recipient animal.

They next went on to isolate the AC-injection-induced splenic regulatory T cells and via elegant adoptive transfer experiments showed that AC-induced CD4+ regulatory T cells could suppress the diseases ONLY at the early stage (also known as the priming phase of the disease). While AC-induced CD8+ regulatory T cells could only suppress the progression of an already initiated diseases (known as the chronic phase of EAE). The CD8s were ineffective at the effector phase and vice versa. This was probably the first report which differentiated between the priming and chronic phase of EAE and showed that effective suppression of autoimmune response in EAE can be achieved by different regulatory T cell populations. This work also revealed that while the suppression of EAE by AC-induced CD8+ regulatory T cells is TGF-β dependent, the AC-induced CD4 T regs, do not use TGF-β.

Characteristically, previous studies have shown that ACAID-CD4 Treg cells do not express FoxP3. And the induction of and the activity of regulatory T cells in ACAID is independent of CD4+FoxP3+ regulatory T cells. AC-induced CD8+ regulatory cells suppress IFN-γ production in vitro and in vivo suppress T cells that effect a DTH reaction in immunized mice. Further, AC-induced CD8+ regulatory cells are restricted by Qa-1 antigens expressed by effector T cells. Since the non-classical MHC class I molecule Qa-1 is known to be expressed only on activated cells, AC-induced CD8+ regulatory T cells specifically suppress activated T cells and hence the effector or chronic phase of an autoimmune disease like EAE. Thus it can be concluded that ACAID suppresses the induction of effector T cells and also the activity of effector T cells by distinct populations of regulatory T cells. Recently it was shown that Type II collagen (CII), a key antigen involved in auto-immune responses during Rheumatoid Arthritis, could induce a similar CII-specific immune suppression via ACAID (Farooq et al 2012). A finding that has opened up possibilities of using this system in an Arthritis model to test its efficacy.

Overall these data indicate that the suppression of an ongoing autoimmune disease by the adoptive transfer of regulatory T cells might only be feasible when the regulatory T cells are specific for the pathogenic antigen. Alternatively, transfer of polyclonal regulatory T cells may cure ongoing disease only in lymphopenic hosts, in which the massive expansion of regulatory T cells may lead to the generation of a sufficient number of antigen-specific regulatory T cells. If this is also the case also in humans, it may represent a significant limitation for the clinical application of regulatory T cells in autoimmune diseases, as, to date, human self-antigen specific regulatory T cells have not been successfully expanded ex vivo. In this regard, the use of Anterior Chamber Associated Immune Deviation (ACAID), can be highly effective as ACAID can generate antigen-specific CD8+ and also CD4+ regulatory T cells.

 






 

Arijit BhowmickArijit Bhowmick is currently a postdoctoral researcher at the Immunology institute of the Mount Sinai Medical Center, NY. He received his PhD in structural immunology from the National Institute of Immunology, New Delhi. His current research interests encompass autoimmunity, Th17 cells and structure based inhibitor designing. 

 






Understanding MFI in the context of FACS data

Understanding MFI in the context of FACS data

The speed, sensitivity and versatility of flow cytometry are things of beauty, but with great power comes great responsibility. The fact is that with potentially millions of data points accrued over the run of a single sample, finding the best way to compare those data can be daunting. One of the more commonly misunderstood and often misleading tools in FACS analysis is a pesky little statistic — MFI.

 

What is MFI?

mean mode median MFIThe first point of confusion is born from the name itself. MFI is often used without explanation, to abbreviate either arithmetic mean, geometric mean, or median fluorescence intensity. In a perfect world, our data would be normally distributed and in that case means, median and mode are all equal. In reality, flow data is rarely normal and never perfect. The more that the data skews, the further the mean drifts in the direction of skew and becomes less representative of the data being analyze as seen on the graphical representation.

Because fluorescent intensity increases logarithmically, arithmetic mean quickly becomes useless to generalize a population of events, as a right-hand skew causes even more exaggeration of the mean. To combat this, geometric mean (gMFI) is often used to account for the log-normal behavior of flow data, however, even gMFI is susceptible to significant shifts. This leaves us with the median or the mid-point of the population. Median is considered a much more robust statistic in that it is less influenced by skew or outliers. Is there a “right” MFI to use to analyze flow data? No. But generally speaking, median is the safest choice and usually most representative of a “typical” cell.

 

Three common mistakes when using MFI

            Characterizing a bi-modal population: Any average only holds true for normal distributions, and a bi-modal population is by definition not normal. Statistics aside, gating each population and presenting percentages will yield data that is both more easily interpretable as well as more statistically significant.

            Comparing data from disparate experiments: Because fluorescent intensity is sensitive to experimental condition (e.g. antibody dilution, tandem dye degradation, laser fluctuations, etc.), it is dangerous to compare intensity of any kind across multiple experiments.

            Blindly using MFI as a quantification of expression: While FACS is more than sensitive enough to provide estimates of ligand abundance, such calculations require normalization and calibration using a standard curve. Additionally, it is tempting to say that a population with a higher MFI has higher expression than one with a lower MFI, however, care must be taken to ensure other factors are not responsible. For example, a large cell with more membrane and consequently more surface protein, can appear brighter than a smaller cell of the same type. Thus, it is important to control carefully for things such as size or compensation that may confound results.

 

So, when should I use MFI?

Not until asked by a reviewer.

Kidding.

MFI has many important uses, but can sometimes be as much a distraction from the data as it is a clarification. Ultimately, like any piece of data, MFI should only be applied if you are absolutely certain that it is the best comparison to make, otherwise it is simply clutter on an otherwise clean histogram.

 

For further reading:

Flowjo’s excellent explanation of the differences between mean, median and mode. http://flowjo.typepad.com/the_daily_dongle/2007/10/mean-median-mod.html

An amazing article explaining when and why to use bi-exponential axes. Importantly, the affect scaling can have on actually visualizing the median value of a population.

http://facs.scripps.edu/ni0706-681.pdf

 






adam bestAdam Best is currently a post-doctoral fellow at the University of California, San Diego where he also received his Ph.D. in Biomedical Sciences. His research focuses on understanding the transcriptional events that govern the formation of memory T cells

 

 




Natural Killer Cell subtypes and markers in human PBMC

Natural killer cellsNatural Killer (NK) cells are a cytotoxic innate immune lymphocyte cell type.  In humans, NK cells comprise up to 15% of peripheral blood mononuclear cells (PBMC), and 5-20% of the PBMC lymphocyte population.  Several subtypes of NK cells exist in humans.  In this post, I will discuss phenotypic properties and markers of NK subtypes present in human PBMC.

Three subtypes of NK cells are recognized: CD56dim CD16+, CD56brightCD16+/- and CD56 CD16+ NK cells. The CD56dim CD16+ and CD56brightCD16+/- subsets are best studied and are phenotypically classified as a more cytotoxic and a more cytokine producing subset of NK cells, respectively.  NK cell activation is mediated by the balance between engagement of activating receptors including NKp46, NKp30, NKp44, NKG2D, CD16, 2B4, NKp80, and DNAM-1, and HLA-I binding inhibitory receptors including killer immunoglobulin-like receptors (KIRs), LIR1/ILT2 and NKG2A/CD94.  NK cells can also be activated in response to cytokines such as IL-2, IL-12, IL-15, and IL-18.

CD56dim CD16+ NK cells:  This subtype comprises the majority, up to 90%, of PBMC NK cells and is considered the most cytotoxic subset.  CD16 is the FCγ receptor III, and can thus bind the FC portion of IgG antibodies and mediate antibody dependant cell-mediated cytotoxicity (ADCC) of antibody-bound target cells.  Expression of inhibitory receptors differs among NK subsets, and this subset exhibits lower expression of KIRs and ILT2 but higher expression of NKG2A/CD94 compared with CD56bright NK cells. Expression of granzyme B and perforin is also high in this subset compared with CD56bright NK cells.  A recent report by De Maria et. al, demonstrated that this subset does in fact robustly produce cytokines including IFNγ early after activation.

CD56brightCD16+/- NK cells: This subtype comprises up to 10% of NK cells in PBMC, but is the major NK subtype in tissues and secondary lymphoid organs.  This subset is conventionally known as the cytokine producing subset of NK cells, and rapidly produces cytokines and chemokines including IFNγ, TNFα, GM-CSF, and RANTES after activation.

Interestingly, in HIV-viremic individuals, a third CD56 CD16+ NK population is significantly expanded in PBMC comprising between 20-55% of NK cells.  This population in healthy individuals and aviremic HIV-infected individuals is rare, under 10% of total NK cells.  Compared with CD56+ NK cells, the CD56 CD16+ NK cells from HIV-viremic patients exhibited lower expression of activating receptors NKp46, NKp30, and NKp44, lower cytotoxic activity, higher expression levels of inhibitory receptors, and lower expression levels of cytokines including IFNγ, TNFα, and GM-CSF.  This subset is also expanded in individuals with chronic HCV infection.  Thus, the expansion of this poorly functional NK subset is likely clinically relevant in chronic viral disease.

In summary, these NK populations can be differentiated by expression of CD16 and CD56.  Of note, NKT (natural killer-like T) cells can also express these markers along with CD3.  Thus, to differentiate these cells from NKT cells, the inclusion of CD3 as a cell identification marker is critical in analysis of these cells by flow cytometry or other methods.

 

Further Reading:

CD56 negative NK cells: origin, function, and role in chronic viral disease.  Björkström NK, Ljunggren HG, Sandberg JK. Trends Immunol. 2010 Nov;31(11):401-6.

The biology of human natural killer-cell subsets. Cooper MA, Fehniger TA, Caligiuri MA. (2001) Trends Immunol 22: 633–640.

Natural killer cell distribution and trafficking in human tissues.  Carrega P, Ferlazzo G. Front Immunol. 2012;3:347.

Revisiting human natural killer cell subset function revealed cytolytic CD56(dim)CD16+ NK cells as rapid producers of abundant IFN-gamma on activation.  De Maria A, Bozzano F, Cantoni C, Moretta L. Proc Natl Acad Sci U S A. 2011 Jan 11;108(2):728-32.

Natural killer cells in HIV-1 infection: dichotomous effects of viremia on inhibitory and activating receptors and their functional correlates.  Mavilio D, Benjamin J, Daucher M, Lombardo G, Kottilil S, Planta MA, Marcenaro E, Bottino C, Moretta L, Moretta A, Fauci AS. Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):15011-6.

Characterization of CD56−/CD16+ natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals. Mavilio D, Lombardo G, Benjamin J, Kim D, Follman D, et al.. (2005) Proc Natl Acad Sci U S A. 102: 2886–2891.