J Biol Regul Homeost Agents. 2004 Jul-Dec;18(3-4):331-4.

Reactive phenotypes after acute and chronic NK-cell activation.
Lima M, Almeida J, Teixeira MA, Santos AH, Queirós ML, Fonseca S, Moura J, Gonçalves M, Orfão A, Pinto Ribeiro AC.

Service of Clinical Hematology, Laboratory of Cytometry, Hospital Geral de Santo António, Porto, Portugal. mmc.lima@clix.pt

Abstract
Several phenotypic changes have been shown to occur after NK-cell stimulation, involving molecules that have been proved to regulate NK-cell migration into tissues and NK-cell activation and proliferation as well as target cell recognition and killing. Here, we review the reactive phenotypes observed in vivo after acute and chronic NK-cell activation.

PMID: 15786700 [PubMed - indexed for MEDLINE

ALMEIDA, J.

FONSECA, S.

GONCALVES, M.

LIMA, M

MOURA, J.

ORFAO, A.

PINTO RIBEIRO, A.C.

QUEIROS, M.L.

SANTOS, A.H.

TEIXEIRA, M.A.

Repositório Científico do Centro Hospitalar do Porto

INTRODUCTIONMost of our knowledge on the modulation of thecellular surface phenotypes associated with NK-cellactivation is based mostly on in vitro studies. Inaccordance, it has been shown that activated NK-cellsexpress a number of molecules that are absent innon-activated NK-cells, and that NK-cell activationresults in either down-regulation or up-regulation ofdifferent receptors constitutively expressed by restingNK-cells, whereas the surface levels of othermolecules remain stable (1-13).In order to define the reactive phenotypes after acuteand chronic NK-cell activation in vivo, we haveanalyzed by four-color flow cytometry the immuno-phenotype of blood CD56+low NK-cells in normalindividuals (14), in patients with acute viral infectionsand in patients with either chronic infections or tumors(15) (Fig. 1). This strategy allowed us to characterizedifferent subsets of normal NK-cells, corresponding toearly and late activation-related stages (15), to comparereactive NK-cell phenotypes with those observed on T-cells (16) and to identify abnormal NK-cell leukemia-associated phenotypes (17).NORMAL AND REACTIVE NK-CELL PHENOTYPESNormal blood CD56+low NK-cells Although a great inter-individual variability is foundin normal individuals due to the presence of differentNK-cell populations in different maturation andactivation states, the majority of the CD56+low NK-cellspresent in normal blood have a CD2-/+low, CD7+high,HLA-DR- and CD45RO- phenotype (14). Most of thesecells are CD11b+/CD38+ and homogeneously expresshigh levels of CD11a and CD45RA, whereas CD11cand CD57 are heterogeneously expressed in avariable fraction of CD56+low cells.CD56+low NK-cells from patients with acute viralinfectionRecently activated CD56+low NK-cells are present athigh frequency in the blood of patients with acute viralinfection (15). They have a pattern of expression ofCD2 and CD7 similar to that observed in restingmature CD56+low NK-cells, except that a higherpercentage of CD2+ NK-cells and a slightly higherintensity of CD7 expression are found. CD2+low/CD7+high CD56+low NK-cells probably originate fromCD2-/CD7+high CD56+low NK-cells upon stimulation,through de novo expression of CD2 molecules on thecell surface. Recently activated CD56+low NK-cellstypically show strong reactivity for CD38 and expresshigh levels of HLA-DR. At that phase, there is alsodown-regulation of CD45RA, a phenomenon that istransiently accompanied by co-expression of CD45ROin variable proportions of cells. CD11a levels areincreased, whereas expression of CD11b isdecreased. Moreover, these CD56+low NK-cellsexpress dimly and heterogeneously CD11c while theyare either negative or dimly positive for CD57.CD56+low NK-cells from patients with chronic infectionsand tumorsChronically activated CD56+low NK-cells shownunique phenotypic features (15), including up-regulation of CD2, which becomes homogeneous(CD2+HIGH), and down-regulation of CD7, whichJournal of Biological Regulators and Homeostatic AgentsReactive phenotypes after acute and chronic NK-cell activationM. LIMA 1, J. ALMEIDA 2, M. d A. TEIXEIRA 1, A.H. SANTOS 1, M.L. QUEIRÓS 1, S. FONSECA 1, J. MOURA 1, M. GONÇALVES 1, A. ORFÃO 2, A.C. PINTO RIBEIRO 11 Service of Clinical Hematology, Laboratory of Cytometry, Hospital Geral de Santo António, Porto, Portugal2 Servicio de Citometría, Universidad de Salamanca and Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, SpainABSTRACT: Several phenotypic changes have been shown to occur after NK-cell stimulation, involving molecules thathave been proved to regulate NK-cell migration into tissues and NK-cell activation and proliferation as well as targetcell recognition and killing. Here, we review the reactive phenotypes observed in vivo after acute and chronic NK-cellactivation. (J Biol Regul Homeost Agents 2004; 18: )KEY WORDS: NK-cells, Activation, Phenotype, Flow cytometryReceived:Revised:Accepted:©Wichtig Editore, 20040393-974X/331-04 $15.00Reactive NK-cell phenotypes332Fig. 1 - Immunophenotypicanalysis of blood CD56+lowNK-cells in normal indivi-duals (A), patients with acuteviral infection (B) and pa-tients with NK-cell lympho-cytosis secondary to in-fections or tumors (C). APC-conjugated anti-CD3 and PE-Cy5- conjugated anti-CD56were used in combinationwith the following pairs ofFITC- / PE- conjugated mo-noclonal antibodies directedagainst T- and NK-associatedantigens: CD2/CD7, CD57/CD11c, CD38/ CD11b, CD11a/HLA-DR, CD45RA/ CD45RO.Results are expressed aspercentage of positive cells,mean fluorescence intensity(arbitrary relative linear unitsscaled from 0 to 10,000) andcoefficient of variation. Barsrepresent the mean valuesand the vertical lines repre-sent the standard deviation.The statistical significanceof differences observedbetween groups was evalua-ted using the Mann-WhitneyU-test. P-values less than0.05 were considered to beassociated with statisticalsignificance. ns, not statisti-cally significant.becomes heterogeneous (CD7-/+low). In contrast torecently activated CD56+low NK-cells, chronicallyactivated CD56+low NK-cells are CD38-/+low andCD11b-/+low and express high and homogeneouslevels of CD57, while CD11c expression is dim andheterogeneous. At this stage, most NK-cells havealready rever ted into their or iginal HLA-DR-/CD45RA+/CD45RO- phenotype, although a variableproportion of HLA-DR+ and/or CD45RO+ NK-cells canstill be found in some individuals.DIFFERENCES AND SIMILARITIES BETWEEN REACTIVENK-CELL AND T-CELL PHENOTYPESPatients with infectious mononucleosis show amassive expansion of CD8+ TCRalpha/beta+ T-cells,the majority of which display an immunophenotypecompatible with recent T-cell activation (16):CD2+high, CD7-/+low, CD11a+high, CD38+high, HLA-DR+,CD45RO+high, CD45RA-/+low, CD11b-/+low, CD11c-/+low,CD57-. Additionally, the levels of CD3, CD5, CD28and CD62L molecules are decreased compared tothose found in normal individuals. Late-activationantigens, such as CD57, were found in smallpropor tions of CD8+ TCRalpha/beta+ T-cells.Increased numbers of activated CD4+ TCRalpha/beta+and TCRgamma/delta+ T-cells are also observed insome patients. Evidence for activation of CD4+TCRalpha/beta+ and TCRgamma/delta+ T-cells rely onchanges similar to those descr ibed for CD8+TCRalpha/beta+ T-cells.Curiously, most of the immunophenotypic changesthat occur after NK-cell activation are similar to thatobserved on T-cells, although different kinetics wereobserved in some markers. For instance up-regulationof CD11c, CD38 and CD45RO are transientlyobserved in early T- and NK-cell activation stages, asdo de novo expression of HLA-DR molecules;modulation of CD2 and CD7 expression occurs inboth cases, consisting in up-regulation of CD2 anddown regulation of CD7 molecules, which seems totake place at a latter stage during NK-cell activation,as compared to that occurring on T-cells; down-regulation of CD7 expression is probably preceded bya transient up-regulation of this molecule in bothcases; CD57 behaves as a late activation marker forboth T- and NK-cells.ABERRANT NK-CELL PHENOTYPESWe have recently repor ted on the immuno-phenotypic features of a particular type of chronic NK-cell leukemia, whose NK-cells were either CD56- orexpressed very low levels of CD56 (CD56-/+low NK-cells), in the context of an aberrant activation-relatedmature phenotype (17). CD56-/+low NK-cells displayseveral unusual immunophenotypic features.Accordingly, besides being CD56-/+low, they areCD11b-/+low (heterogeneous), CD7-/+low (heteroge-neous), CD2+ (homogeneous), CD11c+high (homo-geneous), CD38-/+low (heterogeneous), CD94+high(heterogeneous) and HLA-DR+.Some of the immunophenotypic differences foundbetween normal and CD56-/+dim NK-cells – i.e. over-expression of CD11c and HLA-DR and down-regulation of CD45RA accompanied in some cases byco-expression of CD45RO in a variable fraction of NK-cells - do in fact reproduce the immunophenotypicchanges that typically occur on recently activatedCD56+low NK-cells, whereas other – i.e. increasedCD2 expression and decreased and heterogeneousexpression of CD7, CD11b and CD38 – are similar tothose typically observed in conditions of chronic NK-cell stimulation. In spite of these similarities betweenCD56-/+low NK-cells and either recently- or chronically-activated CD56+low NK-cells, major differences areobserved in the pattern of expression of somemarkers. Accordingly, CD56-/+low NK-cells have higherand more homogenous expression of CD11c, greaterpercentages of CD57+ cells and lower levels of HLA-DR as compared to recently activated NK-cells.Additionally, CD2 expression is more homogeneous,down-regulation of CD7 and CD11b is morepronounced and CD7 expression is even moreheterogeneous on CD56-/+low NK-cells than onchronically activated CD56+low NK-cells. Moreover,disagreement between down-regulation of CD11b andCD38 expression, consisting on a CD11b-/CD38+phenotype, is frequently found in CD56-/+dim NK-cellsbut not on “chronically” activated CD56+low NK-cells.Such aberrant activation-related immunophenotypewas the very first argument supporting the mono-clonal nature of these CD56-/+low NK-cells, which wassubsequently confirmed in female patients using thehuman androgen receptor gene polymerase chainreaction -based assay.CONCLUDING REMARKSThe major ity of changes that have been do-cumented upon NK-cell activation in vitro are alsoreproducible in vivo and define phenotypic patternsassociated to the early/acute- and late/chronic-phases of NK-cell activation.Early activation-related NK-cell phenotypes arefound in patients with acute viral infection whereas inonly a minor proportion of patients with chronicinfections and tumors and normal healthy individuals.Similarly, chronically-activated NK-cells are over-represented in patients with NK-cell lymphocytosisassociated to conditions in which the underlyingstimuli persist for a long time, although they could alsobe found at lower numbers in a few percentage ofpatients with acute infections as well as in normalindividuals.Finally, by understanding the sequence of theimmunophenotypic changes occurring after NK-cell333Lima et alReactive NK-cell phenotypesactivation, we are able to identify aberrantimmunophenotypic patterns and to distinguishreactive NK-cell lymphocytosis from NK-cell leukemiacases.Reprint requests to:Margarida LimaService of HematologyLaboratory of CitometryHospital Geral de Santo AntónioRua D Manuel II, s/n4099-001 Porto, Portugalmmc.lima@clix.ptREFERENCES1. Rabinowich H, Herberman RB, Whiteside TL. Differentialeffects of IL12 and IL2 on expression and function ofcellular adhesion molecules on purified human naturalkiller cells. Cell Immunol 1993; 152: 481-98.2. Rabinowich H, Sedlmayr P, Herberman RB, WhitesideTL. Response of human NK cells to IL-6 alterations ofthe cell surface phenotype, adhesion to fibronectin andlaminin, and tumor necrosis factor-alpha/beta secretion.J Immunol 1993; 150: 4844-55.3. Carbone E, Ruggiero G, Terrazzano G, et al. A newmechanism of NK cell cytotoxicity activation: the CD40-CD40 ligand interaction. J Exp Med 1997; 185: 2053-60.4. Nakarai T, Robertson MJ, Streuli M, et al. Interleukin 2receptor gamma chain expression on resting andactivated lymphoid cells. J Exp Med 1994; 180: 241-51.5. Warren HS, Skipsey LJ. Loss of activation-inducedCD45RO with maintenance of CD45RA expressionduring prolonged culture of T cells and NK cells.Immunology 1991; 74: 78-85.6. Robertson MJ, Caligiuri MA, Manley TJ, Levine H, RitzJR Human natural killer cell adhesion molecules.Differential expression after activation and participationin cytolysis. J Immunol.1990; 145: 3194-201.7. Yamabe T, Takakura K, Sugie K, et al. Induction of the2B9 antigen/dipeptidyl peptidase IV/CD26 on humannatural killer cells by IL-2, IL-12 or IL-15. Immunology1997; 91: 151-8.8. Maenpaa A, Jaaskelainen J, Carpen O, Patarroyo M,Timonen T. Expression of integrins and other adhesionmolecules on NK cells; impact of IL-2 on short- andlong-term cultures. Int J Cancer 1993; 53: 850-5.9. Umehara H, Minami Y, Domae N, Bloom ET. Increasedprocessing of lymphocyte function-associated antigen-1in human natural killer cells stimulated with IL-2. IntImmunol 1994; 6: 1071-80.10. Baume DM, Caligiuri MA, Manley TJ, Daley JF, Ritz J.Differential expression of CD8 alpha and CD8 betaassociated with MHC-restr icted and non-MHC-restricted cytolytic effector cells. Cell Immunol 1990;131: 352-65.11. Anderson P, Caligiuri M, O´Brien C, Manley T, Ritz J,Schlossman SF. Fc gamma receptor type III (CD16) isincluded in the zeta NK receptor complex expressed byhuman natural killer cells. Proc Natl Acad Sci USA 1990;87: 2274-8.12. Vujanovic NL, Rabinowich H, Lee YJ, Jost L, HebermanRB, Whiteside TL. Distinct phenotypic and functionalcharacteristics of human natural killer cells obtained byrapid interleukin 2-induced adherence to plastic. CellImmunol 1993; 151: 133-57.13. Cordero OJ, Sarandeses C, Lopez-Rodriguez JL,Nogueira M. The presence and cytotoxicity of CD16+CD2- subset from PBL and NK cells in long-term IL-2cultures enhanced by prothymosin-alpha. Immuno-pharmacology 1995; 29: 215-23.14. Lima M, Teixeira MA, Queirós ML et al. Immuno-phenotypic characterization of normal blood CD56+loversus CD56+high NK-cell subsets and its impact of theunderstanding of their tissue distribution and functionalproperties. Blood Cells Molecules and Diseases 2001;27: 731-43.15. Lima M, Almeida J, Teixeira MA, Queirós ML, Justiça B,Orfão A. The ex vivo Patterns of CD2/CD7, CD57/CD11c, CD38/CD11b, CD45RA/CD45RO, and CD11a/HLA-DR Expression Identify Acute/Early andChronic/Late NK-Cell Activation States. Blood CellsMolecules and Diseases 2002; 28: 181-90.16. Lima M, Teixeira MA, Queirós ML, et al. Immuno-phenotype and TCR-Vb repertoire of peripheral blood T-cells in acute infectious mononucleosis. Blood CellsMolecules and Diseases 2003; 30: 1-12.17. Lima M, Almeida J, García Montero A, et al. Clinico-biological, immunophenotypic and molecularcharacteristics of monoclonal CD56-/+dim Chronic NK-cell Large Granular Lymphocytosis. American Journal ofPathology (in press)AA PLEASE COMPLETE4

Reactive phenotypes after acute and chronic NK‐cell activation

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Reactive phenotypes after acute and chronic NK‐cell activation

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