Clin. exp. Immunol. (1975) 22, 457-460.
Cell-mediated immunity to microbial antigens in mother and child A. S. R U S S E L L Department of Medicine, University of Alberta, Edmonton, Alberta, Canada (Received 22 April 1975) SUMMARY
We have looked at evidence for the transplacental passage of cell-mediated immunity in man. The lymphocyte response of mother-neonate pairs was assessed to non-specific mitogens, allogeneic cells, and to microbial antigens. Although six mothers were known to be chronic carriers of Herpes simplex virus only one of their neonates responded to this viral antigen. On two occasions a lymphocyte response was induced in cord blood by antigens to which the mother did not respond. This paradox remains unexplained. We suggest that the relative immunity of the neonate to some infectious diseases does not depend on the transplacental passage of cellular immunity. INTRODUCTION The older foetus and the young infant seem to be protected from a number of infectious diseases (Brambell, 1958; Hitzig, 1971; Janeway, 1964). This phenomenon was thought to depend on the transfer of maternal IgG antibodies across the placenta (Hitzig, 1971). This suggestion is less readily acceptable with the realization that a number of viruses can remain infectious in the presence of neutralizing antibodies (Allison, 1972a, b; Benson & Hotchin, 1969; Centifento, Little & Kaufman, 1970). There is in addition an increasing awareness of the importance of cell-mediated immunity (CMI) in the recovery from certain viral infections (Allison, 1972a, b; Blanden, 1971; Isaacson, 1968; Good, 1968). Using in vitro lymphocyte stimulation we have examined the ability of human neonatal cells to express a cell-mediated immune response to common bacterial, viral and fungal antigens. We have also studied the responses to phytohaemagglutinin (PHA), pokeweed mitogen (PWM) and to autologous unrelated cells in the one way mixed leucocyte reaction. To try and better assess the transplacental transfer of the immunity, we have compared the responses of a mother's cells with those from her own child.
MATERIALS AND METHODS Cord blood was obtained immediately post-partum in a heparinized tube,* care being taken to exclude contamination by maternal blood. The maternal specimens were obtained 4 or 5 days post-partum. Leucocyte response to antigens and mitogens was measured by the whole blood culture technique of Junge et al. (1970). 0-1 ml of whole blood was added to 3 ml of Weymouth's medium and to this was added the test * Venoject tubes, Jintan Terumo Co. Ltd, Tokyo, Japan. Correspondence: Dr A. S. Russell, Department of Medicine, 9-1 12A Clinical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2G3.
457
458
A. S. Russell
stimulant in 0-1 ml. The following reagents were used: PHA (P)t (100 ug/mI); poke-weed mitogenj (50 ,ug/ml); streptokinase-streptodornase (SK)% (50 i.u./ml); Herpes simplex virus (HSV)¶ (1/100 dilution of stock suspension); vaccinia (Vacc) inactivated by heating at 60'C for 30 min (105 p.f.u./ml); PPD** (30 pg/ml), Candidatt (2060 PNU/ml); allogeneic cells irradiated with 900 rads. These cells were taken from different, unrelated donors and were introduced as whole blood. The complete inhibition of DNA synthesis by the irradiation was confirmed during each experiment by incubation of these cells with PHA. All cultures were set up in triplicate together with antigen-free controls and were maintained at 37 C in 5%0 CO2 in air for 6 days. Eighteen hours before harvest 2 pCi of [3H]thymidine (specific activity 2 Ci/mmol) were added to each culture. The cells were then washed with 3% acetic acid to lyse the RBC and then with 5% trichloracetic acid and methanol. The DNA was solubilized using hyamine hydroxide, and the incorporated radioactivity was assessed by liquid scintillation counting with compensation for quenching. Results are expressed as a stimulation ratio obtained by dividing the disintegrations per minute (d/min) from test cultures by the d/min incorporated in the antigen-free control. Initial studies of unstimulated cultures showed that a stimulation ratio of 2-6 included 3 s.d. above the mean and represented a significant degree of stimulation (P = 0 01).
RESULTS
The response of maternal and cord lymphocytes are recorded in Table 1. Both groups of cells responded to PHA, PWM and in MLC but only four out of eighteen neonates showed any responses to antigens. The mothers of these four children responded to a total of nine out of nineteen+j antigens, but the neonates responded more selectively and in two cases TABLE 1. A comparison of the maternal cord lymphocyte response to mitogens, allogeneic cells (MLC) and antigens
Source of Mean stimulation index (SI) (and range) lymphocytes Maternal Cord
Number of positive responses/number tested
PHA
PWM
MLC
H/S
SK
Vacc.
PPD
Cand.
1660 (15-6324) 205 (7-1-1225)
433 (43-1225) 112 (9-233)
36 (8-132) 23 (3-87)
14/18
15/18
11/18
5/18
3/15
1/17
1/18
3/14t
0115
3/18*
A stimulation index of over 2 6 was considered positive (P
Cell-mediated immunity to microbial antigens in mother and child A. S. R U S S E L L Department of Medicine, University of Alberta, Edmonton, Alberta, Canada (Received 22 April 1975) SUMMARY
We have looked at evidence for the transplacental passage of cell-mediated immunity in man. The lymphocyte response of mother-neonate pairs was assessed to non-specific mitogens, allogeneic cells, and to microbial antigens. Although six mothers were known to be chronic carriers of Herpes simplex virus only one of their neonates responded to this viral antigen. On two occasions a lymphocyte response was induced in cord blood by antigens to which the mother did not respond. This paradox remains unexplained. We suggest that the relative immunity of the neonate to some infectious diseases does not depend on the transplacental passage of cellular immunity. INTRODUCTION The older foetus and the young infant seem to be protected from a number of infectious diseases (Brambell, 1958; Hitzig, 1971; Janeway, 1964). This phenomenon was thought to depend on the transfer of maternal IgG antibodies across the placenta (Hitzig, 1971). This suggestion is less readily acceptable with the realization that a number of viruses can remain infectious in the presence of neutralizing antibodies (Allison, 1972a, b; Benson & Hotchin, 1969; Centifento, Little & Kaufman, 1970). There is in addition an increasing awareness of the importance of cell-mediated immunity (CMI) in the recovery from certain viral infections (Allison, 1972a, b; Blanden, 1971; Isaacson, 1968; Good, 1968). Using in vitro lymphocyte stimulation we have examined the ability of human neonatal cells to express a cell-mediated immune response to common bacterial, viral and fungal antigens. We have also studied the responses to phytohaemagglutinin (PHA), pokeweed mitogen (PWM) and to autologous unrelated cells in the one way mixed leucocyte reaction. To try and better assess the transplacental transfer of the immunity, we have compared the responses of a mother's cells with those from her own child.
MATERIALS AND METHODS Cord blood was obtained immediately post-partum in a heparinized tube,* care being taken to exclude contamination by maternal blood. The maternal specimens were obtained 4 or 5 days post-partum. Leucocyte response to antigens and mitogens was measured by the whole blood culture technique of Junge et al. (1970). 0-1 ml of whole blood was added to 3 ml of Weymouth's medium and to this was added the test * Venoject tubes, Jintan Terumo Co. Ltd, Tokyo, Japan. Correspondence: Dr A. S. Russell, Department of Medicine, 9-1 12A Clinical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2G3.
457
458
A. S. Russell
stimulant in 0-1 ml. The following reagents were used: PHA (P)t (100 ug/mI); poke-weed mitogenj (50 ,ug/ml); streptokinase-streptodornase (SK)% (50 i.u./ml); Herpes simplex virus (HSV)¶ (1/100 dilution of stock suspension); vaccinia (Vacc) inactivated by heating at 60'C for 30 min (105 p.f.u./ml); PPD** (30 pg/ml), Candidatt (2060 PNU/ml); allogeneic cells irradiated with 900 rads. These cells were taken from different, unrelated donors and were introduced as whole blood. The complete inhibition of DNA synthesis by the irradiation was confirmed during each experiment by incubation of these cells with PHA. All cultures were set up in triplicate together with antigen-free controls and were maintained at 37 C in 5%0 CO2 in air for 6 days. Eighteen hours before harvest 2 pCi of [3H]thymidine (specific activity 2 Ci/mmol) were added to each culture. The cells were then washed with 3% acetic acid to lyse the RBC and then with 5% trichloracetic acid and methanol. The DNA was solubilized using hyamine hydroxide, and the incorporated radioactivity was assessed by liquid scintillation counting with compensation for quenching. Results are expressed as a stimulation ratio obtained by dividing the disintegrations per minute (d/min) from test cultures by the d/min incorporated in the antigen-free control. Initial studies of unstimulated cultures showed that a stimulation ratio of 2-6 included 3 s.d. above the mean and represented a significant degree of stimulation (P = 0 01).
RESULTS
The response of maternal and cord lymphocytes are recorded in Table 1. Both groups of cells responded to PHA, PWM and in MLC but only four out of eighteen neonates showed any responses to antigens. The mothers of these four children responded to a total of nine out of nineteen+j antigens, but the neonates responded more selectively and in two cases TABLE 1. A comparison of the maternal cord lymphocyte response to mitogens, allogeneic cells (MLC) and antigens
Source of Mean stimulation index (SI) (and range) lymphocytes Maternal Cord
Number of positive responses/number tested
PHA
PWM
MLC
H/S
SK
Vacc.
PPD
Cand.
1660 (15-6324) 205 (7-1-1225)
433 (43-1225) 112 (9-233)
36 (8-132) 23 (3-87)
14/18
15/18
11/18
5/18
3/15
1/17
1/18
3/14t
0115
3/18*
A stimulation index of over 2 6 was considered positive (P
