Clin. exp. Immunol. (1974) 16, 535-540.

LYMPHOCYTES BEARING COMPLEMENT RECEPTORS,

SURFACE IMMUNOGLOBULINS AND SHEEP
ERYTHROCYTE RECEPTORS IN PRIMARY
IMMUNODEFICIENCY DISEASES
J. R. LUCKASEN, A. SABAD, K. J. GAJL-PECZALSKA AND
J. H. KERSEY

Departments of Dermatology, Laboratory Medicine and Pathology, and Pediatrics,

University of Minnesota, Minneapolis, Minnesota, U.S.A.

SUMMARY
Nine patients with primary immunodeficiency diseases were studied for the pres-
ence of lymphocytes bearing surface immunoglobulins, complement receptors, and
sheep erythrocyte receptors. All were found to have a normal or increased per-
centage of lymphocytes bearing complement receptors, often despite a deficiency
of immunoglobulin-bearing lymphocytes. The results suggest that these patients
have a deficiency of surface -immunoglobulin synthesis but are not deficient in B
lymphocytes. Unsensitized sheep erythrocyte (E) binding (T) lymphocytes were
normal or decreased; the sum of E-binding (T) plus complement receptor-bearing
(B) lymphocytes was generally within the normal range. One exception was a patient
with combined system immunodeficiency who had a decreased total of T plus B
lymphocytes.

INTRODUCTION
Lymphocytes that originate in bone marrow or the bursa-equivalent are termed B cells.
Thymus-derived or processed lymphocytes are T cells (Good, Biggar & Park, 1971).
T cells are characterized in man by binding to unsensitized sheep erythrocytes (E) (Wybran
& Fudenberg, 1971; Jondal, Holm & Wigzell, 1972). B lymphocytes in normal peripheral
blood carry at least three surface markers: immunoglobulins are detected by immuno-
fluorescence (Pernis, Forni & Amante, 1970; Raff, Sternberg & Taylor, 1970); receptors
for a modified component of complement (C3) are detected using the binding of an E,
antibody, and complement complex (EAC) (Bianco, Patrick & Nussenzweig, 1970; Shevach
et al., 1972); receptors for aggregated immunoglobulins are detected using heat-aggregated
IgG (Dickler & Kunkel, 1972). Investigators in two laboratories have demonstrated that
>9000 of normal peripheral blood lymphocytes with complement receptors also carry sur-
face immunoglobulins (Nussenzweig & Pincus, 1972; Ross et al., 1973).
Patients with several primary immunodeficiency diseases have decreased to absent
Correspondence: Dr John Luckasen, Box 98 Mayo Building, University of Minnesota, Minneapolis
Minnesota 55455, U.S.A.
535
536 J. R. Luckasen et al.
immunoglobulins in serum and on lymphocyte surfaces (Siegal, Pernis & Kunkel, 1971;
Gajl-Peczalska et al., 1973). The present study demonstrates that these patients have normal
or increased lymphocytes with complement receptors, despite low to absent lymphocytes
with surface-associated immunoglobulins. This data might indicate a dissociation between
surface immunoglobulins and complement receptors on B lymphocytes in patients with
primary immunodeficiency diseases.

PATIENTS AND METHODS

Patients were diagnosed according to the classification of the Committee of the World
Health Organization on primary immunodeficiency disorders (Fudenberg et al., 1971).
Age-matched controls had no evidence of immunodeficiency.
Purification of lymphocytes
Venous blood was collected in heparin and sedimented in 500 dextran. Leucocyte-rich
plasma was incubated with an equal volume of iron-containing Lymphocyte Separating
Reagent (Technicon Instruments Corporation, Tarrytown, New York) for 30 min
at 370 C on a rocker platform. This mixture was layered on a Ficoll-Hypaque gradient
(Van Rood, Van Leeuwen and Zweerus, 1970) and centrifuged at 400 g for 45 min. The cells
at the interface were collected and washed three times with Dulbecco's phosphate-buffered
saline (D-PBS) and adjusted to a concentration of 4 x 106/cm3. The cells were incubated
at 370C with latex particles so that any remaining contaminating monocytes and granulo-
cytes, which phagocytize the particles, could be identified while counting. These separated
cells were then incubated on glass for 90 min at 370C to remove any remaining phagocytic
cells. Generally >97% of separated cells were lymphocytes.
EAC rosette assay
Sheep erythrocytes (E) were sensitized with E antibody (A) and complement (C) by a
modification of the method of Shevach et al. (1972). EAC suspension was prepared by
mixing equal volumes of E (0-5/%), A (rabbit anti-E serum, Meloy Laboratories, Spring-
field, Virginia) diluted 1:200 in D-PBS, and C (BI0D2 mouse serum, old or new strains)
diluted 1:10 in D-PBS. This EAC suspension was incubated for 30 min at 370C and washed.
To detect lymphocytes bearing complement receptors, 0-2 ml of EAC suspension was mixed
with 0-2 ml of separated lymphocytes, centrifuged at 500 g for 3 min and incubated at
370 C for 30 min. The pellet was resuspended by vigorous shaking and the rosettes counted.
The tubes were maintained at 370C until reading to avoid E binding to T lymphocytes
(Jondal et al., 1972). Lymphocytes were considered positive when three or more EAC were
firmly bound to the surface of latex-negative cells.
E rosette assay
Lymphocytes were mixed with an equal volume of unsensitized washed E (0.50%) and
incubated for 1 hr at 40C using the method of Jondal et al. (1972). Lymphocytes were con-
sidered to be positive when three or more E were bound to the surface of latex-negative
cells.
Lymphocyte surface-associated immunoglobulins were detached using fluorescein-
conjugated monovalent antisera against u, y, a, and e heavy chains and ; and A light
 Lymphocytes bearing complement receptors 537
chains in a previously described method (Gajl-Peczalska et al., 1973). Results (total) are
the sum of the percentage of lymphocytes stained with anti-heavy or anti-light chain
antisera; generally these values were not significantly different.
RESULTS
Results of assays for lymphocytes bearing complement receptors, lymphocytes with surface-
associated immunoglobulins, and E-binding lymphocytes are presented in Table 1. Fig. 1
TABLE 1. Lymphocytes bearing complement receptors, lymphocytes with surface immunoglobulins, E-
binding lymphocytes, and serum immunoglobulins in immunodeficient patients and controls
Lympho-
Lympho- cytes E-binding
Sex/age cytes with com- E-binding + EAC Serum
with plement lympho- binding immunoglobulins
surface receptors cytes lympho-
immuno- (EAC- cytes
globulins binding)
A. Patients
X-linked (Bruton's)
agammaglobulinemia
D.M. M/2 years <1% 19% 73% 92% Absent
T.S. M/3 years 3% 32% 60% 92% Absent
Variable immuno-
deficiency
T.V. M/15 years 11% 27% 64% 91% IgM, IgA, IgA low
G.R. M/15 years 7% 26% 49% 75% Absent
N.H. F/33 years 9% 28% 51% 79% IgM, IgA absent, IgG
low
S.E. M/26 years 8% 27% 56% 83% IgM, IgA absent, IgG
low
B.S. M/5 years 35% 38% 44% 82% IgA absent, IgM, IgG
low
Severe combined
system immuno-
deficiency
J.W. M/7 months 15% 53% 20% 73% IgM, IgG, IgA low
Ataxia-telangiectasia
L.D. F/5 years 29% 51% 36% 87% IgG, IgA low
B. Controls (deter-
minations on 14-17
individuals)
1-10 yr 16-45% 17-31% 47-73% 84% (mean)
(mean 31%) (mean 24%) (mean 60%)
10-35 yr 14-33% 11-21% 50-78% 80% (mean)
(mean 21%) (mean 16%) (mean 64%)
Lymphoblastoid
cell lines
Raji Not tested 98% 0%
HR-i-K Not tested < 2% 0%
538 J. R. Luckasen el al.
60

50

40

30 0~~~~~~

20-

10 -

I
0 10 20 30
Age in years

FIG. 1. Lymphocytes with complement receptors in patients with(A)X-linked agammaglobulin-

aemia, (D) variable immunodeficiency, (U) combined immunodeficiency and (A) ataxia-telan-
giectasia, and (0) in controls.

shows the percentage of lymphocytes bearing complement receptors in patients and controls.
One patient (D.M.) with X-linked (Bruton's) agammaglobulinemia had a normal per-
centage of lymphocytes bearing complement receptors and the other (T.S.) had increased
lymphocytes with complement receptors. Both had a very low percentage of lymphocytes
which possessed surface immunoglobulins. E-binding lymphocytes were normal and serum
immunoglobulins were absent in both. All patients with variable (late-onset) immuno-
deficiency had increased lymphocytes bearing complement receptors and all but one (B.S.)
had a low percentage of immunoglobulin-bearing lymphocytes. B.S. had increased immuno-
globulin-bearing lymphocytes despite low serum immunoglobulins. The patient with severe
combined system immunodeficiency had a high percentage of lymphocytes bearing com-
plement receptors, a normal percentage of immunoglobulin-bearing lymphocytes, and a low
percentage of E-binding lymphocytes. The serum immunoglobulins were all decreased. The
patient with ataxia-telangiectasia had increased lymphocytes with complement receptors
and a normal percentage of lymphocytes with surface-associated immunoglobulins. E-bind-
ing lymphocytes were low.
Control values for lymphocytes with surface immunoglobulins and lymphocytes with
complement receptors (EAC-binding) for children 1-10 years of age were higher than adults
(mean of 31% vs 2100 and mean of 24% vs 16%, respectively) (Table 1). E-binding lympho-
cytes were somewhat lower in children than adults (mean of 60% vs 6400 respectively).
Mean E-binding plus EAC-binding was 84% in children and 8000 in adults. As reported
previously, Raji lymphoblastoid cells were positive for complement receptors while HR-I K
were negative (Shevach et al., 1972). Neither contained E-binding cells. In our patients the
totals of the complement-bearing lymphocytes and the E-binding lymphocytes were
between 73 and 92% of all lymphocytes. These results are approximately the same as the
totals found in our control populations (80-84Y , mean) (Table 1).
 Lymphocytes bearing complement receptors 539
DISCUSSION
Previous investigations by Nussenzweig & Pincus (1972) and Ross et al. (1973) demon-
strated that the vast majority of normal peripheral blood lymphocytes which carry receptors
for complement also bear surface immunoglobulins. Our result show that patients with
several primary immunodeficiency diseases have normal or increased lymphocytes with
receptors for complement despite low to absent lymphocytes with surface-associated im-
munoglobulins. In these patients the data show a clear dissociation between surface im-
munoglobulins and complement receptors on peripheral blood lymphocytes. Thus, these
individuals apparently have a deficiency of surface immunoglobulin expression without a
deficiency in the total B-lymphocyte population. Further, these results are consistent with
data demonstrating that surface immunoglobulins and complement receptors are separate
molecules (Nussenzweig & Pincus, 1972).
To exclude the possibility of contamination with monocytes and neutrophils which also
bear complement receptors (Lay & Nussenzweig, 1968) extensive purification of lympho-
cytes was carried out. After iron incubation and density gradient separation, the cells were
incubated with latex particles to visually exclude any remaining phagocytic cells during
counting. Cell suspensions were then incubated on glass. The number of EAC-binding
cells was the same or higher but not lower than without glass. Purified cells appeared to be
lymphocytes morphologically and did not phagocytize latex or sensitized E.
In our patients the total of complement-bearing (B) lymphocytes and E-binding (T)
lymphocytes was generally the same as in the control population. This suggests that im-
munodeficient patients do not generally have an increased number of unidentified cells.
The one possible exception is combined system immunodeficiency in which our patient had
a somewhat lower T plus B lymphocyte total than controls.
Yata & Tsukimoto (1972) also reported that patients with X-linked (Bruton's) agamma-
globulinaemia have absent surface-associated immunoglobulins and normal percentages
of lymphocytes bearing complement receptors. Others have also noted the presence of
lymphocytes with complement receptors despite low to absent surface immunoglobulins
in X-linked (Bruton's) agammaglobulinaemia (Schiff et al., 1973).
These results differ somewhat from those of Geha, Rosen & Merler (1973) who found
that patients with X-linked (Bruton's) agammaglobulinemia who had no lymphocytes with
surface immunoglobulins also had no lymphocytes with complement receptors. Our results
and those of Yata & Tsukimoto (1973) and Schiff et al. (1973) showed that patients often
had normal or increased lymphocytes with complement receptors despite low to absent
surface immunoglobulins. The reason for the differences are not clear.
Our results demonstrating an increase in B lymphocytes with complement receptors in
patients with primary immunodeficiency disorders could have several explanations. 'One is
a compensatory increase in B lymphocytes due to a deficiency of T lymphocytes. Another
possible explanation is a deficiency of T lymphocytes whose normal function is to regulate
the proliferation of B lymphocytes (Allison, 1971). A third possibility relates to deficiency of
antibody the normal function of which is to act as a feedback inhibitor of B lymphocyte
proliferation (Uhr & Moller, 1968). Further studies will be necessary to determine whether
any or all of these possibilities are operative.
540 J. R. Luckasen et al.
ACKNOWLEDGMENTS
We thank Mrs Mary Buechele for technical assistance. The authors wish to acknowledge
the continued encouragement and assistance of Dr R. W. Goltz and Dr R. A. Good.
Research supported in part by a U.S. Public Health Service research training grant in
Dermatology (2T01-AM056560) and by Contract number NIH-NCI-NOl-CP-33357
within the Virus Cancer Program of the National Cancer Institute.

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