Clin. exp. Immunol. (1977) 29, 376-384.
Transfer factor therapy in ataxia-telangiectasia A. I. BERKEL, F. ERSOY, LOIS B. EPSTEIN & LYNN E. SPITLERHacettepe University, Institute of Child Health, Department ofPediatrics, Immunology Laboratory, Ankara, Turkey, and the Department of Pediatrics, University of California, San Francisco, and the Cancer Research Institute and Children's Hospital of San Francisco, San Francisco, California, U.S.A.
(Received 31 January 1977) SUMMARY
The effects of weekly doses of transfer factor in four patients with ataxia-telangiectasia were investigated following a total course of 2 months therapy. Transfer factor administration showed no influence on the absolute lymphocyte counts, T-cell rosettes or antibody titres to EBV, but it caused conversion of skin-test reactivity and production of MIF to various antigens. There was a dissociation in blastic transformation response, the skin-test responses and MIF production. Serum interferon levels were low before, and 2, 6 and 24 hr after, therapy. Clinically no improvement in infections was observed following transfer factor therapy.
INTRODUCTION Transfer factor (TF) is a dialysable extract of sensitized lymphocytes which transfers reactivity from a skin-test-positive donor to a skin-test-negative recipient. In normal subjects it also transfers in vitro correlates of delayed hypersensitivity, including lymphocyte stimulation and production of migration inhibition factors in response to a specific antigen (Lawrence, 1969). TF has been used successfully in the treatment of various disorders of cell-mediated immunity (Spitler, Levin & Fudenberg, 1975), in tuberculosis (Whitcomb & Rocklin, 1973), in viral diseases (Spitler et al., 1975; Kirkpatrick, 1975; Rytel et al., 1975), in patients with cancer or malignancies (Levin et al., 1974; Spitler, Levin & Wybran, 1976; Vetto et al., 1976) and in children with infection and malnutrition (Jose, Ford & Welch, 1976). Ataxia-telangiectasia (AT) is a primary immunodeficiency disease characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, chronic sinopulmonary infections, defective cell-mediated immunity, a deficiency in serum IgA, various endocrinological abnormalities in 4-80% of patients and increased susceptibility to malignancy (Ersoy & Berkel, 1974; Boder, 1975). A search for an effective treatment of the immunodeficiency in patients with AT has been a challenging problem for the clinical immunologists. TF therapy is reported to have been applied to seven patients with AT and the results have been equivocal (Spitler et al., 1975; Griscelli, 1975; Buckley, 1975). MATERIALS AND METHODS Patients. Four male patients with AT aged 6, 8, 11 and 11 years respectively, who were diagnosed previously in the Immunoiogy Clinic of Hacettepe Children's Hospital, were subjected to weekly administration of a transfer factor which was prepared from donors who had a positive skin-test response to multiple antigens (Levin, Spitler & Fudenberg, 1975). Each patient was given 1 u of a pooled TF (that material extracted from 5 x 108 lymphocytes) for 8 successive weeks. Before and after the administration of TF therapy the tests described below were performed in each patient. Skin tests. Testing was carried out with a battery of four antigens (phytohaemagglutinin (PHA-P), 10 ,ug/0.1 ml, Burroughs-Wellcome Laboratories, Beckenham, England; Candida, 50 PNU/0-1 ml, Hollister-Stier Laboratories, Ydon, Pennsylvania; Streptokinase-streptodornase (SKSD), 50/12-5 u/0O1 ml, Lederle Labs, Pearl River, N.Y.; PPD, 5 u/01 ml, Refik Saydam Hygiene Institute, Ankara). A positive response was considered to be 5 mm or more induration at 48 hr. Skin sensitization with 0-2 ml of a 5% solution of dinitrofluorobenzene (DNFB) was followed, 14 days later, by challenges of
Correspondence: Dr A. Izzet Berkel, Immunology Laboratory, Hacettepe Children's Hospital, Ankara, Turkey.
Transfer factor therapy in ataxia-telangiectasia
1:10,000-1:100 dilutions of the sensitizing dose of DNFB. Erythema associated with vesiculation was considered as a positive response.
Rosette-forming cells. The percentage of E-rosette forming cells (T-cell rosettes) was measured according to Jondal, Holm
& Wigzell (1972).
Lymphocyte transformation. The in vitro response of the lymphocytes to PHA, Candida, SKSD and allogeneic lympho(MLC) was carried out according to the previously published methods (August et al., 1970; Ersoy & Berkel, 1974; Bach & Voynow, 1966). Incorporation of tritiated thymidine into DNA was determined by a modification of the method of Mans & Novelli (1961). All cultures were performed in duplicates and were harvested on the third day for PHA, and on the seventh day for Candida, SKSD and allogeneic lymphocytes. Results were expressed as 103 ct/min per 106 lymphocytes. A stimulation index (SI = ct/min of stimulated . ct/min of unstimulated) greater than 20 for PHA and greater than 2 for Candida or SKSD was considered as a positive response. For mixed lymphocyte cultures (MLC) an SI index (ct/min of responding x mitomycin C-treated stimulating lymphocytes - ct/min of unstimulated lymphocytes of responding) greater than 2-5 was taken as a positive response. MIF assay. The agarose-plate method described by Astor et al. (1973) was applied and PHA, Candida and SKSD antigens were used. Each specimen was seen in quadruplicate and the control cells were incubated with medium 199 instead of antigen. An index (the area of migration of cells incubated with antigen/ the area of migration of cells incubated without antigen x 100) less than 0-80 was considered as a positive response. Serum-interferon levels. A virus-plaque reduction method described by Epstein (1976) was used. Immunoglobulins and serum complement. Serum immunoglobulins (IgG, IgM, IgA), salivary IgA and serum P1 C levels were determined by the technique of Fahey & McKelvey (1965) by using the immunodiffusion plates of Behring-Werke, Main. Determination of Epstein-Barr virus (EBV) titres* in the serum. The immunofluorescent technique of Henle & Henle (1966) and Henle, Henle & Klein (1971) was used for antibodies to visceral capsid antigen (VCA), early antigen and nuclear antigen (EBNA). The clinical status of the patients before and after the TF therapy was observed and recorded. cytes
RESULTS The clinical findings of the patients are summarized in Table 1. All patients had sinusitis, one also had associated chronic otitis and bronchiectasis. No side effects such as fever, pain in the injection area or malaise were observed after TF administration. Urinalysis was normal in all and no evidence of gammo pathy was seen in the electrophoretic studies of their serum. In two patients (A.E. and H.A.), improvement in appetite and in general well-being was noted by the parents. Immunological findings in individual patients are tabulated in Tables 2-5. In A.E. the absolute lymphocyte count was variable, being low or normal before and after the TF therapy (Table 2). Previously positive skin tests for PHA and SKSD became strongly positive 51 days after the TF therapy, but Candida and DNFB skin tests remained negative. However, PPD was converted to 2+ from negative. T-cell rosettes showed an increase following the TF administration but later fell to the initial value. The lymphocyte-transformation response was normal with PHA and allogeneic lymphocytes before TF and showed no changes with therapy. However, PHA response was low 370 days after TF therapy. There was no in vitro response of lymphocytes to Candida antigen before and after the TF administration. Although the lymphocytes were unresponsive to PHA and Candida in the MIF assay, an SKSD response was observed following the TF administration. Serum IgA was low in this patient and remained so. In K.T. the absolute lymphocyte count remained low and Candida, PPD and DNFB skin tests also remained negative following the administration of TF (Table 3). Previously positive skin tests for PHA and SKSD became more strongly positive and the percentage of T-cell rosettes showed no changes following therapy. The in vitro response of the lymphocytes to PHA and allogeneic lymphocytes was normal and did not change. The previously low Candida response was not influenced by the TF therapy. In the MIF test the PHA response became normal but the lymphocytes were unresponsive to Candida and SKSD. Serum IgA was also low in this patient. In H.A. low values of the absolute lymphocyte count returned to normal 4'5 months after therapy (Table 4). The PHA and SKSD skin tests, which were initially positive, became more strongly so after the treatment. The Candida and PPD skin tests were converted to positive after TF, but the PPD and SKSD were negative on testing on the 447th day. The DNFB skin test remained negative. The percentage * Kindly determined by Dr W. Henle and Dr G. Klein. B
A. Izzet Berkel et al.
+ 4) F
I.o * _
U 't ° ~U
4)0 C) Ci
2 0 U
CU co) :
bo U) 0 0
+1 + .* P.t-
Transfer factor therapy in ataxia-telangiectasia TABLE 2. Immunological findings in A.E.
Absolute lymphocyte numbers/pl Skin tests* PHA Candida PPD SKSD DNFB T-cell rosettes (%) Lymphocyte transformation (SI)"F PHA Candida SKSD MLC Serum interferon MIF PHA Candida SKSD Immunoglobulins (mg/dl) IgG IgM IgA Salivary IgA
n.d. 59-1 n.d. n.d. 2-5 n.d. n.d. n.d. n.d.
1170 129 9 2-1 124
++(12x 12) ++(12x 12) +-(10xl12) +++(15x14) ++(lOx12) _ 39
n.d. 05 n.d. n.d. < 1/10
60-9 0-2 0-6 n.d. < 1/10
1-05 099 1 00
1-07 1-33 0-62
1680 245 Low n.d. 153
1010 90 Low n.d. 153
43 2-1 0.1 0.1 6-5 n.d. 1 00 0 95 n.d.
1250 182 Low n.d. n.d.
n.d. = Not done. * Numbers in parentheses indicate induration in mm at 48 hr. t - = Negative result. + SI = stimulation index.
of T-cell rosettes was normal, but there was some increase with therapy. The low in vitro response of lymphocytes to PHA became normal 2 months after therapy, but 3-5 months later it decreased again. The transformation response to Candida and SKSD was low and was not influenced by the therapy. The MLC response remained normal. In the MIF assay PHA, SKSD and Candida responses became positive 135 and 447 days after the TF administration. In this patient the serum IgA level was normal. In M.B. the absolute lymphocyte counts and the percentage of T lymphocytes were normal, the DNFB skin test was negative and those tests were not influenced by the TF treatment (Table 5). The PHA and SKSD skin tests were initially positive and they remained so. Negative Candida and PPD skin tests became positive within 3 months of the TF therapy. Blastic transformation with PHA become normal, but the low transformation response to Candida and SKSD was not corrected after therapy. The MLC response was normal. In the MIF assay PHA, Candida and SKSD responses became normal 55 to 411 days after the administration of TF. This patient also had a normal serum IgA level. In every patient the salivary IgA level was within the normal range. Serum-interferon levels were undetectable before, and 2, 6 and 24 hr after, TF treatment. Antibody titres for EBV showed that anti-VCA titres were elevated in three patients and anti-EBNA titres were normal in three and the TF administration had no influence on EBV titres (Table 6).
DISCUSSION With the past few years TF therapy has amply been used in many patients with primary immunodeficiencies with defective T-lymphocyte function, in different types of malignancies and in various
A. Izzet Berkel et a!. TABLE 3. Immunological findings in K.T.
Absolute lymphocyte numbers /,pl Skin tests PHA Candida PPD SKSD DNFB T-cell rosettes (%) Lymphocyte transformation (SI) PHA Candida SKSD MLC Serum interferon MIF PHA Candida SKSD Immunoglobulins (mg/dl) IgG IgM IgA Salivary IgA fi1C
+(7 x 7)
13*6 0-6 n.d. 30 n.d.
n.d. n.d. n.d. 1580 25 19 10 126
66 5 n.d. n.d. 4-5 < 1/10
28*5 0-2 05 8-1 < 1/10
077 0-89 0-85
1412 095 840 174 Low n.d. 198
n.d. n.d. n.d. n.d.
See Table 2 for legends and footnotes.
infectious diseases caused by intracellular parasites. Positive effects have been reported, especially in the lethal WAS (Spitler et al., 1975), and in some cases of sarcoma (Levin et al., 1974) and melanomas (Spitler et al., 1976). Generally, in patients treated with TF, the effects could more often be explained by conversion ofskin reactivity or by detection of lymphokines such as MIF rather than by clinical improvement of the patients (Spitler et al., 1975). In the present study, TF was administered to four patients with AT for 8 consecutive weeks and its effects were detected by various parameters. We were able to follow the patients for the following periods of time after the first dose of TF: A.E., 370 days; K.T., 51 days; H.A., 447 days; and M.B., 411 days. We observed that TF had no obvious influence on the absolute lymphocyte counts since it showed some variations in individual patients from time to time. Only one patient (A.E.) had a low total lymphocyte count (972/Ml) during the incubation period of a presumed viral infection, which also caused depression in blastic transformation response to PHA and in percentage of T rosettes. TF therapy caused no significant increases in T-cell rosettes. Skin-test responses showed conversion of skin reactivities following TF administration. The Candida skin test became positive in two patients (in H.A. 2 days after the last dose and in M.B. 33 days after the last dose), but did not change in the other two (A.E. and K.T.) during a follow-up period of 370 and 51 days, respectively. The PPD skin test became positive in three patients (A.E., H.A. and M.B.) and remained negative in one (K.T.). PHA and SKSD skin tests were positive in all patients before TF but the induration became much stronger in three patients (A.E., K.T. and H.A.), and did not change in one (M.B.). PPD and SKSD skin tests were found to be negative in H.A. on the 447th day of follow-up. Each patient was sensitized with DNFB. The skin-test response to this antigen remained negative after TF therapy. We observed no blastic transformation response of the lymphocytes to Candida and SKSD antigens before and after TF therapy. This response for PHA was normal before TF and did not change in two patients (A.E. and K.T.), and the low PHA responses became normal following administration of TF
Transferfactor therapy in ataxia-telangiectasia
TABLE 4. Immunological findings in H.A.
Absolute lymphocyte numbers/jul Skin tests PHA Candida PPD SKSD DNFB T-cell rosettes (%) Lymphocyte transformation (SI) PHA Candida SKSD MLC Serum interferon MIF PHA Candida SKSD Immunoglobulins (mg/dl) IgG IgM IgA Salivary IgA '8i C
+(5x 5) -
++(llx12) ++(llxll) +(8x 10) +(6x7) ++(llx 10) +(5 x 5) ++(lOx12) +++(22x24) ++(lOxlO) n.d. n.d. n.d. 52 53 72
n.d. n.d. n.d. n.d. n.d.
251-5 1.9 0-6 n.d. < 1/10
n.d. n.d. n.d.
079 0-83 0-88
0-88 1P10 1-20
660 113 68 10 1 161
8-8 0-8 07 2-9
900 83 90 n.d. n.d.
n.d. n.d. n.d. n.d. n.d.
16-8 1.0 04 3-6 n.d. 0 70 0-89 0-63 510 160 80 n.d. 93
++(15x 18) +(5x6) -
73 n.d. n.d. n.d. n.d. n.d. 075 0 74 n.d. 850 124 82 n.d. n.d.
See Table 2 for legends and footnotes.
to the other two (H.A. and M.B.). Low PHA responses on the 370th day and 135th day in A.E. and H.A. may be due to presumed infections at the time of this test. The MLC response was present in all patients before and after TF treatment. None of the patients produced MIF before the therapy with TF for three different antigens with the exception of H.A., who was able to make MIF for PHA. Of these four patients, three produced MIF for PHA (K.T., H.A. and M.B.), two began to produce MIF for Candida antigen (H.A. 447 days and M.B. 55 days after the therapy) and three produced MIF for SKSD antigen (A.E. 51 days, H.A. 135 days and M.B. 411 days after therapy) following TF administration. We generally observed a positive correlation between the skin-test reactivity and MIF production in our patients. In fact, the lymphocytes of K.T., H.A. and MB produced MIF for PHA while they had a positive PHA skin test. In A.E. there was no such correlation. For Candida, A.E. and K.T. had negative skin tests and did not produce MIF, while the other two patients (H.A. and M.B.) produced MIF and had skin reactivity for this antigen. This correlation (positive skin tests and MIF production) was also observed for SKSD in three patients (A.E., H.A. and M.B.). K.T. did not produce MIF for SKSD when he had a positive skin test. Our observations on the correlation of skin-test reactivity and the production of MIF for the same antigen confirms the experience of others in the literature (Spitler et al., 1975). The lymphocytes of our patients did not respond to the stimulation with Candida and SKSD antigens in the lymphocyte-transformation test while they had a positive skin-test reactivity and MIF production. This dissociation between MIF production and blastic transformation response of the lymphocytes has been observed in some patients with the Wiskott-Aldrich syndrome or chronic mucocutaneous candidiasis treated with TF (Spitler et al., 1972; Valdimarsson et al., 1972; Kirkpatrick & Smith, 1974). The presence of T-lymphocyte populations responsible for different functions may be a logical explanation for this dissociation. We observed long-lasting skin-test reactivity (from 370 to 411 days of follow-up) for PPD and SKSD,
A. Izzet Berkel et al. TABLE 5. Immunological findings in M.B.
Absolute lymphocyte numbers/,ul Skin tests PHA Candida PPD SKSD DNFB T-cell rosettes (0 ) Lymphocyte transformation (SI) PHA Candida SKSD MLC Serum interferon MIF PHA Candida SKSD Immunoglobulins (mg/dl) IgG IgM IgA Salivary IgA ,B1 C
++(lOx 10) -
++- (lox 10)
(5x 5) + + (10 x 10)
A-(8x 10) 4(6x 6) +(8x 7) + + (10x 10)
+-(9x 10) -
+ + (6 x 7) -
12 6 0-4 n.d. n.d. n.d. n.d. n.d. n.d. 710 75 74 10-3 87
13 6 04 1-3 2-8 < 1/10
29 8 04 0.1 3-6 < 1/10
121 0 84 0 97
140 0 50 1 12
1470 80 100 n.d. 135
73 1 0-6 1.9 4-1 n.d. 064 0 70 1 24
n.d. n.d. n.d. n.d. n.d.
510 160 80 n.d. 93
- (8 x 10) + +(lOx 10) + +(lox 10) n.d. 49
n.d. n.d. n.d. 075 0 74 0 56
1010 115 103 n.d. 74
See Table 2 for legends and footnotes. TABLE 6. Anti-EBV titres before and after TF
A.E. K.T. H.A. M.B.
Before 24 hr later 49 days later Before 6 hrlater 63 days later Before 24 hr later 49 days later Before 19 days later
10 n.d. 10 < 10 n.d. n.d. 10 n.d. 10 < 10 < 10
320 640320 160 160 160 320 640 640 40 40
10 10 10 10 10 10 20 20 20 < 10 < 10
< < < < <