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Leukemia & Lymphoma Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ilal20
Human NK Cells in Health and Disease: Clinical, Functional, Phenotypic and DNA Genotypic Characteristics a
a
Stephen J. Richards & Colin Stephen Scott a
Yorkshire Leukaemia Diagnostic Unit, Department of Haematology, Cookridge Hospital, Leeds, England Published online: 01 Jun 2015.
To cite this article: Stephen J. Richards & Colin Stephen Scott (1992) Human NK Cells in Health and Disease: Clinical, Functional, Phenotypic and DNA Genotypic Characteristics, Leukemia & Lymphoma, 7:5-6, 377-399, DOI: 10.3109/10428199209049794 To link to this article: http://dx.doi.org/10.3109/10428199209049794
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
0 1992 Harwood Academic Publishers GmbH
Leukemia and Lymphoma, Vol. 7, pp. 377-399 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Human NK Cells in Health and Disease: Clinical, Functional, Phenotypic and DNA Genotypic Characteristics STEPHEN J. RICHARDS and COLIN STEPHEN SCOTT Downloaded by [Deakin University Library] at 04:26 12 August 2015
Yorkshire Leukaemia Diagnostic Unit, Department of Haematology, Cookridge Hospital, Leeds, England (Received 20 December 1991)
Natural killer ( N K ) cells are the subject of great current interest because of their possible (in uiuo) role in tumour cell surveillance and killing, and because of the potential application of cytokine-modulated N K cells in cancer immunotherapy. In addition, clonal proliferations of NK-associated (NKa) cell populations represent a high proportion of chronic (non-B) lymphoid malignancies and abnormal (both clonal and non-clonal)NKa components are being increasingly reported in association with diverse clinical pictures such as autoimmune disease. This communication extensively reviews what is presently known regarding normal and leukaemic NKa phenotypic diversity, the mechanisms of NK-mediated cytolysis, the role of N K cells in malignancy, and the diagnostic and cellular aspects of malignant N K a proliferations. KEY WORDS:
Natural Killer DNA genotypes
NK lmmunophenotyping Review Disease
INTRODUCTION Contemporary studies of immune system biology have shown that lymphocytes comprise various subpopulations characterised by the expression of well-defined immunophenotypic membrane markers and by distinct functional properties. This current perspective recognises T and B lymphocyte fractions, together with a third component widely referred to as natural killer (NK) cells'. Natural killer cells were initially defined in functional terms as the cell population capable of spontaneous cytotoxic activity against a range of both autologous and allogeneic target cells, a phenomenon discovered fortuitously during the course of investigations designed to examine the susceptibility of tumour target cells to lysis by the cell-mediated immune system. From these studies, it Address for correspondence: Dr C..S. Scott. Yorkshire Leukaemia Diagnostic Unit, Department of Haematology, Cookridge Hospital, Leeds LS16 6QB, England.
Function
became apparent that this natural cytotoxicity could be characterised by spontaneous lysis of 'Cr-labelled tumour target cells in uitro by lymphocytes from healthy individual^^.^. Although initially considered to be an experimental artefact, it was later shown to be a distinct form of lymphocyte cytotoxicity which differed from the cell-mediated immunity (CMI) typically associated with cytotoxic T lymphocytes (CTL). However in contrast to CMI, which requires prior exposure to target cell antigen (i.e. acquisition of immunological memory), NK cell natural cytotoxicity appears to occur independently of major histocompatibility (MHC) antigen expression by the target cell. To date, three main types of cytotoxicity have been associated with various phenotypic populations4; (a) classical cytotoxic CD3+CD4+ or CD3 'CD8' lymphocytes (CTL), that are specific for MHC Class I1 and Class I antigens respectively5s6;(b) NK cells that operate independently of MHC restriction; and (c) CD3+ cytotoxic cells which show apparent unrestricted MHC reactivity'. This latter
311
378
S. J. RICHARDS AND C. S. SCOTT
population is present at low levels in normal peripheral blood and includes both TCRyG cells described by Brenner et a1.,8 and a CD3+CD16+ fraction identified by Lanier et d 9 . In addition to these, a highly efficient form of cytotoxicity is displayed by lymphokine-activated killer (LAK) cellslO.Although LAK cells are not present to any great extent in normal peripheral blood, they may increase in a variety of inflammatory disorders and can be readily produced by in vitro activation with interleukin-2(IL-2). An important early observation in NK cell biology was the demonstration" that NK activity was associated with a morphologically distinct population of lymphocytes known as large granular lymphocytes (LGL). These cells, with a high cytop1asmic:nuclear ratio and abundant azurophilic cytoplasmic granules, constitute a relatively minor (1&15%) but discrete component of normal peripheral blood lymphocytes12. However, the association between NK activity and LGL morphology is not absolute as not all NK cells are LGLs and, conversely, LGL do not invariably have an NK cytotoxic capability". In addition to NK cell activity, it was found some LGL can mediate a different form of cytotoxicity known as antibody-dependent cellular cytotoxicity (ADCC)14*15.The effector cells of ADCC, often referred to as killer (K) cells, have a characteristic functional receptor (designated as CD16 or FcRIII) for the Fc portion of IgG antibody molecules. As blocking of this CD16 molecule or IgG receptor with protein A or anti-CD16 antibodies eliminates ADCC activity without affecting NK function16, this suggested that these two functions of the same cell were distinct and consistent with the view that NK and K cells are largely overlapping populations4. The property of ADCC is however not restricted to NK cell fractions as other haemopoietic cells, particularly granulocytes and in vitro activated monocytes, also express membrane CD16 molecules and exhibit similar types of cytolytic activity"-l8.
Downloaded by [Deakin University Library] at 04:26 12 August 2015
+
PHENOTYPIC CHARACTERISTICS OF NORMAL NK CELLS
NK cells were first defined immunophenotypically by their lack of reactivity with T and B cell markers and were termed 'N' or Null cells". The absence of a definitive NK cell marker led to attempts to produce NK-specific monoclonal antibodies and these efforts have produced three groups of antibodies (which
define the CD16, CD56 and CD57 determinants) that have been fully characterised by the Third and Fourth Leucocyte Typing Workshops2032 '. These studies showed that whereas there is no pan-NK cell antigen, analogous to CD3 and CD19 which broadly define T and B lymphocyte populations respectively, a number of markers were found to be NK-associated (NKa). The first of these was the FcRIII receptor which was initially defined by EA (erythrocyte-antibody) rosetting procedures and later by CD16 monoclonal antibodie~~ NK ~ .cells ~ ~ .can now be readily identified phenotypically on a multiparameter basis by the concomitant expression of CD16 and CD56 membrane determinants together with a lack of CD3 expression24 although these phenotypic definitions are not absolute as a small subset of CD3+ lymphocytes can exhibit non-MHC restricted cytotoxicity' and some NK cells are CD3CD16-CD56' 2 5 . Membrane CD16 is expressed by the majority of NK cells and is a low affinity receptor, associated with the ( chain subunit of CD3I3, that binds antigenassociated IgG immune complexes but not monomeric IgG molecules. Many monoclonal antibodies to CD16 have been produced (e.g. B73.1, 3G8 and Leu1 1) although their reactivities vary according to epitope specificities and cellular expression' 6 * 2 6 . For example, whilst normal granulocytes express membrane CD16, molecular studies have revealed that this is biochemically different from CD16 present on NK cells and is devoid of a transmembrane section. For NK cells, this membrane- integrated part of the CD16 molecule appears to play a functional role in signalling and The leucocyte CD56 determinant, as defined by monoclonal antibodies such as Leu19 and NKHl, is expressed by most NK cells30*31and has been to be identical to one of the family of cellular adhesion molecules associated with neural cells (N-CAM). Although these molecules are not directly involved in the cytolysis of target cells, and may not even mediate homophilic binding between NK cells and N-CAM' tumour cell targets32,it is postulated that they participate in interactions critical to the generation of an effective immune response33.A study detailing the patterns of CD16 and CD56 antigen co-expression by peripheral blood cytotoxic cells suggested three apparently distinct subpopulations. Two of these, characterised by CD3-CD16+CD56' and CD3+CD16-CD56' composite phenotypes, could mediate NK activity and morphologically were LGL, whereas the third (CD3-CD16-CD56')
Downloaded by [Deakin University Library] at 04:26 12 August 2015
NK CELLS IN HEALTH AND DISEASE
comprised both granular and agranular forms25. Consequently, it was concluded that these morphological and phenotypic subpopulations could represent possible maturational stages for LGL. A further monoclonal antibody, which was generated against the human lymphoblastoid tumour line HSB-2 and initially designated HNK-1, identifies the CD57 NK-associated membrane determinant. At first, the reactivity of this antibody was described as being NK cell specific34, but subsequent studies35 showed that it identified two populations of cytotoxic lymphocytes; one CD3' with low NK activity and a paucity of cytoplasmic granules, and the other a CD3- typical LGL with high NK activity. In addition to the distinctive presence of NKa determinants, NK cells may also express various T-cell associated antigens. For example, a proportion (2@50%) of NK cells express low density membrane CD836,and most NK cells express membrane CD2 and CD7, but not CD5 ". With respect to myeloid-associated determinants, normal N K cells lack membrane CD13, CD14 and CD3337-39but do express a numbers of antigens belonging to the family of functional receptors known as leucocyte adhesion molecule^^^^^'. In particular, the three variable cx chain subunits of the CDI l/CD18 antigen complex (CDI la, CD11b and CDI lc) are all expressed by NK cells; C D l l b is found on approximately 80% of NK cells with a higher relative expression compared to CDl la or C D I I C ~Mem~. brane CD11b, which effectively represents the C3bi complement receptor, is widely defined by monoclonal antibodies such as OKM1, although this determinant is also expressed by T cells, monocytes and granulocyte^^^. In addition, recent studies have further shown that the granulocyte-associated lactoN-fucopentaose carbohydrate CD 15 determinant is expressed by a minor proportion of morphologically defined LGL and is co-expressed by approximately 50% of CD16' blood lymphocyte^^^. Detailed immunophenotyping studies using multiparameter flow cytometry have revealed many apparent subsets of NK cells which have been identified not only on the basis of positive/negative antigen expression but also on their relative levels of staining. For example, the expression of membrane CD8 by different NKa fractions can be broadly subdivided into three levels of staining intensity (CD8-, CDSd'"'+ and CD8 +). Whereas the difference between CD8di"+ and CD8' staining has long been considered to be largely quantitative, recent studies have shown that CD3- cells expressing membrane
319
CD8 (i.e. most CD8di"f lymphocyte^^^ exclusively express CD8a gene products (as a/u homodimers) whereas CD8 is expressed as both a/u homodimers and a/P heterodimers on typical CD3 'CD8' lymphoc y t e ~ Furthermore, ~~. the patterns of NKa determinant expression also appear to show some variation with respect to these different levels of membrane CD8 staining. Lanier and L ~ k e nand ~ ~Lanier et aLZ5, found that CD16+CD57+cells could be either CD8or CD8+, compared to the CD8dim+CD16+ fraction which was mainly CD3-CD5-, and that the CD16+CD56+ subset was CD2+CD3-CD4-CD5CD8dim + in contrast to the CD16-CD56' fraction which was predominantly CD2+CD3+CD4-CD5+ CD8 . Recent studies that have examined, by combining immunomagnetic manipulation with multicolour flow cytometry, have determined relationships between the expression of CDllb, CD16, CD56 and CD57 NKa antigens, and CD2, CD3, CD5, CD7, CD38 and TCR T cell d e t e r m i n a n t ~ ~ ~These , ~ ' . reveal that a minor proportion of normal CD3+CD8+ lymphocytes express NKa determinants (mainly CD56 and CD57) even though the major NKa+ components are associated with CD3-CD8dim+and CD3-CD8- cell fractions (Table 1). In addition, highly significant correlations were found between the expression of individual NKa determinants by CD3+CD8+, CD3-CD8dim+ and CD3-CD8- populations in +
Table 1 Composite NK-associated (NKa) phenotypes of normal peripheral blood CD8+, CD8di"+and CD3-CD8- fractions" N K a ,fraction'
NKu determinant e.upression"
CD16 CD8 (n = 29)
-
-
CD8dim+(n = 29)*
+ + V
V V
CD3-CDK(n = 1 I)'
+
CDSb
Frequency'
CD57 vor + vor+ vor + -
+ vor + - or + vor
-
79% 17% 4% 62% 3% 10% 7 yo 18% 100%
Composite NKa phenotypes as detailed previously [44.471. Patterns of CD16. CDS6 and CDS7 expression where +, v and indicate > 60%. 30-60% and 5 years) have normal NK activity, compared to low NK activities in patients in remission for < 5 years. Furthermore, defective target cell conjugation and abnormal cytolytic mechanisms have also been ~ ~ impaired +~~~ reported in hairy cell l e ~ k a e m i a ' and NK activity has been reported in patients with pre-leukaemia/myelodysplasia and chronic B cell proliferations160*'61.In the latter diagnostic group however, these observations of reduced blood cell NK activity could largely be due to a 'dilution' effect of NK cells by leukaemic cells in the mononuclear fraction t e ~ t e d ' ~ ~Such . ' ~ limitations ~. in interpretation illustrate the need to express cytolytic activity per unit number of phenotypically-defined NKa cells rather than to meaningless mononuclear cell numbers which show considerable inter-patient variation.
385
(reactive, pre-malignant or malignant) T-cell proliferations. Reactive polyclonal LGL/NKa expansions have been reported in viral infections, hepatitis, nephrotic syndrome and l y m p h ~ m a ~ ~ , ' "whereas ~'~" both persistent non-clonal (chronic reactive) and clonal LGL/NKa+ abnormalities may be associated with various autoimmune diseases. In contrast to the relatively well defined B cell chronic lymphocytic leukaemias, the clinical, morphological and immunophenotypic features of mature or chronic T-cell proliferations show great variability. This is particularly true for abnormal persistent LGL/NKa+ proliferations which have been referred to as (a) chronic granular lymphocytosis, (b) T-y lymphoproliferative disease, (c) chronic T-lymphocytosis, (d) T-suppressor CLL or (e) persistent lymphocytosis with n e ~ t r o p e n i a l " * ' ~Although ~. many cases of LGL/NKa proliferation have been reported, there is little doubt that the literature and its conclusions are biased towards those cases that have presented with significantly increased lymphocyte counts and/or the occurrence of distinct leukaemia-associated symptomology. In addition, it is evident that abnormal increases in NKa' lymphocytes is not invariably associated with morphological increases in lymphocytes with cytoplasmic granulation. Whether LGL- NKa' expansions are ontogenetically related to or distinct from LGL'NKa' (or even LGL'NKa-) expansions is not known, but diagnostic categorisation based upon a single morphological feature such as cytoplasmic granulation could further contribute to underestimating the frequency of such disorders and could cause further bias in both clinical and immunophenotypic evaluations. Accepting these limitations, this current review has examined the clinical, haematological and cellular characteristics of many representative studies of abnormal LGL expansions in an attempt to highlight perhaps some of the more consistent features associated with this spectrum of diseases. LGL AND NK-ASSOCIATED (NKa) The haematological and clinical features of 130 such PROLIFERATIONS patients with LGL expansions abstracted from the Clinical and haematological features In recent years, literature are summarised in Tables 2 and 3 an increasing number of reports describing patients respectively. Analysis of haematological features with abnormally increased proportions and/or abso- reveals anaemia in 42% of patients, thrombocytopelute numbers of large granular lymphocytes (LGL) nia in 23% and neutropenia in 78%. The degree of have appeared in the l i t e r a t ~ r e ' ~ *169. ' ~ ~ -These lymphocytosis is usually mild, with more than half of morphologically-defined abnormalities, most of which the patients having lymphocyte counts in the range express various NKa determinants (i.e. LGL +/NKa +) 4.1-15.0 x lo9/] and only 14% of patients having as well as those cases where NKa marker is found in lymphocyte counts exeeding this. There is n o apparent sex predominance and even though all ages may be the absence of LGL morphology (LGL-NKa'), variously comprise transient (reactive) or chronic affected, most cases occur in patients over the age of +
+
S. J. RICHARDS AND C. S . SCOTT
386
Table 2 Haematological features of 130 patients with large granular lymphocyte proliferations Lymphocyte c o u n d
Number of patients with
Study Anaemia"
Neurropenia'
Thrombocytopenia'
12121
511 1
0112
117 19/38 8/34 O/l 2
4/11 617 18/38 19/34 8/12
312I 117 2/l1 017 1/38 7/34 4/12
22/94 23%
391130 30%
13/130 56%
18/130 14%
13/21 517 6111 117 11/38' 14/34 5/12
18/21 617 9111 517 33/38 23/34 7112
9121 217 3/11 017 8/36
Observed totals: Percentages:
551130 42yo
lO1/130 :I8 % ~
> 15.0
4.1-15.0
6121 017
Newland et a/., 1984 McKenna et a/., 1985 Chan et al., 1986 Lauria et al., 1987 Loughran & Starkebaum, 1987 Semenzato et a/., 1987 Oshimi et a/., 1988
~~
0.54.0
nt
617
~~
Patient considered anaemic if haemoglobin < 13.5 gjdl (males) or < 11.5 g/dl (females). Patient considered neutropenic if absolute count
Leukemia & Lymphoma Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ilal20
Human NK Cells in Health and Disease: Clinical, Functional, Phenotypic and DNA Genotypic Characteristics a
a
Stephen J. Richards & Colin Stephen Scott a
Yorkshire Leukaemia Diagnostic Unit, Department of Haematology, Cookridge Hospital, Leeds, England Published online: 01 Jun 2015.
To cite this article: Stephen J. Richards & Colin Stephen Scott (1992) Human NK Cells in Health and Disease: Clinical, Functional, Phenotypic and DNA Genotypic Characteristics, Leukemia & Lymphoma, 7:5-6, 377-399, DOI: 10.3109/10428199209049794 To link to this article: http://dx.doi.org/10.3109/10428199209049794
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
0 1992 Harwood Academic Publishers GmbH
Leukemia and Lymphoma, Vol. 7, pp. 377-399 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Human NK Cells in Health and Disease: Clinical, Functional, Phenotypic and DNA Genotypic Characteristics STEPHEN J. RICHARDS and COLIN STEPHEN SCOTT Downloaded by [Deakin University Library] at 04:26 12 August 2015
Yorkshire Leukaemia Diagnostic Unit, Department of Haematology, Cookridge Hospital, Leeds, England (Received 20 December 1991)
Natural killer ( N K ) cells are the subject of great current interest because of their possible (in uiuo) role in tumour cell surveillance and killing, and because of the potential application of cytokine-modulated N K cells in cancer immunotherapy. In addition, clonal proliferations of NK-associated (NKa) cell populations represent a high proportion of chronic (non-B) lymphoid malignancies and abnormal (both clonal and non-clonal)NKa components are being increasingly reported in association with diverse clinical pictures such as autoimmune disease. This communication extensively reviews what is presently known regarding normal and leukaemic NKa phenotypic diversity, the mechanisms of NK-mediated cytolysis, the role of N K cells in malignancy, and the diagnostic and cellular aspects of malignant N K a proliferations. KEY WORDS:
Natural Killer DNA genotypes
NK lmmunophenotyping Review Disease
INTRODUCTION Contemporary studies of immune system biology have shown that lymphocytes comprise various subpopulations characterised by the expression of well-defined immunophenotypic membrane markers and by distinct functional properties. This current perspective recognises T and B lymphocyte fractions, together with a third component widely referred to as natural killer (NK) cells'. Natural killer cells were initially defined in functional terms as the cell population capable of spontaneous cytotoxic activity against a range of both autologous and allogeneic target cells, a phenomenon discovered fortuitously during the course of investigations designed to examine the susceptibility of tumour target cells to lysis by the cell-mediated immune system. From these studies, it Address for correspondence: Dr C..S. Scott. Yorkshire Leukaemia Diagnostic Unit, Department of Haematology, Cookridge Hospital, Leeds LS16 6QB, England.
Function
became apparent that this natural cytotoxicity could be characterised by spontaneous lysis of 'Cr-labelled tumour target cells in uitro by lymphocytes from healthy individual^^.^. Although initially considered to be an experimental artefact, it was later shown to be a distinct form of lymphocyte cytotoxicity which differed from the cell-mediated immunity (CMI) typically associated with cytotoxic T lymphocytes (CTL). However in contrast to CMI, which requires prior exposure to target cell antigen (i.e. acquisition of immunological memory), NK cell natural cytotoxicity appears to occur independently of major histocompatibility (MHC) antigen expression by the target cell. To date, three main types of cytotoxicity have been associated with various phenotypic populations4; (a) classical cytotoxic CD3+CD4+ or CD3 'CD8' lymphocytes (CTL), that are specific for MHC Class I1 and Class I antigens respectively5s6;(b) NK cells that operate independently of MHC restriction; and (c) CD3+ cytotoxic cells which show apparent unrestricted MHC reactivity'. This latter
311
378
S. J. RICHARDS AND C. S. SCOTT
population is present at low levels in normal peripheral blood and includes both TCRyG cells described by Brenner et a1.,8 and a CD3+CD16+ fraction identified by Lanier et d 9 . In addition to these, a highly efficient form of cytotoxicity is displayed by lymphokine-activated killer (LAK) cellslO.Although LAK cells are not present to any great extent in normal peripheral blood, they may increase in a variety of inflammatory disorders and can be readily produced by in vitro activation with interleukin-2(IL-2). An important early observation in NK cell biology was the demonstration" that NK activity was associated with a morphologically distinct population of lymphocytes known as large granular lymphocytes (LGL). These cells, with a high cytop1asmic:nuclear ratio and abundant azurophilic cytoplasmic granules, constitute a relatively minor (1&15%) but discrete component of normal peripheral blood lymphocytes12. However, the association between NK activity and LGL morphology is not absolute as not all NK cells are LGLs and, conversely, LGL do not invariably have an NK cytotoxic capability". In addition to NK cell activity, it was found some LGL can mediate a different form of cytotoxicity known as antibody-dependent cellular cytotoxicity (ADCC)14*15.The effector cells of ADCC, often referred to as killer (K) cells, have a characteristic functional receptor (designated as CD16 or FcRIII) for the Fc portion of IgG antibody molecules. As blocking of this CD16 molecule or IgG receptor with protein A or anti-CD16 antibodies eliminates ADCC activity without affecting NK function16, this suggested that these two functions of the same cell were distinct and consistent with the view that NK and K cells are largely overlapping populations4. The property of ADCC is however not restricted to NK cell fractions as other haemopoietic cells, particularly granulocytes and in vitro activated monocytes, also express membrane CD16 molecules and exhibit similar types of cytolytic activity"-l8.
Downloaded by [Deakin University Library] at 04:26 12 August 2015
+
PHENOTYPIC CHARACTERISTICS OF NORMAL NK CELLS
NK cells were first defined immunophenotypically by their lack of reactivity with T and B cell markers and were termed 'N' or Null cells". The absence of a definitive NK cell marker led to attempts to produce NK-specific monoclonal antibodies and these efforts have produced three groups of antibodies (which
define the CD16, CD56 and CD57 determinants) that have been fully characterised by the Third and Fourth Leucocyte Typing Workshops2032 '. These studies showed that whereas there is no pan-NK cell antigen, analogous to CD3 and CD19 which broadly define T and B lymphocyte populations respectively, a number of markers were found to be NK-associated (NKa). The first of these was the FcRIII receptor which was initially defined by EA (erythrocyte-antibody) rosetting procedures and later by CD16 monoclonal antibodie~~ NK ~ .cells ~ ~ .can now be readily identified phenotypically on a multiparameter basis by the concomitant expression of CD16 and CD56 membrane determinants together with a lack of CD3 expression24 although these phenotypic definitions are not absolute as a small subset of CD3+ lymphocytes can exhibit non-MHC restricted cytotoxicity' and some NK cells are CD3CD16-CD56' 2 5 . Membrane CD16 is expressed by the majority of NK cells and is a low affinity receptor, associated with the ( chain subunit of CD3I3, that binds antigenassociated IgG immune complexes but not monomeric IgG molecules. Many monoclonal antibodies to CD16 have been produced (e.g. B73.1, 3G8 and Leu1 1) although their reactivities vary according to epitope specificities and cellular expression' 6 * 2 6 . For example, whilst normal granulocytes express membrane CD16, molecular studies have revealed that this is biochemically different from CD16 present on NK cells and is devoid of a transmembrane section. For NK cells, this membrane- integrated part of the CD16 molecule appears to play a functional role in signalling and The leucocyte CD56 determinant, as defined by monoclonal antibodies such as Leu19 and NKHl, is expressed by most NK cells30*31and has been to be identical to one of the family of cellular adhesion molecules associated with neural cells (N-CAM). Although these molecules are not directly involved in the cytolysis of target cells, and may not even mediate homophilic binding between NK cells and N-CAM' tumour cell targets32,it is postulated that they participate in interactions critical to the generation of an effective immune response33.A study detailing the patterns of CD16 and CD56 antigen co-expression by peripheral blood cytotoxic cells suggested three apparently distinct subpopulations. Two of these, characterised by CD3-CD16+CD56' and CD3+CD16-CD56' composite phenotypes, could mediate NK activity and morphologically were LGL, whereas the third (CD3-CD16-CD56')
Downloaded by [Deakin University Library] at 04:26 12 August 2015
NK CELLS IN HEALTH AND DISEASE
comprised both granular and agranular forms25. Consequently, it was concluded that these morphological and phenotypic subpopulations could represent possible maturational stages for LGL. A further monoclonal antibody, which was generated against the human lymphoblastoid tumour line HSB-2 and initially designated HNK-1, identifies the CD57 NK-associated membrane determinant. At first, the reactivity of this antibody was described as being NK cell specific34, but subsequent studies35 showed that it identified two populations of cytotoxic lymphocytes; one CD3' with low NK activity and a paucity of cytoplasmic granules, and the other a CD3- typical LGL with high NK activity. In addition to the distinctive presence of NKa determinants, NK cells may also express various T-cell associated antigens. For example, a proportion (2@50%) of NK cells express low density membrane CD836,and most NK cells express membrane CD2 and CD7, but not CD5 ". With respect to myeloid-associated determinants, normal N K cells lack membrane CD13, CD14 and CD3337-39but do express a numbers of antigens belonging to the family of functional receptors known as leucocyte adhesion molecule^^^^^'. In particular, the three variable cx chain subunits of the CDI l/CD18 antigen complex (CDI la, CD11b and CDI lc) are all expressed by NK cells; C D l l b is found on approximately 80% of NK cells with a higher relative expression compared to CDl la or C D I I C ~Mem~. brane CD11b, which effectively represents the C3bi complement receptor, is widely defined by monoclonal antibodies such as OKM1, although this determinant is also expressed by T cells, monocytes and granulocyte^^^. In addition, recent studies have further shown that the granulocyte-associated lactoN-fucopentaose carbohydrate CD 15 determinant is expressed by a minor proportion of morphologically defined LGL and is co-expressed by approximately 50% of CD16' blood lymphocyte^^^. Detailed immunophenotyping studies using multiparameter flow cytometry have revealed many apparent subsets of NK cells which have been identified not only on the basis of positive/negative antigen expression but also on their relative levels of staining. For example, the expression of membrane CD8 by different NKa fractions can be broadly subdivided into three levels of staining intensity (CD8-, CDSd'"'+ and CD8 +). Whereas the difference between CD8di"+ and CD8' staining has long been considered to be largely quantitative, recent studies have shown that CD3- cells expressing membrane
319
CD8 (i.e. most CD8di"f lymphocyte^^^ exclusively express CD8a gene products (as a/u homodimers) whereas CD8 is expressed as both a/u homodimers and a/P heterodimers on typical CD3 'CD8' lymphoc y t e ~ Furthermore, ~~. the patterns of NKa determinant expression also appear to show some variation with respect to these different levels of membrane CD8 staining. Lanier and L ~ k e nand ~ ~Lanier et aLZ5, found that CD16+CD57+cells could be either CD8or CD8+, compared to the CD8dim+CD16+ fraction which was mainly CD3-CD5-, and that the CD16+CD56+ subset was CD2+CD3-CD4-CD5CD8dim + in contrast to the CD16-CD56' fraction which was predominantly CD2+CD3+CD4-CD5+ CD8 . Recent studies that have examined, by combining immunomagnetic manipulation with multicolour flow cytometry, have determined relationships between the expression of CDllb, CD16, CD56 and CD57 NKa antigens, and CD2, CD3, CD5, CD7, CD38 and TCR T cell d e t e r m i n a n t ~ ~ ~These , ~ ' . reveal that a minor proportion of normal CD3+CD8+ lymphocytes express NKa determinants (mainly CD56 and CD57) even though the major NKa+ components are associated with CD3-CD8dim+and CD3-CD8- cell fractions (Table 1). In addition, highly significant correlations were found between the expression of individual NKa determinants by CD3+CD8+, CD3-CD8dim+ and CD3-CD8- populations in +
Table 1 Composite NK-associated (NKa) phenotypes of normal peripheral blood CD8+, CD8di"+and CD3-CD8- fractions" N K a ,fraction'
NKu determinant e.upression"
CD16 CD8 (n = 29)
-
-
CD8dim+(n = 29)*
+ + V
V V
CD3-CDK(n = 1 I)'
+
CDSb
Frequency'
CD57 vor + vor+ vor + -
+ vor + - or + vor
-
79% 17% 4% 62% 3% 10% 7 yo 18% 100%
Composite NKa phenotypes as detailed previously [44.471. Patterns of CD16. CDS6 and CDS7 expression where +, v and indicate > 60%. 30-60% and 5 years) have normal NK activity, compared to low NK activities in patients in remission for < 5 years. Furthermore, defective target cell conjugation and abnormal cytolytic mechanisms have also been ~ ~ impaired +~~~ reported in hairy cell l e ~ k a e m i a ' and NK activity has been reported in patients with pre-leukaemia/myelodysplasia and chronic B cell proliferations160*'61.In the latter diagnostic group however, these observations of reduced blood cell NK activity could largely be due to a 'dilution' effect of NK cells by leukaemic cells in the mononuclear fraction t e ~ t e d ' ~ ~Such . ' ~ limitations ~. in interpretation illustrate the need to express cytolytic activity per unit number of phenotypically-defined NKa cells rather than to meaningless mononuclear cell numbers which show considerable inter-patient variation.
385
(reactive, pre-malignant or malignant) T-cell proliferations. Reactive polyclonal LGL/NKa expansions have been reported in viral infections, hepatitis, nephrotic syndrome and l y m p h ~ m a ~ ~ , ' "whereas ~'~" both persistent non-clonal (chronic reactive) and clonal LGL/NKa+ abnormalities may be associated with various autoimmune diseases. In contrast to the relatively well defined B cell chronic lymphocytic leukaemias, the clinical, morphological and immunophenotypic features of mature or chronic T-cell proliferations show great variability. This is particularly true for abnormal persistent LGL/NKa+ proliferations which have been referred to as (a) chronic granular lymphocytosis, (b) T-y lymphoproliferative disease, (c) chronic T-lymphocytosis, (d) T-suppressor CLL or (e) persistent lymphocytosis with n e ~ t r o p e n i a l " * ' ~Although ~. many cases of LGL/NKa proliferation have been reported, there is little doubt that the literature and its conclusions are biased towards those cases that have presented with significantly increased lymphocyte counts and/or the occurrence of distinct leukaemia-associated symptomology. In addition, it is evident that abnormal increases in NKa' lymphocytes is not invariably associated with morphological increases in lymphocytes with cytoplasmic granulation. Whether LGL- NKa' expansions are ontogenetically related to or distinct from LGL'NKa' (or even LGL'NKa-) expansions is not known, but diagnostic categorisation based upon a single morphological feature such as cytoplasmic granulation could further contribute to underestimating the frequency of such disorders and could cause further bias in both clinical and immunophenotypic evaluations. Accepting these limitations, this current review has examined the clinical, haematological and cellular characteristics of many representative studies of abnormal LGL expansions in an attempt to highlight perhaps some of the more consistent features associated with this spectrum of diseases. LGL AND NK-ASSOCIATED (NKa) The haematological and clinical features of 130 such PROLIFERATIONS patients with LGL expansions abstracted from the Clinical and haematological features In recent years, literature are summarised in Tables 2 and 3 an increasing number of reports describing patients respectively. Analysis of haematological features with abnormally increased proportions and/or abso- reveals anaemia in 42% of patients, thrombocytopelute numbers of large granular lymphocytes (LGL) nia in 23% and neutropenia in 78%. The degree of have appeared in the l i t e r a t ~ r e ' ~ *169. ' ~ ~ -These lymphocytosis is usually mild, with more than half of morphologically-defined abnormalities, most of which the patients having lymphocyte counts in the range express various NKa determinants (i.e. LGL +/NKa +) 4.1-15.0 x lo9/] and only 14% of patients having as well as those cases where NKa marker is found in lymphocyte counts exeeding this. There is n o apparent sex predominance and even though all ages may be the absence of LGL morphology (LGL-NKa'), variously comprise transient (reactive) or chronic affected, most cases occur in patients over the age of +
+
S. J. RICHARDS AND C. S . SCOTT
386
Table 2 Haematological features of 130 patients with large granular lymphocyte proliferations Lymphocyte c o u n d
Number of patients with
Study Anaemia"
Neurropenia'
Thrombocytopenia'
12121
511 1
0112
117 19/38 8/34 O/l 2
4/11 617 18/38 19/34 8/12
312I 117 2/l1 017 1/38 7/34 4/12
22/94 23%
391130 30%
13/130 56%
18/130 14%
13/21 517 6111 117 11/38' 14/34 5/12
18/21 617 9111 517 33/38 23/34 7112
9121 217 3/11 017 8/36
Observed totals: Percentages:
551130 42yo
lO1/130 :I8 % ~
> 15.0
4.1-15.0
6121 017
Newland et a/., 1984 McKenna et a/., 1985 Chan et al., 1986 Lauria et al., 1987 Loughran & Starkebaum, 1987 Semenzato et a/., 1987 Oshimi et a/., 1988
~~
0.54.0
nt
617
~~
Patient considered anaemic if haemoglobin < 13.5 gjdl (males) or < 11.5 g/dl (females). Patient considered neutropenic if absolute count
