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Cell-surface immunoglobulins in chronic lymphocytic leukemia and allied disorders
Cell-Surface lmmunoglobulins in Chronic Lymphocytic Leukemia and Allied Disorders
ALAN
C. AISENBERG,
KURT J. BLOCH, JOHN
C. LONG,
M.D.,
Ph.D.
M.D. M.D.
lmmunoglobulin on the surface of peripheral blood lymphocytes from 57 patients with chronic lymphocytic leukemia (CLL) and allied disorders was investigated by fluorescence microscopy and correlated with circulating immunoglobulin. In 38 of 48 patients with CLL, the predominant surface immunoglobulin identified on peripheral blood lymphocytes was M (IgM) of either kappa or lambda light chain type. In five patients, the predominant surface protein was immunoglobulin G (IgG) of either kappa or lambda type. In three others, the lymphocyte surface immunoglobulin could not be definitely identified and in two, no surface immunoglobulin was detected. Circulating immunoglobulin levels, particularly IgM, were depressed in the majority of patients with CLL. In three subjects with IgM-bearing lymphocytes, the serum contained a circulating IgM M component and
Boston, Massachusetts
three of the five subjects with IgG-bearing cells, had a circulating IgG M component. In three patients with CLL, immunoglobulin disappeared from the cell surface with progression of the disorder, although neoplastic cells remained in the circulation. The amount of immunoglobulin on the surface of cells from patients with chronic lymphosarcoma cell leukemia was much greater than that on cells from patients with CLL, and the surface immunoglobulin pattern in hairy cell leukemia also appeared distinctive. Study of immunoglobulin on the surface of
From the John Collins Warren Laboratories, Huntington Memorial Hospital, Harvard University, and the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformity, Massachusetts General Hospital, Boston, Massachusetts 02114. This work was supported by research grants from the Massachusetts Division of the American Cancer Society, the Damon Runyon Memorial Fund for Cancer Research, the Massachusetts Chapter of the Arthritis Foundation, the L.H. Bendit Foundation, and the U. S. Public Health Service (GM 02212, AM 3564 and AM 5067). Requests for reprints should be addressed to Dr. Alan C. Aisenberg, Huntington Laboratories, Massachusetts General Hospital. Boston, Massachusetts 02114. Manuscript accepted March 20, 1973.
184
August 1973
lymphocytes has helped to define the cellular origin and monoclonal nature of CLL, the source of circulating M components in this disease, and the relationship of CLL to other lymphoproliferative disorders. Although technically demanding, the study of surface immunoglobulin should prove useful in clinical medicine. In the past several years investigations in experimental animals have established the existence of two classes of small lymphocytes [l], B cells of bone marrow origin, which have not been processed in the thymus, and T cells, also of bone marrow derivation, which have been thymus-processed. The two classes of lymphocytes can be distinguished by several characteristics. Thus B cells bear surface-bound immunoglobulin, which is demonstrable by immunofluorescence [2-41, and receptors for complement [5] and antibody [6], whereas T cells do not, and B cells are more adherent than T cells to Nylon@ fibers [7]. T cells can also be distinguished from B cells by the presence of thymus-specific surface markers on the former [2] and by their
The American Journal of Medicine
Volume 55
CELL-SURFACE
TABLE
Cell Surface and Serum lmmunoglobulin in Chronic Lymphocytic Leukemia
I
IMMUNOGLOBULINS
(lg)
CellSurfacb Ig*
Case No. G
M
‘K
x
P
IgG Surface 1 87 2 38 3 89 4 84 523
0 6 0
0 4
98 77 80 30 5
3 19 4 9 29
94 75 90 46 35
IgM Surface 6
5
54
61
7 8
6 5 7 3 1 4 3 6 4 14 6 4
82 87 92 90 88 40 48 96 65 60 71 67
79 94 93 84 83 41 57 84 95 70 21 77
11 20 8 9 6 2 3 3 9 9 5 8 12
19
1
89
90
10
93
20 21 22 23 24 25 26 27 28
0 0 0 8 0 2 6 5 12 8 7 4 6 6 6 8 0 2 1 2 19 11 1 0
69 79 93 26 85 73 88 84 57 74 63 83 60 80 63 60 77 949 22 32 47 35 950 92s
68 74 79 23 94 82 12 6 7 25 4 16 7 12 9 9 1 7 2 2 28 1 1 4
4 9 12 3 0 2 59 75 85 80 57 83 58 82 57 90 47 90s 44 16 80 46 974 95s
65 51 88 43 96 77 86 81 83 89 70 79 57 88 75 90 77 939 20 44 60 55 964 915
9
10 11 12 13 14 15 16 17 18
29
30 31 32 33 34 35 36 37 38 39 40
41 42 43
Surface
88 77 ... ... 95 86 81 49 86 95 86 50 71
Interpretation
Determinants GK GK GK GK GX
1,200t 9OOt 1,120 440 8,OOOt
70 48 128 60 140
110
105 60 ... ... <20 200 20 41 51 115 51 51 370 48 <50 <50 220 140 <60 <61 44 20 20 78 84 115 <20 <20 60 <20 <20 74 120 <50 66 <50 350 204
58 55 ... ... 32 27 19 76 38 27 60 215$ 200 18 18 15 80 52 <20 <20 190$ 1653 18 12
28 27 72 32
Determinants
760 900 ... ... 430 900 550 660 550 400 550 660 560 500 920 180 950 560 1,000 680 500 120 120 265 660 710 190 100 550 430 200 1,000 440 600 560 1,000 1,500 860
MK
MK MK MK MK MK MK MK MK MK MK MK? MK MK MK MK MK MK MK MK MX MX MX MX MX MX MX MX MX MX MX MX MX MX MX MX MX MX
lg Incompletely
44 634 390 739 125 73 45 0 4 2 32 43 460 0 0 0 48
SerumIg* _______ G A M
Characterized
IgK ?type Igx ?type Ig ?type
1,000 420 420
170 39 <61 <20 <50 <15
1,300 400
230 <50
No Surface lg 47 48
0 0
0 0
2 9
2 0
10 5
No Ig Nolg
16 0
IN LYMPHOCYTIC
LEUKEMIA-AISENBERG
ET AL.
ability to form rosettes with sheep erythrocytes [8]. These methods are applicable to the study of human lymphocytes, and this application has led to increased understanding of neoplastic lymphocytes of man. A number of investigators [g-12] have described immunoglobulin of M (IgM) specificity on the surface of cells from patients with chronic lymphocytic leukemia (CLL), a finding which has been used to support the B cell origin of these neoplastic lymphocytes. Our report is based on studies of 57 patients with CLL and allied disorders. These observations permit certain generalizations to be made about the amount and type of cell surface immunoglobulin in CLL, the relationship of surface immunoglobulin to circulating im*munogRrbulin and M components, and the relationship of CLL to lymphosarcoma cell leukemia,
hairy cell leukemia PATIENTS
AND
and Mycosis fungoides.
METHODS
The diagnosis of CLL, chronic lymphosarcoma cell leukemia [13], acute lymphosarcoma cell leukemia [13], hairy cell leukemia [14] and Mycosis fungoides [15] was based on accepted clinical, laboratory and pathologic criteria, including examination of the Wright’sstained peripheral blood smear. With the single exception noted in the text, consecutive patients with these disorders and lymphocyte counts above 10,000/mm3 were studied; leukemic blood with lymphocyte counts below 10,000/mm3 was not used to avoid contamination of lymphocyte preparations with non-neoplastic cells. Serum concentrations of immunoglobulin G (IgG), A (IgA) and M (IgM) were measured with “immunoplates” Calif.),
and
electrophoresis Living blood
(Hyland each
serum
Laboratories, was
and agarobe-gel
lymphocytes
were
by the Ficoll-Hypaque
examined
Los
Angeles,
by
immuno-
electrophoresis
prepared gradient
from
[ll].
defibrinated
method
previous-
* Surface lg results are expressed as the per cent of lymphocytes staining with fluorescein-conjugated antiserums specific for G heavy chain, M heavy chain, K (kappa) light chain andX(lambda)lightchain of human lg. Pisa POlyValent antiserum directed against both light chains, and the heavy chains of IgG, IgA and IgM. Range for 10 normal subjects is G, 2 to 6 per cent; M, 7 to 12 per cent; kappa, 5 to 15 per cent: lambda 4 to 7 per cent; and P, 8 to 17 per cent. Serum lg is expressed in mg/lOO ml. Range in 50 normal donors is IgG, 720 to 1,500; IgA, 90 to 325; and IgM, 45 to 150. f Low concentration of IgG M component detected by immunoelectrophoresis. $ Low concentration of IgM M component detected by immunoelectrophoresis. Q Intense surface staining. More than 50 per cent of stained cells continue to fluoresce when the light intensity is reduced to 10 per cent by the interposition of a neutral filter in the optical system.
August 1973
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185
CELL-SURFACE
TABLE
I;ase No. 1
2 3 4 5 6 7 8 9
II
IMMUNOGLOBULINS
IN LYMPHOCYTIC
LEUKEMIA-AISENBERG
ET AL.
Cell Surface and Serum lmmunoglobulin (lg) in Chronic Lymphosarcoma Cell Leukemia (CLSL), Acute Lymphosarcoma Cell Leukemia (ALSL), Hairy Cell Leukemia (HCL) and Mycosis Fungoides (MF) .____.. __ ~~~~ Cell-Surface
~~~_._ Diagnosis
CLSL CLSL CLSL CLSL CLSL ALSL HCL HCL MF
i-
M
5 0 9 7 17 41 76 0 0
915 950 875 815 15 0 0 12 0
K
715 895 8 5 58 2 0 73 0
~_
Ig*
Serum Ig* P
A
20 24 954 949 7 66 80 3 0
73§ 98s 91§ 90§ 539 68 63 78 1
Interpretation MK MU MX MX IgK ?type GA Gh ?DKI. No Ig
G 920 1,500 660 900 1,400 860 1,400 1,960 1,270
-A 84 105 96 78 430 140 190 394 286
M 37 150: 2151 100 64 42 64 110 98
*$Q See footnotes for Table I. t See text. ly described [li]. Lymphocyte purity (assessed with Wright’s stain) and viability (assessed with trypan blue) of all preparations used exceeded 98 per cent, erythrocytes having been removed by osmotic shock with ammonium chloride [l I]. The lymphocytes were incubated for 1 hour at 37°C with fluorescein-conjugated antiserums, washed three times, suspended in buffered glycerine and examined with a Zeiss ultraviolet microscope equipped with a mercury arc lamp and an interference primary filter. A minimum of 200 lymphocytes were examined with each fluor and the number of fluorescent cells recorded; thereafter a 10 per cent transmitting neutral filter was interposed in the optical system and an additional 200 cells were examined. Fluorescein-conjugated goat antiserums specific for each heavy chain class (IgG, IgA, IgM, IgD and IgE) and for kappa and lambda light chains were obtained (Meloy Laboratories, Springfield, Va.), and used routinely without dilution. A polyvalent antiserum to the heavy chains of IgG, IgA and IgM, and kappa and lambda light chains was obtained from the same source. The specificity of the fluorescent reagents, a critical problem in all fluorescence microscopy, was controlled by appropriate inhibition with unlabeled IgG (Schwarz/Mann, Orangeburg, N. Y.), IgA of kappa type [ll], IgM of kappa type [ll] and Bence Jones proteins of kappa and lambda types (Meloy Laboratories). In the course of these investigations we found that the fluorescein-conjugated anti-IgG, anti-IgA and anti-IgD reagents had some anti-kappa and antilambda activity (as do almost all commercial antiserums [16]): this activity could usually be neutralized by the addition of the appropriate unlabeled light chain proteins. Although the addition of unlabeled proteins did not give rise to visible precipitation, it may be presumed that soluble antigen-antibody complex formation did occur. This process did not interfere with the fluorescence method, i.e., there was no increase in surface immunoglobulin attributable to complexes under the conditions employed. Ambiguous findings, i.e., staining with both light chain reagents or staining with more than one heavy chain reagent, were occa-
188
August 1973
The American Journal of Medicine
sionally observed, particularly with the brilliantly staining lymphosarcoma cells. These ambiguities were readily resolved, with two exceptions (Case 44, Table I and Case 5, Table II), by one of the following manipulations: retesting the cells after standing overnight at 4°C [12]; retesting the cells after incubation in 0.25 per cent trypsin [12] in isotonic saline solution (Microbiological Associates, Bethesda, Maryland) for 15 minutes at 37°C followed by washing twice with isotonic saline solution; or retesting the cells with a 1 : 10 dilution of the fluorescent antiserums. In CLL, it was difficult to classify the surface immunoglobulin on lymphocytes unless at least 15 per cent of cells stained with a given fluorescent conjugate. If fewer cells were stained, then the determination could frequently not be reproduced in a subsequent test. A cell was considered to show positive staining if three or more fluorescent aggregates were present on the surface. In the present experiments, all cells were tested with an antiserum to the heavy chain of IgA, the results obtained were not included in Tables I and II because the percentage of cells stained did not exceed 10 per cent in any case tested. Thymus-specific surface antigens were evaluated with the globulin fraction of a rabbit antiserum to human fetal thymus prepared as previously described [17]. This globulin fraction was absorbed with A-positive erythrocytes and with chronic lymphocytic leukemia cells. The nonfluorescent antithymus globulin was reacted with viable lymphocytes, and a fluoresceinconjugated goat antirabbit globulin antiserum and the fluorescence microscope were used to demonstrate fixation of the globulin to the cell surface (i.e., the indirect or sandwich technic was used). RESULTS Surface immunoglobulins are demonstrable on only a small fraction of lymphocytes obtained from the peripheral blood of healthy control subjects. In a series of 10 cases, 2 to 6 per cent reacted with the fluorescein-conjugated antiserum specific for the heavy chain of IgG, 0 to 3 per
Volume 55
CELL-SURFACE
lMMUNOGLOl3ULlNS
cent with anti-IgA, 7 to 12 per cent with anti-IgM, 5 to 16 per cent with anti-kappa, 4 to 7 per cent with anti-lambda and 8 to 17 per cent with the polyvaient anti-immunoglobulin antiserum [ll]. The major fraction of positive normal cells shows punctate fluorescence in a granular or aggregated pattern on the surface of the cell, a minor population shows crescents of fluorescent material [18]. The cells from most patients with CLL showed surface immunoglobulin in the granular pattern (Figure 1). Of the 48 patients with CLL studied (Tables I and I I I), it was possible to assign 38 to the IgM group since the lymphocytes reacted predominantly with the reagent specific for the heavy chain of IgM, and with either the reagent for the kappa or lambda light chain. The lymphocytes from five patients reacted predominantly with the reagent specific for the heavy chain of IgG; of these, four reacted predominantly with anti-kappa and one with anti-lambda reagent. The surface immunoglobulin on the cells of the remaining five patients with CLL could not be completely characterized. Cells from one patient (Case 44) reacted with both anti-IgG and anti-IgM reagents and could not be restudied after the trypsin-wash technic, which helps resolve ambiguous results, became available. The surface immunoglobulin of cells from a second patient (Case 45) reacted with the polyvalent antiserum and the anti-lambda reagent, and those from a third patient (Case 46) which reacted only with the polyvalent reagent, were not definitely identified, perhaps because of the insensitivity of certain fluorescent antiserums. Alternatively, lymphocytes from the second patient (Case 45) may only display the light chain of immunoglobulin; similar observations have been reported by Grey et al. [9]. We cannot state wheth-
TABLE
III
Summary
of Findings in Chronic.Lymphocytic
IN LYMPHOCYTIC
LEUKEMIA-AISENBERG
ET AL.
Figure 1. Case 43. Appearance of chronic lymphocytic leukemia cells stained with fluorescein-conjugates anti-lambda antiserum. Original magnification X 400.
er cells from two of our patients (Cases 47 and 48), which reacted with none of the antiserums, in fact bore no surface immunoglobulin or also represent failure of the antiserums to detect small amounts of immunoglobulin. It should be noted that cells from these last five patients also failed for the heavy to react with antiserums specific the machains of IgA, IgD and IgE. Furthermore, jority
Leukemia
of
lymphocytes
and Allied
from
these
two
patients
Disorders
Typeof SurfaceIg Total No.
Chronic lymphocytic leukemia Intense surface staining Circulating M component Chronic lymphosarcoma cell leukemia Intense surface staining Circulating M component Acute lymphosarcoma cell leukemia Intense surface staining Circulating M component Hairy cell leukemia Intense surface staining Circulating M component
Mycosisfungoides
48 4 6 5 5 2 1 0 0 2 cl 0 1
(K !r X) 38 3
G (K or X)
0
lg (7 type)
No Ig
2 (W’U 0
0
3 1 0 1 1 0 0
0
0
1 (?lgD)
0
0
0 0 0
1
3 (MW 4 4
0
August 1973
5 0 3UgG) 0
A
0
The American Journal 01 Medicine
2 0 0 0
Volume 55
0
187
CELL-SURFACE
TABLE
IV
IMMUNOGLOBULINS
IN LYMPHOCYTIC
Loss of Cell Surface
lmmunoglobulin
Patients with Chronic Lymphocytic ~__.~ _
Case No.* 27 29
31
M
U/24/71 3129172 11/08/71 11124171 2101172 5103172 6112172 12/03/71 4/06/72 7107172
K
(lg) in
Leukemia
x
P
Globulint
84
6
75
81
._.
...
...
...
74 85 89 ... 2 83 ... ...
25 23 11 4 0 16 ... ...
49 80 80 0 0 83 ... I. .
11 89 ... ... 38 0 79 2 5
11 ... ... ... ... 11 ... 2 4
47 and globulin
48) did not react with an antithyor form rosettes with sheep eryth-
ponents, the serum concentration of IgG was reduced (<720 mg/dl) in 33 of 43 patients and in the low normal range (720 to 1,000 mg/dl) in 7 of the remaining 10 patients. The concentration of IgA was decreased (<90 mg/dl) in 33 of 46 patients tested. Three patients had IgM M components in low concentration; of the remainder, serum IgM was decreased (<45 mg/dl) in 32 of 43 and in the low normal range (45 to 80 mg/dl) in 8 of the remaining 11. It is of interest that serum M components of IgG heavy chain were observed in 3 of 5 patients with CLL and surface IgG, but M components of IgM heavy chain specificity were found in only 3 of the 38 with surface IgM. In chronic lymphosarcoma cell leukemia (Tables I I and II I), the nature of the surface immunoglobulin was established as IgM (type kappa or type lambda) in four of the five patients studied. In one patient (Case 5) the light chain was of the kappa type, but we were not able to retest this patient’s cells to resolve the heavy chain type. The cells in these five patients were notable for the brilliance of the surface fluorescence. Thus, more than 50 per cent of lymphosarcoma cells continued to fluoresce after a neutral filter, which re-
August
1973
The American Journal of Medicine
the
incidence in the
light
light path.
to 10 per cent, In contrast,
was only
in4 of
the 48 patients with CLL showed fluorescence of comparable brilliance. Two of the patients with chronic lymphosarcoma cell leukemia had a circulating IgM M component. In general, the levels of circulating immunoglobulin were higher in pa-
The single cell leukemia
rocytes [ 191, properties usually associated with T cells [8,19]. Hence these patients are hot considered to have a T cell variant of CLL. Circulating immunoglobulin levels were depressed in most patients with CLL. Excluding consideration of the three patients with IgG M com-
188
ET AL
tients with chronic lymphosarcoma than in patients with CLL.
* See Table I. f Results are expressed as per cent of cells staining with a l/50 dilution of an antihuman thymocyte globulin prepared in rabbit followed by fluorescein-conjugated goat antirabbit antiserum. From 85 to 92 per cent of normal circulating lymphocytes stain with this reagent.
(Cases mocyte
duced terposed
Antithymocyte
Cell-Surface Ig* Date
LEUKEMIA-AISENBERG
patient with acute was found to have
cell
leukemia
lymphosarcoma lymphocyte sur-
face IgG of lambda type (not of the brilliance seen in chronic lymphosarcoma cell leukemia), as was one of the patients with hairy cell leukemia. The lymphocytes of a second patient with hairy cell leukemia reacted with an anti-kappa antiserum, and 51 per cent of these cells reacted with an anti-IgD heavy chain reagent. The staining with the anti-IgD reagent was not inhibited by unlabeled kappa light chain, but the surface immunoglobulin was only tentatively assigned to the IgD class, since unlabeled IgD protein was not available for specific inhibition. Circulating immunoglobulin levels were normal in the two patients with hairy cell leukemia. No cell surface immunoglobulin was detected on the lymphocytes of the single patient with Mycosis fungoides who had 34,000 circulating lymphocytes/mm3. In the eight instances in which circulating M components were detected by immunoelectrophoresis, the heavy chain of the M component corresponded to the predominant heavy chain identified on the surface of neoplastic cells. In four patients, the light chain type of the serum M component was also identified and found to correspond to the predominant light chain type identified on the surface of the patients’ lymphocytes. In the course of these studies it was noted that in several patients cell surface immunoglobulin was easily demonstrated on initial study, but was not detected in subsequent tests. Table IV documents this observation in three subjects. In each, the loss of surface immunoglobulin was associated with an unfavorable evolution of the disorder which required chemotherapy (two of the three patients died within 3 months of the final study listed in Table IV), but in each case there were neoplastic cells (above abundant circulating 20,000/mm3) at the time of study. The non-immunoglobulin bearing cells in these patients were considered to be abnormal because they resembled CLL cells (and contrasted with 85 to 92 per cent of normal lymphocytes [li’] in their failure to react with antithymocyte globulin. Five patients with CLL whose lymphocyte
Volume 55
CELL-SURFACE
IMMUNOGLOBULINS
counts were less than 5,000/mm3 were studied. In two of these, identification of the surface immunoglobulin on lymphocytes was difficult, most likely because of the presence of a significant number of normal B cells. In three cases, no surface immunoglobulin was detected. These patients had been extensively treated with chemotherapy, and the appearance of “null” cells may reflect a phenomenon similar to that cited. COMMENTS Until the recent introduction of methods which permit studies of the lymphocyte cell surface, medical investigators showed little interest in CLL. However, with the new technics available, there has been a revival of interest in this “monotonous proliferation of small lymphocytes,” and progress has been made in understanding the disorder and its relationship to other types of lymphoproliferative disease. On the basis of the work we have presented and that of other investigators [9,10,12], it is evident that lymphocytes from about 95 per cent of patients with CLL bear quantities of surface immunoglobulin which can be detected by immunofluorescence. The clonal character of this neoplastic proliferation is established by the finding of predominantly one type of immunoglobulin heavy chain and one type of light chain on the neoplastic lymphocytes of each patient, and is further supported by the demonstration of restriction of the surface immunoglobulin to one IgG subclass and one Gm allotype in the study of Froland and Natvig [20]. In the present series, patients whose cells bear IgM exceed those whose cells bear IgG by a ratio of 9:l. The five patients with CLL whose cells showed IgG rather than IgM heavy chains are of special interest; in three instances an M component of the same heavy and light chain type was found in the serum. In contrast, IgM M components, in small amounts, were found in only 3 of 38 patients with CLL whose lymphocytes bore surface IgM molecules. These serum M components (which have been described by earlier investigators [21]) most likely represent the secretion of immunoglobulin, similar to that displayed on the surface of the neoplastic cells, by at least some members of the same neoplastic clone. The low levels of circulating .immunoglobulin found in most patients with CLL has been the subject of earlier studies [22], but the very low concentration of serum IgM found in the majority of patients in the present series, has not received much attention. Clearly, the formation of IgM, as well as that of the other immunoglobulins, is cur-
IN LYMPHOCYTIC
LEUKEMIA-AISENBERG
ET AL.
tailed in CLL, perhaps through a displacement of normal precursor cells by the effete neoplastic clone. Cells from patients with chronic lymphosarcoma cell leukemia resemble CLL cells in that both contain surface immunoglobulin; cells from the former group tend to show surface immunoglobulin in considerably greater concentration. The single patient with acute lymphosarcoma cell leukemia and the two with hairy cell leukemia examined do not allow generalizations, but the pattern of serum and surface immunoglobulin observed in both disorders differed from CLL and chronic lymphosarcoma cell leukemia. The cells from the patient with acute lymphosarcoma cell leukemia and from one of the patients with hairy cell leukemia bore IgG (the cells of the other patient with hairy cell leukemia appeared to bear IgD), but unlike the IgG-bearing patients with CLL, this surface IgG was not associated with circulating IgG M components. It is reassuring that the surface immunoglobulin data substantiate the separation into clinical compartments which the hematologist has based on Wright’s stain morphology and clinical course. A number of findings support the contention that CLL is a disorder of B lymphocytes. Thus, in common with normal B cells, and in contrast to normal T cells, CLL lymphocytes bear demonstrable surface immunoglobulin [9-l 21. Furthermore, as discussed, immunoglobulin levels (a B cell function) are reduced in CLL, and other investigators [23,24] have characterized the immune impairment of CLL as predominantly involving antibody formation (a B cell function). Elsewhere we have reported that CLL cells lack a thymus-specific antigen [17], and studies in progress [19] indicate that CLL cells resemble B lymphocytes in their inability to form rosettes with sheep erythlrocytes and in their adherence to Nylon@ fiber columns. In addition, others have shown that CLL cells, in common with B lymphocytes, possess complement receptors [25,26]. The sluggish reaction of CLL cells to phytohemagglutinin [27] may be adduced as further evidence for their B cell nature. However, since both T and B cells are presumed to arise from a common bone marrow stem cell [28], the possibility that CLL cells arise from T lymphocytes by neoplastic derepression cannot be completely excluded despite the many similarities between the CLL lymphocyte and the normal B cell. At present it seems best to investigate the neoplastic disorders of lymphocytes with a variety of technics and to delay firm assignment into B or T
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199
CELL-SURFACE IMMUNOGLOBULINS
cell
categories.
that there
Indeed,
are multiple
IN LYMPHOCYTIC LEUKEMIA-AISENBERG
recent subspecies
evidence
suggests
of normal
tion
T and
B lymphocytes [29,30]. In this connection, it should again be noted that chronic lymphosarcoma leukemia cells bear more surface immunoglobulin than do CLL cells, hence it is possible that the lymphosarcoma cell belongs to a different subspecies of B cells. Furthermore, the cells in hairy cell leukemia may represent an additional subspecies, and perhaps even CLL cells should be divided into IgM and IgG subtypes. It seems appropriate to point out here the relative rarity of cases of CLL bearing surface IgA; only a single case appears to have been reported [12]. At present the experimental data are complex, but they promise eventually to contribute to the understanding and classification of both lymphoid neoplasms and the cellular basis of normal immune function. The limitations of the technic of surface immunoglobulin study should not be overlooked. Thus we could not detect immunoglobulin on the surface of the cells from about 5 per cent of our patients with CLL, either because the ceils were devoid of these proteins or because the methods used were not sufficiently sensitive. Furthermore, disease progression or associated vigorous antitumor therapy may result in the depletion of surface immunoglobulin in patients whose cells previously bore ample surface immunoglobulin. These patients had large numbers of neoplastic cells in their circulation. Whether the cells observed possessed a nonfunctional mechanism for producing surface immunoglobulin or represented the evolu-
clone
ET AL
or selection cannot
of a nonimmunoglobulin-forming
yet
be distinguished.
It is also
ap-
parent that the surface immunoglobulin data become increasingly difficult to interpret as the lymphocyte count falls and the neoplastic cells become a minor population immersed among the normal lymphocytes. Another potential source of difficulty, which does not need elaboration here, stems from the fluorescent antibody method itself and the potential nonspecificity of the antiserums employed. Provided that allowances are made for the various technical limitations cited, the study of cell surface immunoglobulins provides an approach to CLL and other lymphoproliferative disorders which is similar to the approach to multiple myeloma afforded by the study of serum M components. In the patient with lymphocytosis, the demonstration of surface immunoglobulin of a single light and a single heavy chain type suggests a clonal, and most likely a neoplastic, proliferation. The possibility that a cellular equivalent of benign monoclonal gammopathy exists remains to be shown. Once the technical limitations have been recognized, lymphocyte surface immunoglobulins can be determined with speed and reliability: the technic should
be useful
in clinical
medicine.
ACKNOWLEDGMENT We are indebted to Doctors Zareh Demerjian, Allan Sandler, Robert Carey, Bernard Jacobson, Leonard Ellman and Richard Stein for referring patients to us for study, and to Mrs. Barbara Wilkes for excellent technical assistance.
REFERENCES 1. 2.
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August 1973
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CELL-SURFACE
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IMMUNOGLOBULINS
acterization of circulating mononuclear cells. Amer J Med 47: 149, 1969. Epstein EH, Levin DL, Croft JD, et al.: Mycosis fungoides. Survival, prognostic features, response to therapy, and autopsy findings. Medicine 51: 61, 1972. Reimer CB, Phillips DJ, Maddison SE, et al.: Comparative evaluation of commercial precipitating antisera against human IgM and IgG. J Lab Clin Med 76: 949, 1970. Aisenberg AC, Bloch KJ, Long JC, et al.: Reaction of normal human lymphocytes and chronic lymphocytic leukemia cells with an anti-thymocyte antiserum. Blood 41: 417, 1973. Froland SS, Natvig JB: Surface-bound immunoglobulin on lymphocytes from normal and immunodeficient humans. Stand J lmmunol 1: 1,1972. Aisenberg AC, Long JL, Wilkes B: Chronic lymphocytic leukemia cells: rosette formation and adherence to Nylon fiber columns. In preparation. Froland SS, Natvig JB: Class, subclass and allelic exclusion of membrane-bound lg of human B lymphocytes. J Exp Med 136: 409,1972. Moore DF, Migliore PJ, Shellenberger CC, et al.: Monoclonal macroglobulinemia in malignant lymphoma. Ann Intern Med 72: 43, 1970. Miller DG: Immunological disturbances in lymphoma and leukemia, Immunological Diseases, vol I, 2nd ed
23.
24.
25.
26.
27.
28.
29.
30.
August 1973
IN LYMPHOCYTIC
LEUKEMIA-AISENBERG
ET AL.
(Samter M, ed), Boston, Little, Brown & Co.. 1971, p 548. Cone L, Uhr JW: Immunological deficiency disorders associated with chronic lymphocytic leukemia and multiple myeloma. J Clin Invest 43: 2241, 1964. Block JB, Haynes HA, Thompson WL, et al.: Delayed hypersensitivity in chronic lymphocytic leukemia. J Nat Cancer lnst 42: 973,1969. Shevach EM, Herberman R, Frank MM, et al.: Receptors for complement and immunoglobulins on human leukemic cells and human lymphoblastoid cell lines. J Clin Invest 51: 1933, 1972. Pincus S, Bianco C, Nussenzweig V: Increased proportion of complement-receptor lymphocytes in the peripheral blood of patients with chronic lymphocytic leukemia. Blood 40: 303, 1972. Rubin AD: In vitro evaluation of lymphocyte proliferation in lymphoproliferative disorders, Proceedings of the Fifth Leukocyte Culture Conference (Harris JE, ed), New York, Academic Press, 1970, p 239. Yoffey JM, Courtice FC: Lymphatics, Lymph and the Lymphomyeloid Complex, New York, Academic Press, 1970, p 761. Raff MC: Surface antigenic markers for distinguishing T and B lymphocytes in mice. Transplant Rev 6: 52, 1971. Good RA: Heterogeneity of lymphocyte populations. Rev Europ Etud Clin Biol 17: 11, 1972.
The American Journal of Medicine
Volume 55
191
ALAN
C. AISENBERG,
KURT J. BLOCH, JOHN
C. LONG,
M.D.,
Ph.D.
M.D. M.D.
lmmunoglobulin on the surface of peripheral blood lymphocytes from 57 patients with chronic lymphocytic leukemia (CLL) and allied disorders was investigated by fluorescence microscopy and correlated with circulating immunoglobulin. In 38 of 48 patients with CLL, the predominant surface immunoglobulin identified on peripheral blood lymphocytes was M (IgM) of either kappa or lambda light chain type. In five patients, the predominant surface protein was immunoglobulin G (IgG) of either kappa or lambda type. In three others, the lymphocyte surface immunoglobulin could not be definitely identified and in two, no surface immunoglobulin was detected. Circulating immunoglobulin levels, particularly IgM, were depressed in the majority of patients with CLL. In three subjects with IgM-bearing lymphocytes, the serum contained a circulating IgM M component and
Boston, Massachusetts
three of the five subjects with IgG-bearing cells, had a circulating IgG M component. In three patients with CLL, immunoglobulin disappeared from the cell surface with progression of the disorder, although neoplastic cells remained in the circulation. The amount of immunoglobulin on the surface of cells from patients with chronic lymphosarcoma cell leukemia was much greater than that on cells from patients with CLL, and the surface immunoglobulin pattern in hairy cell leukemia also appeared distinctive. Study of immunoglobulin on the surface of
From the John Collins Warren Laboratories, Huntington Memorial Hospital, Harvard University, and the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformity, Massachusetts General Hospital, Boston, Massachusetts 02114. This work was supported by research grants from the Massachusetts Division of the American Cancer Society, the Damon Runyon Memorial Fund for Cancer Research, the Massachusetts Chapter of the Arthritis Foundation, the L.H. Bendit Foundation, and the U. S. Public Health Service (GM 02212, AM 3564 and AM 5067). Requests for reprints should be addressed to Dr. Alan C. Aisenberg, Huntington Laboratories, Massachusetts General Hospital. Boston, Massachusetts 02114. Manuscript accepted March 20, 1973.
184
August 1973
lymphocytes has helped to define the cellular origin and monoclonal nature of CLL, the source of circulating M components in this disease, and the relationship of CLL to other lymphoproliferative disorders. Although technically demanding, the study of surface immunoglobulin should prove useful in clinical medicine. In the past several years investigations in experimental animals have established the existence of two classes of small lymphocytes [l], B cells of bone marrow origin, which have not been processed in the thymus, and T cells, also of bone marrow derivation, which have been thymus-processed. The two classes of lymphocytes can be distinguished by several characteristics. Thus B cells bear surface-bound immunoglobulin, which is demonstrable by immunofluorescence [2-41, and receptors for complement [5] and antibody [6], whereas T cells do not, and B cells are more adherent than T cells to Nylon@ fibers [7]. T cells can also be distinguished from B cells by the presence of thymus-specific surface markers on the former [2] and by their
The American Journal of Medicine
Volume 55
CELL-SURFACE
TABLE
Cell Surface and Serum lmmunoglobulin in Chronic Lymphocytic Leukemia
I
IMMUNOGLOBULINS
(lg)
CellSurfacb Ig*
Case No. G
M
‘K
x
P
IgG Surface 1 87 2 38 3 89 4 84 523
0 6 0
0 4
98 77 80 30 5
3 19 4 9 29
94 75 90 46 35
IgM Surface 6
5
54
61
7 8
6 5 7 3 1 4 3 6 4 14 6 4
82 87 92 90 88 40 48 96 65 60 71 67
79 94 93 84 83 41 57 84 95 70 21 77
11 20 8 9 6 2 3 3 9 9 5 8 12
19
1
89
90
10
93
20 21 22 23 24 25 26 27 28
0 0 0 8 0 2 6 5 12 8 7 4 6 6 6 8 0 2 1 2 19 11 1 0
69 79 93 26 85 73 88 84 57 74 63 83 60 80 63 60 77 949 22 32 47 35 950 92s
68 74 79 23 94 82 12 6 7 25 4 16 7 12 9 9 1 7 2 2 28 1 1 4
4 9 12 3 0 2 59 75 85 80 57 83 58 82 57 90 47 90s 44 16 80 46 974 95s
65 51 88 43 96 77 86 81 83 89 70 79 57 88 75 90 77 939 20 44 60 55 964 915
9
10 11 12 13 14 15 16 17 18
29
30 31 32 33 34 35 36 37 38 39 40
41 42 43
Surface
88 77 ... ... 95 86 81 49 86 95 86 50 71
Interpretation
Determinants GK GK GK GK GX
1,200t 9OOt 1,120 440 8,OOOt
70 48 128 60 140
110
105 60 ... ... <20 200 20 41 51 115 51 51 370 48 <50 <50 220 140 <60 <61 44 20 20 78 84 115 <20 <20 60 <20 <20 74 120 <50 66 <50 350 204
58 55 ... ... 32 27 19 76 38 27 60 215$ 200 18 18 15 80 52 <20 <20 190$ 1653 18 12
28 27 72 32
Determinants
760 900 ... ... 430 900 550 660 550 400 550 660 560 500 920 180 950 560 1,000 680 500 120 120 265 660 710 190 100 550 430 200 1,000 440 600 560 1,000 1,500 860
MK
MK MK MK MK MK MK MK MK MK MK MK? MK MK MK MK MK MK MK MK MX MX MX MX MX MX MX MX MX MX MX MX MX MX MX MX MX MX
lg Incompletely
44 634 390 739 125 73 45 0 4 2 32 43 460 0 0 0 48
SerumIg* _______ G A M
Characterized
IgK ?type Igx ?type Ig ?type
1,000 420 420
170 39 <61 <20 <50 <15
1,300 400
230 <50
No Surface lg 47 48
0 0
0 0
2 9
2 0
10 5
No Ig Nolg
16 0
IN LYMPHOCYTIC
LEUKEMIA-AISENBERG
ET AL.
ability to form rosettes with sheep erythrocytes [8]. These methods are applicable to the study of human lymphocytes, and this application has led to increased understanding of neoplastic lymphocytes of man. A number of investigators [g-12] have described immunoglobulin of M (IgM) specificity on the surface of cells from patients with chronic lymphocytic leukemia (CLL), a finding which has been used to support the B cell origin of these neoplastic lymphocytes. Our report is based on studies of 57 patients with CLL and allied disorders. These observations permit certain generalizations to be made about the amount and type of cell surface immunoglobulin in CLL, the relationship of surface immunoglobulin to circulating im*munogRrbulin and M components, and the relationship of CLL to lymphosarcoma cell leukemia,
hairy cell leukemia PATIENTS
AND
and Mycosis fungoides.
METHODS
The diagnosis of CLL, chronic lymphosarcoma cell leukemia [13], acute lymphosarcoma cell leukemia [13], hairy cell leukemia [14] and Mycosis fungoides [15] was based on accepted clinical, laboratory and pathologic criteria, including examination of the Wright’sstained peripheral blood smear. With the single exception noted in the text, consecutive patients with these disorders and lymphocyte counts above 10,000/mm3 were studied; leukemic blood with lymphocyte counts below 10,000/mm3 was not used to avoid contamination of lymphocyte preparations with non-neoplastic cells. Serum concentrations of immunoglobulin G (IgG), A (IgA) and M (IgM) were measured with “immunoplates” Calif.),
and
electrophoresis Living blood
(Hyland each
serum
Laboratories, was
and agarobe-gel
lymphocytes
were
by the Ficoll-Hypaque
examined
Los
Angeles,
by
immuno-
electrophoresis
prepared gradient
from
[ll].
defibrinated
method
previous-
* Surface lg results are expressed as the per cent of lymphocytes staining with fluorescein-conjugated antiserums specific for G heavy chain, M heavy chain, K (kappa) light chain andX(lambda)lightchain of human lg. Pisa POlyValent antiserum directed against both light chains, and the heavy chains of IgG, IgA and IgM. Range for 10 normal subjects is G, 2 to 6 per cent; M, 7 to 12 per cent; kappa, 5 to 15 per cent: lambda 4 to 7 per cent; and P, 8 to 17 per cent. Serum lg is expressed in mg/lOO ml. Range in 50 normal donors is IgG, 720 to 1,500; IgA, 90 to 325; and IgM, 45 to 150. f Low concentration of IgG M component detected by immunoelectrophoresis. $ Low concentration of IgM M component detected by immunoelectrophoresis. Q Intense surface staining. More than 50 per cent of stained cells continue to fluoresce when the light intensity is reduced to 10 per cent by the interposition of a neutral filter in the optical system.
August 1973
The American Journal of Medicine
Volume 55
185
CELL-SURFACE
TABLE
I;ase No. 1
2 3 4 5 6 7 8 9
II
IMMUNOGLOBULINS
IN LYMPHOCYTIC
LEUKEMIA-AISENBERG
ET AL.
Cell Surface and Serum lmmunoglobulin (lg) in Chronic Lymphosarcoma Cell Leukemia (CLSL), Acute Lymphosarcoma Cell Leukemia (ALSL), Hairy Cell Leukemia (HCL) and Mycosis Fungoides (MF) .____.. __ ~~~~ Cell-Surface
~~~_._ Diagnosis
CLSL CLSL CLSL CLSL CLSL ALSL HCL HCL MF
i-
M
5 0 9 7 17 41 76 0 0
915 950 875 815 15 0 0 12 0
K
715 895 8 5 58 2 0 73 0
~_
Ig*
Serum Ig* P
A
20 24 954 949 7 66 80 3 0
73§ 98s 91§ 90§ 539 68 63 78 1
Interpretation MK MU MX MX IgK ?type GA Gh ?DKI. No Ig
G 920 1,500 660 900 1,400 860 1,400 1,960 1,270
-A 84 105 96 78 430 140 190 394 286
M 37 150: 2151 100 64 42 64 110 98
*$Q See footnotes for Table I. t See text. ly described [li]. Lymphocyte purity (assessed with Wright’s stain) and viability (assessed with trypan blue) of all preparations used exceeded 98 per cent, erythrocytes having been removed by osmotic shock with ammonium chloride [l I]. The lymphocytes were incubated for 1 hour at 37°C with fluorescein-conjugated antiserums, washed three times, suspended in buffered glycerine and examined with a Zeiss ultraviolet microscope equipped with a mercury arc lamp and an interference primary filter. A minimum of 200 lymphocytes were examined with each fluor and the number of fluorescent cells recorded; thereafter a 10 per cent transmitting neutral filter was interposed in the optical system and an additional 200 cells were examined. Fluorescein-conjugated goat antiserums specific for each heavy chain class (IgG, IgA, IgM, IgD and IgE) and for kappa and lambda light chains were obtained (Meloy Laboratories, Springfield, Va.), and used routinely without dilution. A polyvalent antiserum to the heavy chains of IgG, IgA and IgM, and kappa and lambda light chains was obtained from the same source. The specificity of the fluorescent reagents, a critical problem in all fluorescence microscopy, was controlled by appropriate inhibition with unlabeled IgG (Schwarz/Mann, Orangeburg, N. Y.), IgA of kappa type [ll], IgM of kappa type [ll] and Bence Jones proteins of kappa and lambda types (Meloy Laboratories). In the course of these investigations we found that the fluorescein-conjugated anti-IgG, anti-IgA and anti-IgD reagents had some anti-kappa and antilambda activity (as do almost all commercial antiserums [16]): this activity could usually be neutralized by the addition of the appropriate unlabeled light chain proteins. Although the addition of unlabeled proteins did not give rise to visible precipitation, it may be presumed that soluble antigen-antibody complex formation did occur. This process did not interfere with the fluorescence method, i.e., there was no increase in surface immunoglobulin attributable to complexes under the conditions employed. Ambiguous findings, i.e., staining with both light chain reagents or staining with more than one heavy chain reagent, were occa-
188
August 1973
The American Journal of Medicine
sionally observed, particularly with the brilliantly staining lymphosarcoma cells. These ambiguities were readily resolved, with two exceptions (Case 44, Table I and Case 5, Table II), by one of the following manipulations: retesting the cells after standing overnight at 4°C [12]; retesting the cells after incubation in 0.25 per cent trypsin [12] in isotonic saline solution (Microbiological Associates, Bethesda, Maryland) for 15 minutes at 37°C followed by washing twice with isotonic saline solution; or retesting the cells with a 1 : 10 dilution of the fluorescent antiserums. In CLL, it was difficult to classify the surface immunoglobulin on lymphocytes unless at least 15 per cent of cells stained with a given fluorescent conjugate. If fewer cells were stained, then the determination could frequently not be reproduced in a subsequent test. A cell was considered to show positive staining if three or more fluorescent aggregates were present on the surface. In the present experiments, all cells were tested with an antiserum to the heavy chain of IgA, the results obtained were not included in Tables I and II because the percentage of cells stained did not exceed 10 per cent in any case tested. Thymus-specific surface antigens were evaluated with the globulin fraction of a rabbit antiserum to human fetal thymus prepared as previously described [17]. This globulin fraction was absorbed with A-positive erythrocytes and with chronic lymphocytic leukemia cells. The nonfluorescent antithymus globulin was reacted with viable lymphocytes, and a fluoresceinconjugated goat antirabbit globulin antiserum and the fluorescence microscope were used to demonstrate fixation of the globulin to the cell surface (i.e., the indirect or sandwich technic was used). RESULTS Surface immunoglobulins are demonstrable on only a small fraction of lymphocytes obtained from the peripheral blood of healthy control subjects. In a series of 10 cases, 2 to 6 per cent reacted with the fluorescein-conjugated antiserum specific for the heavy chain of IgG, 0 to 3 per
Volume 55
CELL-SURFACE
lMMUNOGLOl3ULlNS
cent with anti-IgA, 7 to 12 per cent with anti-IgM, 5 to 16 per cent with anti-kappa, 4 to 7 per cent with anti-lambda and 8 to 17 per cent with the polyvaient anti-immunoglobulin antiserum [ll]. The major fraction of positive normal cells shows punctate fluorescence in a granular or aggregated pattern on the surface of the cell, a minor population shows crescents of fluorescent material [18]. The cells from most patients with CLL showed surface immunoglobulin in the granular pattern (Figure 1). Of the 48 patients with CLL studied (Tables I and I I I), it was possible to assign 38 to the IgM group since the lymphocytes reacted predominantly with the reagent specific for the heavy chain of IgM, and with either the reagent for the kappa or lambda light chain. The lymphocytes from five patients reacted predominantly with the reagent specific for the heavy chain of IgG; of these, four reacted predominantly with anti-kappa and one with anti-lambda reagent. The surface immunoglobulin on the cells of the remaining five patients with CLL could not be completely characterized. Cells from one patient (Case 44) reacted with both anti-IgG and anti-IgM reagents and could not be restudied after the trypsin-wash technic, which helps resolve ambiguous results, became available. The surface immunoglobulin of cells from a second patient (Case 45) reacted with the polyvalent antiserum and the anti-lambda reagent, and those from a third patient (Case 46) which reacted only with the polyvalent reagent, were not definitely identified, perhaps because of the insensitivity of certain fluorescent antiserums. Alternatively, lymphocytes from the second patient (Case 45) may only display the light chain of immunoglobulin; similar observations have been reported by Grey et al. [9]. We cannot state wheth-
TABLE
III
Summary
of Findings in Chronic.Lymphocytic
IN LYMPHOCYTIC
LEUKEMIA-AISENBERG
ET AL.
Figure 1. Case 43. Appearance of chronic lymphocytic leukemia cells stained with fluorescein-conjugates anti-lambda antiserum. Original magnification X 400.
er cells from two of our patients (Cases 47 and 48), which reacted with none of the antiserums, in fact bore no surface immunoglobulin or also represent failure of the antiserums to detect small amounts of immunoglobulin. It should be noted that cells from these last five patients also failed for the heavy to react with antiserums specific the machains of IgA, IgD and IgE. Furthermore, jority
Leukemia
of
lymphocytes
and Allied
from
these
two
patients
Disorders
Typeof SurfaceIg Total No.
Chronic lymphocytic leukemia Intense surface staining Circulating M component Chronic lymphosarcoma cell leukemia Intense surface staining Circulating M component Acute lymphosarcoma cell leukemia Intense surface staining Circulating M component Hairy cell leukemia Intense surface staining Circulating M component
Mycosisfungoides
48 4 6 5 5 2 1 0 0 2 cl 0 1
(K !r X) 38 3
G (K or X)
0
lg (7 type)
No Ig
2 (W’U 0
0
3 1 0 1 1 0 0
0
0
1 (?lgD)
0
0
0 0 0
1
3 (MW 4 4
0
August 1973
5 0 3UgG) 0
A
0
The American Journal 01 Medicine
2 0 0 0
Volume 55
0
187
CELL-SURFACE
TABLE
IV
IMMUNOGLOBULINS
IN LYMPHOCYTIC
Loss of Cell Surface
lmmunoglobulin
Patients with Chronic Lymphocytic ~__.~ _
Case No.* 27 29
31
M
U/24/71 3129172 11/08/71 11124171 2101172 5103172 6112172 12/03/71 4/06/72 7107172
K
(lg) in
Leukemia
x
P
Globulint
84
6
75
81
._.
...
...
...
74 85 89 ... 2 83 ... ...
25 23 11 4 0 16 ... ...
49 80 80 0 0 83 ... I. .
11 89 ... ... 38 0 79 2 5
11 ... ... ... ... 11 ... 2 4
47 and globulin
48) did not react with an antithyor form rosettes with sheep eryth-
ponents, the serum concentration of IgG was reduced (<720 mg/dl) in 33 of 43 patients and in the low normal range (720 to 1,000 mg/dl) in 7 of the remaining 10 patients. The concentration of IgA was decreased (<90 mg/dl) in 33 of 46 patients tested. Three patients had IgM M components in low concentration; of the remainder, serum IgM was decreased (<45 mg/dl) in 32 of 43 and in the low normal range (45 to 80 mg/dl) in 8 of the remaining 11. It is of interest that serum M components of IgG heavy chain were observed in 3 of 5 patients with CLL and surface IgG, but M components of IgM heavy chain specificity were found in only 3 of the 38 with surface IgM. In chronic lymphosarcoma cell leukemia (Tables I I and II I), the nature of the surface immunoglobulin was established as IgM (type kappa or type lambda) in four of the five patients studied. In one patient (Case 5) the light chain was of the kappa type, but we were not able to retest this patient’s cells to resolve the heavy chain type. The cells in these five patients were notable for the brilliance of the surface fluorescence. Thus, more than 50 per cent of lymphosarcoma cells continued to fluoresce after a neutral filter, which re-
August
1973
The American Journal of Medicine
the
incidence in the
light
light path.
to 10 per cent, In contrast,
was only
in4 of
the 48 patients with CLL showed fluorescence of comparable brilliance. Two of the patients with chronic lymphosarcoma cell leukemia had a circulating IgM M component. In general, the levels of circulating immunoglobulin were higher in pa-
The single cell leukemia
rocytes [ 191, properties usually associated with T cells [8,19]. Hence these patients are hot considered to have a T cell variant of CLL. Circulating immunoglobulin levels were depressed in most patients with CLL. Excluding consideration of the three patients with IgG M com-
188
ET AL
tients with chronic lymphosarcoma than in patients with CLL.
* See Table I. f Results are expressed as per cent of cells staining with a l/50 dilution of an antihuman thymocyte globulin prepared in rabbit followed by fluorescein-conjugated goat antirabbit antiserum. From 85 to 92 per cent of normal circulating lymphocytes stain with this reagent.
(Cases mocyte
duced terposed
Antithymocyte
Cell-Surface Ig* Date
LEUKEMIA-AISENBERG
patient with acute was found to have
cell
leukemia
lymphosarcoma lymphocyte sur-
face IgG of lambda type (not of the brilliance seen in chronic lymphosarcoma cell leukemia), as was one of the patients with hairy cell leukemia. The lymphocytes of a second patient with hairy cell leukemia reacted with an anti-kappa antiserum, and 51 per cent of these cells reacted with an anti-IgD heavy chain reagent. The staining with the anti-IgD reagent was not inhibited by unlabeled kappa light chain, but the surface immunoglobulin was only tentatively assigned to the IgD class, since unlabeled IgD protein was not available for specific inhibition. Circulating immunoglobulin levels were normal in the two patients with hairy cell leukemia. No cell surface immunoglobulin was detected on the lymphocytes of the single patient with Mycosis fungoides who had 34,000 circulating lymphocytes/mm3. In the eight instances in which circulating M components were detected by immunoelectrophoresis, the heavy chain of the M component corresponded to the predominant heavy chain identified on the surface of neoplastic cells. In four patients, the light chain type of the serum M component was also identified and found to correspond to the predominant light chain type identified on the surface of the patients’ lymphocytes. In the course of these studies it was noted that in several patients cell surface immunoglobulin was easily demonstrated on initial study, but was not detected in subsequent tests. Table IV documents this observation in three subjects. In each, the loss of surface immunoglobulin was associated with an unfavorable evolution of the disorder which required chemotherapy (two of the three patients died within 3 months of the final study listed in Table IV), but in each case there were neoplastic cells (above abundant circulating 20,000/mm3) at the time of study. The non-immunoglobulin bearing cells in these patients were considered to be abnormal because they resembled CLL cells (and contrasted with 85 to 92 per cent of normal lymphocytes [li’] in their failure to react with antithymocyte globulin. Five patients with CLL whose lymphocyte
Volume 55
CELL-SURFACE
IMMUNOGLOBULINS
counts were less than 5,000/mm3 were studied. In two of these, identification of the surface immunoglobulin on lymphocytes was difficult, most likely because of the presence of a significant number of normal B cells. In three cases, no surface immunoglobulin was detected. These patients had been extensively treated with chemotherapy, and the appearance of “null” cells may reflect a phenomenon similar to that cited. COMMENTS Until the recent introduction of methods which permit studies of the lymphocyte cell surface, medical investigators showed little interest in CLL. However, with the new technics available, there has been a revival of interest in this “monotonous proliferation of small lymphocytes,” and progress has been made in understanding the disorder and its relationship to other types of lymphoproliferative disease. On the basis of the work we have presented and that of other investigators [9,10,12], it is evident that lymphocytes from about 95 per cent of patients with CLL bear quantities of surface immunoglobulin which can be detected by immunofluorescence. The clonal character of this neoplastic proliferation is established by the finding of predominantly one type of immunoglobulin heavy chain and one type of light chain on the neoplastic lymphocytes of each patient, and is further supported by the demonstration of restriction of the surface immunoglobulin to one IgG subclass and one Gm allotype in the study of Froland and Natvig [20]. In the present series, patients whose cells bear IgM exceed those whose cells bear IgG by a ratio of 9:l. The five patients with CLL whose cells showed IgG rather than IgM heavy chains are of special interest; in three instances an M component of the same heavy and light chain type was found in the serum. In contrast, IgM M components, in small amounts, were found in only 3 of 38 patients with CLL whose lymphocytes bore surface IgM molecules. These serum M components (which have been described by earlier investigators [21]) most likely represent the secretion of immunoglobulin, similar to that displayed on the surface of the neoplastic cells, by at least some members of the same neoplastic clone. The low levels of circulating .immunoglobulin found in most patients with CLL has been the subject of earlier studies [22], but the very low concentration of serum IgM found in the majority of patients in the present series, has not received much attention. Clearly, the formation of IgM, as well as that of the other immunoglobulins, is cur-
IN LYMPHOCYTIC
LEUKEMIA-AISENBERG
ET AL.
tailed in CLL, perhaps through a displacement of normal precursor cells by the effete neoplastic clone. Cells from patients with chronic lymphosarcoma cell leukemia resemble CLL cells in that both contain surface immunoglobulin; cells from the former group tend to show surface immunoglobulin in considerably greater concentration. The single patient with acute lymphosarcoma cell leukemia and the two with hairy cell leukemia examined do not allow generalizations, but the pattern of serum and surface immunoglobulin observed in both disorders differed from CLL and chronic lymphosarcoma cell leukemia. The cells from the patient with acute lymphosarcoma cell leukemia and from one of the patients with hairy cell leukemia bore IgG (the cells of the other patient with hairy cell leukemia appeared to bear IgD), but unlike the IgG-bearing patients with CLL, this surface IgG was not associated with circulating IgG M components. It is reassuring that the surface immunoglobulin data substantiate the separation into clinical compartments which the hematologist has based on Wright’s stain morphology and clinical course. A number of findings support the contention that CLL is a disorder of B lymphocytes. Thus, in common with normal B cells, and in contrast to normal T cells, CLL lymphocytes bear demonstrable surface immunoglobulin [9-l 21. Furthermore, as discussed, immunoglobulin levels (a B cell function) are reduced in CLL, and other investigators [23,24] have characterized the immune impairment of CLL as predominantly involving antibody formation (a B cell function). Elsewhere we have reported that CLL cells lack a thymus-specific antigen [17], and studies in progress [19] indicate that CLL cells resemble B lymphocytes in their inability to form rosettes with sheep erythlrocytes and in their adherence to Nylon@ fiber columns. In addition, others have shown that CLL cells, in common with B lymphocytes, possess complement receptors [25,26]. The sluggish reaction of CLL cells to phytohemagglutinin [27] may be adduced as further evidence for their B cell nature. However, since both T and B cells are presumed to arise from a common bone marrow stem cell [28], the possibility that CLL cells arise from T lymphocytes by neoplastic derepression cannot be completely excluded despite the many similarities between the CLL lymphocyte and the normal B cell. At present it seems best to investigate the neoplastic disorders of lymphocytes with a variety of technics and to delay firm assignment into B or T
August 1973
The American Journal 01 Medicine
Volume 55
199
CELL-SURFACE IMMUNOGLOBULINS
cell
categories.
that there
Indeed,
are multiple
IN LYMPHOCYTIC LEUKEMIA-AISENBERG
recent subspecies
evidence
suggests
of normal
tion
T and
B lymphocytes [29,30]. In this connection, it should again be noted that chronic lymphosarcoma leukemia cells bear more surface immunoglobulin than do CLL cells, hence it is possible that the lymphosarcoma cell belongs to a different subspecies of B cells. Furthermore, the cells in hairy cell leukemia may represent an additional subspecies, and perhaps even CLL cells should be divided into IgM and IgG subtypes. It seems appropriate to point out here the relative rarity of cases of CLL bearing surface IgA; only a single case appears to have been reported [12]. At present the experimental data are complex, but they promise eventually to contribute to the understanding and classification of both lymphoid neoplasms and the cellular basis of normal immune function. The limitations of the technic of surface immunoglobulin study should not be overlooked. Thus we could not detect immunoglobulin on the surface of the cells from about 5 per cent of our patients with CLL, either because the ceils were devoid of these proteins or because the methods used were not sufficiently sensitive. Furthermore, disease progression or associated vigorous antitumor therapy may result in the depletion of surface immunoglobulin in patients whose cells previously bore ample surface immunoglobulin. These patients had large numbers of neoplastic cells in their circulation. Whether the cells observed possessed a nonfunctional mechanism for producing surface immunoglobulin or represented the evolu-
clone
ET AL
or selection cannot
of a nonimmunoglobulin-forming
yet
be distinguished.
It is also
ap-
parent that the surface immunoglobulin data become increasingly difficult to interpret as the lymphocyte count falls and the neoplastic cells become a minor population immersed among the normal lymphocytes. Another potential source of difficulty, which does not need elaboration here, stems from the fluorescent antibody method itself and the potential nonspecificity of the antiserums employed. Provided that allowances are made for the various technical limitations cited, the study of cell surface immunoglobulins provides an approach to CLL and other lymphoproliferative disorders which is similar to the approach to multiple myeloma afforded by the study of serum M components. In the patient with lymphocytosis, the demonstration of surface immunoglobulin of a single light and a single heavy chain type suggests a clonal, and most likely a neoplastic, proliferation. The possibility that a cellular equivalent of benign monoclonal gammopathy exists remains to be shown. Once the technical limitations have been recognized, lymphocyte surface immunoglobulins can be determined with speed and reliability: the technic should
be useful
in clinical
medicine.
ACKNOWLEDGMENT We are indebted to Doctors Zareh Demerjian, Allan Sandler, Robert Carey, Bernard Jacobson, Leonard Ellman and Richard Stein for referring patients to us for study, and to Mrs. Barbara Wilkes for excellent technical assistance.
REFERENCES 1. 2.
3.
4.
5.
6.
7.
8.
190
Gopd RA: Structure-function relations in the lymphoid system. Clin lmmunobiol 1: 1028, 1972. Raff MC: Two distinct populations of peripheral lymphocytes in mice distinguishable by immunofluorescence. Immunology 19: 637, 1970. Pernis B, Forni L, Amante L: lmmunoglobulin spots on the surface of rabbit lymphocytes. J Exp Med 132: 1001,1970. Unanue ER, Grey HM, Rabellino E, et al.: Immunoglobulins on the surface of lymphocytes. II. The bone marrow as the main source of lymphocytes with detectable surface-bound immunoglobulin. J Exp Med 133: 1188,197l. Bianco C, Nussenzweig V: Theta-bearing and complement-receptor lymphocytes are distinct populations of cells. Science 173: 154, 1971. Basten A, Miller JFAP, Sprent J, et al.: A recipient for antibody on lymphocytes. I. Method of detection and functional significant. J Exp Med 135: 610, 1972. Boldt D, Skinner AM, Kornfeld S: Studies of two subpopulations of human lymphocytes differing in responsiveness to conconavalin A. J Clin Invest 51: 3225,1972. Jondal M. Holm G, Wigzell H: Surface markers on
August 1973
The American Journal of Medicine
9.
10.
11.
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