This document describes a clinical trial using interleukin-2 (IL-2) to treat a 17-month-old boy with combined immunodeficiency. IL-2 was purified from leukocytes and administered subcutaneously over 50 days. Within 3 weeks, IL-2 treatment normalized the patient's lymphocytosis and improved his eosinophilia. Most striking was the normalization of the patient's abnormal OKT4/OKT8 ratio and increase in OKT3+ cells. Infectious episodes decreased during treatment. Some effects were transient, returning to pathological levels after treatment ended.

1. Immunobiol., vol. 167, pp. 452-461 (1984)
1 Universitats-Kinderklinik Tiibingen, Tiibingen,
2 Medizinische Hochschule Hannover, Hannover, and
3 GBF _ Gesellschaft fur Biotechnologische forschung mbH, Braunschweig, Federal Republic
of Germany
In vivoEffects ofInterleukin 2 on Lymphocyte
Subpopulations in a Patient with a Combined
Immunodeficiency*
R. DOPFER', D. NIETIIAMMER', H. H. PETER', E.-M. KNIEP',
D. A. MONNER" and P. F. MDHLRADT'
Received April 17, 1984 . Accepted May 29, 1984
Abstract
This report describes a clinical trial with Imerleukin 2 (IL-2) on a 17-month old male child
with combined immunodeficiency (Nezelof's syndrome). IL-2 was prepared from conditioned
media of phytohemagglutinin-stimulated leukocytes from huffy coats. The purification of IL-
2 involved chromatography on Matrex Blue A sepharose and gel filtration chromatography.
The preparation was free of macrophage cytotoxicity factor, macrophage migration inhibition
factor and colony-stimulating factor. It contained negligible activity of interferon-y_ lL-2
activity was adjusted to 1600 U/ml, which corresponds to about 0.8 Ilg homogeneous IL-2/ml.
The patient was treated over a 50-day period with a total dose of 20,000 U IL-2, which was
injected subcutaneously. IL-2 was well tolerated. Within 3 weeks, the treatment led to a
normalization of a lymphocytosis which had prevailed for the previous 3 months. A pro­
nounced eosinophilia also improved but did not reach normalleve1s. The most striking effect
was a normalization of the OKT4-tIOKT8-t ratio with a concomitant relative increase in
OKT3-t cells in the peripheral blood. No effects were seen on E rosette formation, B cell
counts or serum Ig levels. Also NK or ADCC activity remained high, as before the treatment.
Infectious episodes and requirement for antibiotic treatment were less frequent during IL-2
therapy. Some effects of IL-2 were transient, e.g., the counts of OKT4-t and OKT3-t cells
which returned to pathological values a few weeks after the treatment was discontinued.
Introduction
Interleukin-2 (IL-2) is a lymphokine produced by stimulated helper T
cells. IL-2 binds to T cells which have, after antigen or mitogen contact,
, Parlially supported by DFG Sachbeihilfe Pe 151 /7-6 (H.H.P.), by BMFT pmject PTB
8372 (E.M.K.), and by BMFT project 8426.
Abbreviations: ADCC = antibody-dependent cellular cytotoxicity; ADA = adenosine
deaminase; AIDS = acquired immunodeficiency syndrome; BSA = bovine serum albumin;
eIg = cellular immunoglobulin; ConA = Concanavalin A; DiY = diphtheria toxin; IL-2 =
Interleukin 2; IFN = interferon; LGL = large granular lymphocytes; NK = natural killer;
PBMC = peripheral blood mononuclear cells; PHA = phytohemagglutinin; PNP = purine
nucleoside phosphorylase; SIg = surface immunoglobulin.

2. In vivo Effects of Interleukin 2 . 453
acquired the appropriate receptor. This provides the necessary signal for
proliferation of T cells (for reviews see 1,2). IL-2 is, therefore, a prerequis­
ite for expansion of antigen-specific regulatory and effector T cell clones.
Helper T cells playa central role because they provide help for humoral as
well as cellular defense mechanisms, and consequently IL-2 also indirectly
affects the humoral response by expansion of helper clones. Thus, defects of
the pre- or intra-thymic T cell development lead to a severe combined
immunodeficiency syndrome affecting both cellular and humoral defense.
A number of pathological conditions have been reported in which deficits
in IL-2 production are associated with grave immune disorders. In children,
defective IL-2 production was found in various primary immunodeficien­
cies (3), in a child with Nezelof's syndrome (4) and in an infant with
pediatric acquired immunodeficiency syndrome (AIDS) (4). In adults, a
decreased or absent IL-2 production has been reported in systemic lupus
erythematosus (5), angioimmunoblastic lymphadenopathy (6) and in AIDS
(7).
We describe here the case of a child with a cellular immunodeficiency
syndrome, characterized by an elevated number of apparently immature T
cells, an abnormally low percentage of mature T cells and, at an advanced
stage, a low OKT4/0KT8 ratio. The effects of a 50-day treatment with IL-2
on the T lymphocyte subpopulations are reported.
Materials and Methods
Production of fL-2 from buffy coat leukocytes
Human IL-2 was produced in bioreactors, basically as described (8), but with the following
modifications. Lymphocytes were prepared from 24 h buffy coats obtained from the blood
bank in Springe, FRG. The isolation of lymphocytes, which will be described in detail
elsewhere, included a step in a Hemoneucs blood wash centrifuge to remove the bulk of
erythrocytes, precipitation with a plasma expander (Plasmasteril, Fresenius, Bad Homburg,
FRG) similar to described procedures (9), and removal of adherent cells with carbonyl iron
(10). The resulting leukocyte mixture consists of 70 % lymphocytes, 20 %granulocytes, and
10 % monocytes. These leukocytes were sumulated for 20 h with 2 % PHA (Gibco, Grand
lsland, N.Y., USA) in serum-free RPMI 1640 in bioreactors at a cell concentration of 5 x 10'1
ml. Routine preparations contain about 20 U/ml of IL-2 (for definition sec below).
Partial purific3.uon of IL-2
2% vlv n-butanol was added as a preservative, media (3000 ml) were adjusted to pH 6.5
with 1 M phosphoric acid, and the mixture passed over a 3 x 12 em column of Matrex Gel Blue
A (Amicon, Witten, FRG) at a rate of 100 mllh. 2 %n-butanol was also added to all other
buffers used except in the last dialysis step. The column was washed with 300 ml of 150 mM
NaCI in 10 mM N';K phosphate buffer pH 6.5, and 300 ml of0.4 M NaCI in the same buffer.
This procedure removes the mitogen and about 90 % of the protein. IL-2 activity was then
washed off in about 200 ml of buffer containing 1 M NaCI. This eluate was concentrated to
10 mt on PM 10 membranes (Amicon), and subjected to gel chromatography on a
2.6 x 100 em Ultrogel AcA 44 (LKB, Bromma. Sweden) column equilibrated with 0.2 M Tris.
05 M glycine. 2 % n-butanol, adjusted to pH 7.6 with HCl. The flow rate was 15 mllh.

3. 454 . R. DOPFER et a1.
Fractions eluting at a mol. wt. range of 18,000--22,000 contained IL-2 and were pooled,
concentrated on PM 10 membranes to 12 ml after addition of 1% human AB serum, and
dialyzed against PBS. The preparations were free of macrophage migration inhibition factor,
macrophage cytotoxicity factor and colony stimulating factor, but occasionally contained
interferon in titers corresponding to those in untreated conditioned media. The IL-2 activity
was adjusted to approx. 1500 U/ml. The preparations were only moderately pyrogenic in the
rabbit fever-test.
IL-2 rest, defim'tion ofa unir
IL-2 activity was tested with murine Con A blasts as responder cells as described (11). An
internal standard was always included in the tests. 2000 U, as defined by us, correspond to
about 1 Ilg of pure IL-2, as we estimated from highly purified, partially-sequenced IL-2
prepared in our laboratory.
Laboratory tests to establish jmmune competence
Differential red and white blood cell counts were performed at least twice a week. Peripheral
blood mononuclear cells (PBMC) were isolated from heparinized blood by FicolUHypaque
density gradient centrifugation. The analysis of lymphocyte markers and functions have been
detailed in a previous paper (12), which also contains first admission data of the infant (No.2)
described in this report.
Briefly, lymphocytes expressing surface membrane Ig (SIg+) were characterized with
fluoresceinated F(abh goat anti-human IgG, IgA, IgM antiserum (Behringwerke AG, Mar­
burg, fRG). B cells showing terminal differentiation were identified by strong cytoplasmic Ig
(CIg+) reactivity on methanol-fixed cytocentrifuged smears. The T cell markers included
spontaneous rosette formation with neuraminidase-treated sheep erythrocytes (E-rosettes) and
staining with the monoclonal reagents OKT3 (pan T cell marker), OKT4 (T helper/inducer
phenotype), OKT8 (T suppressor/killer phenotype) and OKTlO (early thymocytes (13), some
bone marrow cells (14), and NK cells (15». Fe-receptor bearing lymphocytes were detected by
rosette formation with IgG-coated ox erythrocytes (EA-IgG-rosettes). Mitogen and alloanti­
gen responsiveness were determined in flat-boHom-microculture plates using optimal concen­
trations of phytohemagglutinin (PHA), concanavalin A (Con A), a lectin-free IL-2, Can A +
IL-2 and irradiated allogeneic PBMC (12). Natural killing (NK) and antibody dependent
cellular cytotoxicity (ADCC) were measured in 12 hr ~ICr release assays against K562 and
Ll210 target cells, respectively (12).
The evaluation of the humoral immune response included measurements of serum Ig
concentrations by laser nephelometry, isoagglutinin titers and specific antibodies .to candida,
bovine serum albumin (BSA) and diphtheria toxin (DiT) (12). Adenosine deaminase (ADA)
and purine nucleoside phosphorylase (PNP) were determined in whole blood to exclude
genetic defects of the purine salvage pathway known to be associated with severe combined
immunodeficiency (16).
Results
Clinical symptoms and immunological status of the patient A. O.
The patient, a boy whose parents were first degree cousins with no family
history of immunodeficiency, was hospitalized when five months old
because of recurrent infections of the gastro-intestinal and upper and lower
respiratory tract. He was slightly underweight and his skin showed signs of
eczema and dermatitis. Cervicallymphnodes were enlarged, and X-ray of
the thorax revealed no thymus shadow. Biopsy of the skin showed infection

4. In vivo Effects of Interleukin 2 . 455
with candida and rod-shaped bacteria. Biopsy of one lymph node revealed
granulomas consisting of mast cells, eosinophils and plasma cells, and signs
of infection with bacillus of Calmette and Guerin with which the patient
had been vaccinated as a newborn.
Platelet counts, hemoglobin and serum calcium levels were within normal
ranges, which makes the diagnosis of a Wiscott-Aldrich or DiGeorge
syndrome unlikely. ADA and PNP blood enzyme activities were normal as
Table I. Immunological state of patient A.b. 2 months after hospitalization, January 1982, age
7 months
Patient Age-matched healthy controls
Blood cells/~l celiS/ILl
leukocytes 28000 12200 (6000-17500)
lymphocytes 23000 7600 (4000-13500)
eosinophils 3600 300 (70-750)
platelets 220000 340000 (220 000-460 000)
hemoglobin (gil) 122 122 (100-150)
CaH
(mvalll) 4.7 4.9 (4.6-5.2)
Immunoglobulins
IgG (gil) 2.8 3.1 (1.2-8.3)
IgM (gil) 2.0 0.6 (0.3-1.3)
IgA (gil) 1.0 0.3 (0.1-0.6)
IgE (IUlml) 38 49
Lymphocyte surface markers % pos. cells % pos. cells
SIg <I 2-12
CIg < I 1- 3
OKT 10 63 8-36
OKT3 34 60-80
OKT4 28 4s-{'0
OKT 8 10 18-30
EA-IgG-rosettes 46 3-36
E-rosettes 75 56-85
In viero immune response cpm cpm
medium control 400 1500 (300-4600)
PHA (5 ~lIml) 2200 170000 (33 000-450 000)
Con A (10 ~l!ml) 2400 120000 (16000-470 000)
IL-2 6300 12000 (3400-17000)
Con A + IL-2 23 300 96000 (40000-200000)
allo MLC 300 28000 (3 500-85 000)
% spec. ~ICr release % spec. 5lCr release
NK a K 562 (16,1. 501, 1,1) 74/58/21 54/25/6
ADCC a L 1210 (25,1, 801, 2,1) 95/86/76 81/59127

5. 456 "R, DoPFE> etal.
was a chromosomal analysis performed on the patient's PBMe. Leukocytes
were high and differential blood counts showed a lymphocytosis and
elevated numbers of eosinophils, Serum IgG was low, while IgM, IgA and
IgE levels were within the age-matched normal range, Low isoagglutinin
titers were present but specific antibodies against candida, BSA or DiT, the
latter} months after vaccination with DiT, could not be detected, The
majority of the circulating lymphocytes formed E-rosettes and an elevated
proportion carried Fc-IgG receptors (46 % EA-IgG rosettes), Serotyping
with monoclonal antibodies showed an abnormally high fraction of
OKTlO+ cells, whereas the percentage of OKT}+, OKT4+ and OKT8+
cells was diminished, Immature B cells (SIg+) and B cells undergoing
terminal differentiation (CIg+) were well below 1% of the isolated PBMe.
A similar impairment was found for the lymphoproliferative response
towards polyclonal T cell mitogens and alloantigens, However, the strongly
diminished Con A-induced lymphoproliferation could be improved by
external supply of IL-2, The response to IL-2 without additional mitogens
was at the lower margin of the response of healthy controls, Finally, NK
and ADCC activities of the patient'S PBMC were both markedly elevated
as compared to a control group, These laboratory parameters are sum­
marized in Table 1. Taken together they all point to a T cell defect as the
underlying cause for the patient's combined immunodeficiency. The condi­
tion may best be classified as a variant of Nezelof's syndrome with
lymphocytosis (Fig, 1).
- ll - 2 Il - 2 Normal
.. Range
'- .-----.
20000
.~
i'
~
g
".0
15000w
0
,,,0 , 0
, , • ".- 10000
! :
,
" ""~
" "
J
,0 • . " "g- , 1,1 f , , , , :L , :~ , 0
, , ,
~
:
,
°'0,
, ,
E " I' ., ,~ I' oJ • 0 0, ,~
5000 q ~' '0,
j 0
0
7/1/&2 811 911 1011 II/I 12'1 1/1/83 211 3/1 ' " 5/1
DQte
Fig. 1. Lymphocyte and eosinophil counts of patient A. O. over a lO-month period which
included twO phases of IL-2 treatment.

6. In vivo Effects of Interleukin 2 . 457
Treatment with II-2
Throughout the time of hospitalization the patient received prophylactic
and therapeutical antibiotic treatment including Isoniazid, Colistinsulfate,
Flucloxacillin, Ketoconazol, and Trimethoprim-Sulfamethoxazol. A treat­
ment with thymosin (10 mg twice a week) over 5 months produced no
clinical improvement and had neither effects on leukocyte countS nor on
lymphocyte markers and functions. Three weeks after thymosin treatment
had been stopped, thymus epithelium was transplanted from a child who
had undergone cardiac surgery. Again no improvement of the immunologi­
calor clinical status was noted. A bone marrow transplantation was
considered but an HLA-matched sibling was not available and a haplo­
identical transplantation of bone marrow from the father was refused by the
parents. In this situation, informed consent was obtained from the parents
to start IL-2 treatment.
The treatment was begun when the patient was 17 months old, 4 months
after thymosin treatment had been stopped and 3 months after the thymus
epithelium transplantation had failed. By this time, the patient had an
abnormally low ratio of OKT4+ versus OKTS+ T cells (Fig. 2). 40 % of the
lymphocytes were of large granular morphology (LGL), and 69 % of the
lymphocytes exhibited the NK cell marker HNKI (17). IL-2 was injected
subcutaneously in the inguinal region since it had been shown in animal
experiments that IL-2, when injected intravenously, is cleared rapidly from
100
... 80
~
•u
•>
;;
o
..
60
40
20
Status
before Il-2
•
•
•
•
•
•
••
Il-2 Injections
!IIIIIIIII
•,
"I, /0---0 OKT 10
~
.___/ . OKT 3
/X'~'OKT'
/ ------.~ ___.OKra
-.---
Status
after Il-2
•
••
Normal
Range
L--L~'~'~~_-L__-L_ _~_,"r-_~___~
1/14/82 7127 I 10/26 118 11123 12114 211/83
Date
20 •
,
,
•
r
15 '3
10
~
7
g
~
;;
.
x
Fig. 2. Lymphocyte counts and percentages of OKT3, OKT4, OKT8, and OKTtO positive
lymphocytes during the first IL-2 treatment period. Differential blood cell counts were done
with capillary blood. Lymphocyte markers were analyzed on leukocytes isolated from venous
blood by a Ficoll/Hypaque gradient centrifugation.

7. 458 . R. DOPFER et ,I.
the blood (18, 19). 1 ml doses of 1600 U IL-2 were given every 3 to 4 days
over a period of 50 days. The only side effect was a mild but inconsistent
fever reaction. Within three weeks after beginning of IL-2 treatment,
lymphocyte counts, which had been high for the previous 3 months,
decreased to and stayed at normal levels. The percentage of LGL had
dropped to 14 %. Also eosinophils decreased, but remained at elevated
numbers (Fig. I).
Before, during, and 50 days after IL-2 treatment, humoral and cellular
immune parameters were determined. Part of the results are shown in
Fig. 2. It appeared that IL-2 treatment led to an increase of the percentage
of OKT4+ and OKT3+ cells and a slight increase of mitogen responsiveness
(maximal PHA response 55,400 cpm, maximal ConA response
25,400 cpm). No effect was seen on E- and EA-IgG-rosette formation nor
on B cell counts and terminal B cell differentiation. Similarily, serum Ig
levels and specific antibody production did not notably change during the
IL-2 treatment. The same was true for NK and ADCC activities which
remained high during the entire observation period.
Several weeks after discontinuation of IL-2 treatment, the percentage of
circulating mature T cells was again very low (Fig. 2) and the number of
eosinophils had risen (Fig. 2). Upon resumption of IL-2 treatment,
eosinophils dropped as during the first period of administration, but rose
again towards the end of the 6-week treatment. No immunological data
were gathered this time as the drawing of larger volumes of blood was
considered too great an inconvenience to the patient. Although difficult to
quantitate, there were signs of general improvement of the patient's clinical
status while under IL-2 treatment. The infectious episodes, and conse­
quently the requirement for antibiotic treatment were less frequent during
IL-2 therapy. However, the effects of IL-2 appeared of a transient nature.
Three months after the last IL-2 treatment the patient's condition wors­
ened. He died from pneumonia at the age of 26 months. An autopsy was
refused by the parents.
Discussion
All available evidence suggested that the patient A. b. suffered from a
combined immunodeficiency (Nezelof's syndrome) due to a T cell defect
accompanied by an abnormal proliferation of an immature pre-Tor NK­
like cell type. Thus, the proportion of lymphocytes showing an immature T
or NK cell phenotype (OKTIO+, HNKI +, Fc-IgG receptor+, LGL mor­
phology) was clearly elevated and functional NK cell activity of the
patient's PBMC was also high. In contrast, we observed low percentages of
mature T cells expressing OKT3, OKT4, or OKT8 phenotypes. In agree­
ment with this finding was the severely impaired T cell response to

8. In vivo Effects of Interleukin 2 . 459
mitogens which could, however, be partially restored in vitro by the
addition of IL-2. This observation and the low percentage of OKT4+ T
helper/inducer cells suggested that the defective T cell response was at least
partially due to a lack of the patient's IL-2 producing capacity.
Since the therapeutic approaches to overcome the T cell differentiation
defect by medication with thymosin or transplantation of thymic
epithelium had not shown any promising effects, we decided to treat the
patient with human IL-2, in the hope of expanding T cells that had acquired
IL-2 receptors in response to antigen. Over the first treatment period of
50 days, the pathologically high number of circulating lymphocytes
decreased to nearly normal values whereas the percentages, yet not the
absolute numbers, of OKT4+ and OKT3+ cells increased in the blood and
the OKT4/0KT8 ratio became almost normal. As DE SOUSA has pointed
out, the lymphocytes in a sample of peripheral blood represent only a
millionth of the total lymphocytes in the body (20), and it is, therefore,
impossible to precisely interpret these effects of IL-2 as an expansion of
OKT4+ cells, a normalization of a prior maldistribution of lymphocytes
(20) or both. In any event, a modest normalization of the immune status is
evident, and is further supported by the transient improvement of the
responsiveness to T cell mitogens. It has been reported that IL-2 induces in
vitro growth and enhancement of activity of murine NK cells (21). Such
effects were not observed in our patient, possibly because NK-like,
OKTIO+ cells were numerous, and NK activity was high even before IL-2
treatment.
IL-2 was well tolerated at the given dose and the subcutaneous route of
injection. The patient, weighing 5 kg, was given a total dose of 20,000 U of
IL-2 for 50 days. According to our estimation based on the specific activity
of a preparation of IL-2 from our laboratory which was pure enough to
yield a partial amino acid sequence, 2000 U corresponded to about I I!g of
pure IL-2. At a first glance, it appears unlikely that a dose of 10 I!g of a
peptide given over a period of 50 days should have any effects at all. It is,
however, known that other peptides are biologically active at pg levels (22).
Such levels may well have been reached in the lymphatic system under our
conditions. Similar doses were used in a brief in vitro trial by FLOMENBERG
et al. (4). In this case, post mortem histological data were reported,
suggesting that the draining lymph node near the IL-2 injection site showed
nests of lymphocytes which were not seen in the other lymph nodes.
An interesting side effect of the IL-2 therapy was the decrease in
circulating eosinophils. It is difficult to interpret this finding and correlate it
with known effects of IL-2. Eosinophilia, however, has been reported in
immunodeficient patients (23). In these cases, eosinophilia was correlated
with Pneumocystis carinii infections which frequently occur in immunode­
ficient patients. Since post mortem inspection of our patient was not
feasible, a safe diagnosis of such infections could not be obtained in this
case. We think, however, that the observed decrease of eosinophils following

9. 460 . R. DOPFER et al.
IL-l administration may be a further, though indirect, sign of transient
improvement of the immunological.status.
We realize that many questions, in particular that of the appropriate dose
of IL-2, are still open, but we are hopeful that IL-2 therapy can assist
conventional medication of life-threatening infections in immunodeficient
patients and may, if not restore a normal T cell response, at least prolong
the life of such patients until a HLA-matched bone marrow donor can be
found.
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