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Hypothesis: Non-hodgkin lymphomas are abnormal immune responses

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Page 1: Hypothesis: Non-hodgkin lymphomas are abnormal immune responses

Cancer Immunol. Immunother. 7, 37-42 (1979)" ancer mmunol9gyand mmunotherapy

© Springer-Verlag 1979

Hypothesis: Non-Hodgkln Lymphomas Are Abnormal Immune Responses

J. A. Habeshaw

I. c. R. F. Gordon Hamilton Fairley Laboratories, St. Bartholomew's Hospital, West Smithfield, London EC 1A 7BE, England

Summary. Experimental evidence allows deduction of the degree of differentiation of lymphoid cells from the surface phenotype. When marker techniques are applied to tumours of lymphoid cells, the phenotypes of the par- ticipating cells indicate that the majority of NHL (non- Hodgkin lymphomas) are derived from reactive B lym- phocytes of germinal follicle, blast cell, or proplasma cell type. These findings indicate that NHLs are abnor- mal responses to persistent and as yet unidentified an- tigens, and show features more akin to immune deficien- cy states than 'true' neoplasms.

Introduction

Proponents of both the Lukes and Collin [27] and the Kiel [10] classifications of NHL have drawn attention to the cytological similarities between the normal cellu- lar components of the germinal centre and the proliferat- ing lymphocytes found in the majority of lymphoid tu- mours. Surface marker studies have confirmed that many lymphomas of B cells express C3d receptors, as do B lymphocytes present in the germinal follicles of reactive lymphoid tissue [43]. These apparently trivial facts raise important questions bearing on the aetiology and pathogenesis of NHL, namely, why so many lym- phomas are derived from germinal centre B lympho- cytes, and what the stimulus is that initiates the transfor- mation of a 'resting' B lymphocyte into a continuously proliferating clone? Surface marking techniques encour- age speculation about the mechanisms by which lym- phomas are produced, and could alter the current con- cepts of malignancy in lymphoid tissue.

In 1974 Salmon and Seligmann [41] proposed that lymphoid tumours were produced by an 'oncogenic event' occurring in a population of lymphocytes stimu- lated by anantigen. The oncogenic event produced mat- uration arrest of the reacting lymphocytes, and, by pre- venting terminal differentiation of reactive B cells into plasma cells (which would normally terminate cell pro-

liferation), allowed clonal expansion of the affected B cells. The resulting neoplasm, which involved the entire population of lymphocytes from one clone, would, it was predicted, express a single class (idiotype) of immu- noglobulin and would be composed largely of cells 'held up' at one stage of their differentiation.

Since 1974 a great amount of experimental work has resulted in a much better understanding of B and T cell differentiation in both mouse and man [23, 6, 31, 11, 18, 8, 44, 20, 4]. The identification of surface antigens ex- pressed by stem cells [11], B cells [18] , activated lym- phocytes [8], and plasma cells [44], together with the correlation of Fc and C3d receptors with the functional capacity of the developing B cell [1, 15, 40, 36, 16, 25], provides a phenotypic profile of the B cell at each stage of its differentiation and maturation in an immune re- sponse. Further evidence of B cell maturation can be deduced from sequential changes in the isotype of sur- face immunoglobulin heavy chain, in which switching fromM to M + D or D to M + G(+D) or M + A (+D) has been described with increasing maturity of the B cell [37, 45, 46]. These phenotypic correlates with B cell differentiation and maturation are shown in Table 1. This oversimplified representation can serve as a model, which can be used to assess the maturity of B lympho- cytes in human lymphomas. Most human lymphomas are derived from B cells with the follicular phenotye Slg + C3d +, which indicates an origin from the post- primary (follicular) phase of a secondary immune re- sponse. An understanding of the events concerned in follicular evolution is therefore essential to the interpre- tation of B-cell phenotype in lymphoma.

Antigen-dependent B Lymphocyte Differentiation

B lymphocytes exist in several forms following contact with antigen. Both B and T lymphocytes that have un- dergone activation (triggering) by antigen acquire a new surface antigen (in mouse ALA-I, activated lymphocyte

0340-7004/79/0007/0037/$ 01.20

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3 8 J . A . Habeshaw: Hypothesis: Non-Hodgkin Lymphomas Are Abnormal Immune Responses

Table 1. Correlation of surface phenotype with differentiation and maturation of the B lymphocyte

B cell subset Phenotypic features

ALL Ia Slg class Cylg Fcy Fc# C3d

Pre-B cell + +

Pre-primary response - - +

Primary immune response - + (IgM secretor)

Follicular 13 cell - + M + D -- - -

Post-follicular (memory B cell) -- + M + D - + --

Proplasma cell - + M + D _+ G -- - + G +A

Plasma cell - - - G or A -- -

Immature 13 cell - + + M -- + +

m M m _ _

M - + + +

M M - - -

+

+

m

P l a s m a c e l l

P l a s m a c e l l

/ ? ~ - X / ~ - - ' ~ ~ ~ ~ I ce l l ~ ~ / , , " ~ - ~ / ~ ' ~ ( y }~ t ~..-'j

I ~ Viregin[] ] c e c e l l s A - ~ / t

',, BONETMARROWI " x S t e m c e l l !

+ A g

M e m o r y c e l l

X \

I

I

I

I

/ /

)*Ag

A g I g G ( B )

C o m p l e x

Fig. 1. Events in the germinal centre, showing how competition for antigen between bone marrow-derived virgin B cells and germinal centre- derived plasma cells produces memory cells, with clonal restriction and amplification of the immune response. (Based on data from Nieuwen- huis and Keuning [34]). Explanatory notes: K, J, L, M, X, Y, Z, A, B, C represent virgin B cells (surface phenotype S I g + F c + C 3 + ) with differ- ing immunoglobulin idiotypes produced by a stochastic process from bone marrow stem cells. The cells K, J, meet an antigen Ag to which cell J responds by proliferation to produce antibody IgM(J), and J memory cells. The resulting primary follicle acquires virgin B ceils LM from mar- row. Since cell L has higher affinity for antigen Ag than cell J, the antigen remaining in the follicle triggers blastogenesis and mitosis in cell L, resulting in the production of L plasma cells and L memory cells. With T cell help, L plasma cells secrete IgG(L), which combines with avail- able antigen Ag to form AglgG(L)complex. The cycle then continues with cell Y and cell B producing progressively higher-affinity memory cells, and antibody. The virgin B cell clones K, M, X, Z, A, C, do not proliferate and are eliminated. J, L, Y, 13 memory cells circulate and survive. T cell help is envisaged as triggering conversion of a proplasma cell (SIg + C3 - Fc --) into a plasma cell in the presence of anti- gen. The whole sequence is dependent upon the presence of antigen, and its decreasing availability to cells of low affinity. Most human lympho- mas are derived from germinal centre B cells, blast B cells or proplasma cells, which implies that antigen plays an important role in sustaining the neoplastic process

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J. A. Habeshaw: Hypothesis: Non-Hodgkin Lymphomas Are Abnormal Immune Responses

antigen -I) [8]. Antigen-binding B cells (virgin B cells) from unimmunised mice are antigenically distinct from cells binding the same antigen in immunised mice (? memory B cells), and both are antigenially distinct from antibody-secreting B cells (PFC) and plasma cells [24]. Plasma cells express an antigen PEA-1 (plasma cell an- tigen 1), which is also expressed on PFC (plaque-form- ing cells) but not on virgin B cells. [44]. B cells forming germinal centres express C3d but not Fc receptors [43], and they also express Ia antigen (but not ALL or T cell antigens) [12]. Plasma cells produce cytoplasmic and secreted immunoglobulin, but do not usually express SIg, Fc, or C3 receptors [39]. Presumptive 'memory cells' arising in vitro after several cycles of mitogen-in- duced proliferation express surface Ig strongly, but lack intracytoplasmic Ig [48], showing that the plasma cell precursor and the memory cell belong to separate differ- entiation 'compartments,' a conclusion supported by the capacity of the memory B cell to recirculate [30] while the proplasma cell and plasma cell do not. The forma- tion of germinal centres is of major importance: the B cells in germinal centres have SIg and C3 but no Fc receptor, and from this population are produced memo- ry cells [3] and precursors of antibody-forming plasma cells [2]. The production of memory cells can be en- hanced by antigen-antibody complexes [22]. Only com- plement-fixing antibodies are capable of localising anti- gen to germinal centres [7], and the presence of comple- ment is also required for the localisation of cells to the germinal centres [47] and for the generation of B cell memory clones [21]. The proliferative and synthetic re- sponses of B cells derived from germinal centres (as in tonsil) to pokeweed mitogen do not occur in the absence of T cell help [ 17]. The production of a germinal centre in response to antigenic stimulation requires the con- junction of several variables (antigen-specific T cells, an- tigen, preformed antibody, primed B cells, and comple- men0 before the production of memory cells and plasma cell precursors from SIg + C3 + B cells can occur. The exact function of the germinal centres is not clear, but it has been suggested that they act as amplification centres for generation of antigen-reactive cells [34] and as re- gions where selection of high-affinity B cells occurs with deletion of clones with low-affinity antigen receptors, thus limiting the immune response to a few clones of B cells bearing high-affinity receptors for antigen [42]. Certainly memory ceU production is associated with ger- minal centre development in germ-free rats [3], and they are one of the major sites of B cell transformation and proliferation in normal circumstances [34]. The cell pop- ulation of the germinal centre is derived from bone mar- row precursors [34] and these give rise to B cells, which recirculate and populate the marginal zones of follicles in spleen and other sites. These marginal zone cells are probably memory B cells, and they do not appear to

39

form follicular structures themselves, Germinal centre reactions are summarised in Figure 1 and the accom- panying legend.

B-Cell Phenotype in Lymphoma

Materials and Methods

Lymph node biopsies from 174 patients with an established diagno- sis of NHL were examined. In 23 patients more than one biopsy was studied over an extended interval, repeat biopsy being necessary for assessment of incomplete remission or recurrence. Cell suspensions made from the affected nodes were studied for expression of ALL, Ia, and T cell-associated (HTLA) surface antigens by immunoflores- cence with specific antisera. Surface Ig expression was studied by means of specific anti-Ig light and heavy chain antisera on acetate buffer-(pH 5.5)-washed cells. Receptors for sheep erythrocyte (E), and for FcF (ox RBC + rabbit anti-ox RBC IgG) Fc# (ox RBC + rabbit anti-ox RBC IgM) and C3d (ox RBC + rabbit anti-ox IgM + human R3 reagent) were assessed concurrently. Cytoplasmic Ig was determined on ethanol-fixed cytocentrifuge preparations, spe- cific antisera against M, G, A, D, K, and L chains being used. A more detailed account of the techniques used is to be published else- where [14].

Results

Tumours of undifferentiated cells (lymphoblastic lym- phoma) accounted for 16 cases. The phenotypes were those of stem cells (ALL + Ia +) in four cases, and of HTLA-positive T cells in seven cases. Five cases of B lymphoblastic lymphoma expressed the phenotypes SIgM + (3 cases) or SIgM + CyIgM + (2 cases), con- firming an origin from B cells early in the maturation sequence (primary or pre-primary immune response). Fifty-four patients had lymphocytic lymphoma, often associated with chronic lymphocytic leukaemia, and 20 of these expressed SIg only (IgM or K light chain), 13 expressed SIgM with Fcy Fc# and C3d receptors, 8 expressed SIgM with Fc F and C3d receptors, 7 ex- pressed SIgM with C3d receptors, 4 expressed SIgM with Fcy and Fc# receptors, and 2 were SIg - Ia + "null" cell tumours. Most of these tumours ap- peared to arise from pre-primary immune response B lymphocytes.

Tumours derived from follicular cells (centroblastic, centrocytic, and centrocytic and centroblastic turnouts) were studied in 72 patients. In 29 cases the B cell pheno- type was SIg + C3d+. In addition, 14 cases showed a predominance of T cells in the lesion studied, associated with a monoclonal B cell component. In 25 cases, the B cells expresssed SIg only. Of the remaining four cases, two expressed the phenotype of null cells (SIg -- Ia +) and two SIg with Fc~; receptors. Immunoblastic and plasmablastic tumours were examined in 19 patients, 13

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40 J. A. Habeshaw: Hypothesis: Non-Hodgkin Lymphomas Are Abnormal Immune Responses

showing SIg only (often of M + G, or G class), three showing T cell predominance, two SIg - Cylg + phe- notype, and one SIg with Fc F receptor. Lymphoplas- macytoid tumours (in which mature lymphocytic components and plasma cells occur together) showed a follicular phenotype (SIg + C3d +) in 4 cases, SIg + phenotype (M + G or (3) in 3 cases, SIg with Fc F and C3d receptors in 13 cases, SIg with cytoplasmic Ig (2 cases), or T cell predominance with monoclonal B cells (1 case). The study ofphenotype in sequential biop- sies (23 cases) provided evidence for switching from IgM to IgG (5 cases) and for T cell predominance pre- ceding or succeeding a follicular B cell phenotype or conversion to an immunoblastic tumour (5 cases). Con- version of a follicular phenotype (SIg + C3d +) to an SIg +, or SIg + Cylg + phenotype was seen in three pa- tients. A summary of these data will be published else- where [ 13].

It therefore seems that most cases of N H L in man are derived from (a) primary or pre-primary immune response B lymphocytes (as in lymphocytic lymphoma, (b) germinal centre B cells, including a T cell-predomi- nant phase in the evolution of these tumours, or (c) post- follicular memory or proplasma cells, which may be blast-transformed (as in immunoblastic lymphoma) or quiescent (as in lymphoplasmacytoid lymphoma). Sur- face marking shows that in the majority of turnouts the 'neoplastic' B cell population is derived from a clone that is undergoing antigen-driven expansion in a follicu- lar or post-follicular immune reaction. There are other important indicators that reveal striking similarities be- tween the lymphoma lymphocytes and the normal course of an immune response. Early reactions to anti- gen in lymph nodes are characterised by T cell accumu- lation and division, which precedes B lymphoid hyper- plasia and antibody formation [5]. Certain aspects of isotype diversity, namely IgM-to-IgG switching, are also predictably seen in the evolution of certain classes of N H L [13, 14]. These findings indicate that NHLs show the features of an abnormal immune response [14]. Non-Hodgkin lymphomas are common neoplasms in certain immune-deficiency states [26], showing that there is no clear distinction between true lymphoid neo- plasms and extreme forms of immune dysfunction. Chromosomal abnormalities similar to those described in immune-deficiency states [28] also occur in lympho- ma cells (particularly aberrations of chromosome 14 [26, 29]. Monoclonality of the immunoglobulin ex- pressed by neoplastic B cells is generally regarded as being a marker of malignancy. I f the role of the germinal centre is to produce clonal restriction of responsive B cells during the antigen-driven clonal expansion of mem- ory cells and plasma cell precursors, a block to B cell maturation beyond this stage could result in failure to produce an effective secondary response, and produce a

perpetually expanding antigen-reactive clone. Similarly, failure of maturation of memory cells or plasma cell precursors might lead to accumulation of these cell types if antigen stimulation continues. The alternative view, that the stimulus to neoplastic B cell proliferation is 'intrinsic' and not 'antigenic' in nature, fails to account for the high frequency of secondary response-associated tumours, which require an intact IgM response to occur before they can develop, and for the observed patterns of T cell predominance and isotype diversity occurring in tumours of this class. Although much remains to be understood about the nature of stimuli provoking B cell division and differentiation, observations of NHLs have gone a long way to confirm the original hypothesis of Salmon and Seligmann [41]. It is now possible to extend this concept by proposing that NHLs represent abnor- mal immune responses in which the participating cells become frozen in an attitude of proliferation by failing to terminate the response in the normal way. I f this is true, functional studies of lymphoma cells may indicate deficiencies in T helper or T suppressor subsets [32, 33], the continued presence of the stimulating antigen [26], or underlying selective immune-deficiency disease [26, 33] as the true causes of this class of malignancy. It is to be hoped that further advances in the investigation of N H L will improve the therapeutic effectiveness and range of the currently inadequate treatments for these conditions.

Acknowledgements: The author would like to thank Dr. J. Malpas, ICRF Medical Oncology Unit, St. Bartholomew's Hospital, and M. F. Greaves, ICRF Membrane Immunology Group, Lincolns Inn Fields, London, for their helpful comments and suggestions. JAH is a Medical Research Council training fellow.

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Received November 16, 1978/Accepted June 1, 1979