Characterisation of CTL directed towards non-inherited maternal alloantigens in human cord blood.

Allogeneic cord blood transplantation (CBT), especially from unrelated donors, is being increasingly used for treating paediatric patients with both malignant and non-malignant disorders. Recent clinical and experimental evidence suggests that human cord blood mononuclear cells (CBMC) may acquire in utero a state of tolerance towards non-inherited maternal antigens (NIMA). In order to better define this phenomenon, we measured, by means of a limiting dilution assay (LDA), the frequency of NIMA-specific CTL precursors (CTLp) in cord blood samples obtained from 13 healthy neonates. The immunophenotype of the effector cells recovered from LDA was also analysed. Data concerning both CTLp frequency and phenotype of effector cells were compared with those obtained stimulating CBMC with cells of paternal origin (NIPA) and adult PBMC with allogeneic targets. Results showed that cytotoxic cells directed towards cells of maternal origin could be detected in all cord blood samples tested. Phenotype analysis demonstrated that NIPA elicit the expansion of CD3+/CD8bright T cells, a phenotype associated with alloreactive CTL. By contrast, NIMA preferentially stimulated the expansion of CD3-/CD8dim+ cells, a phenotype associated with NK cells, which are known to be able, in certain clinical conditions, to kill allogeneic haematopoietic cells without causing GVHD. Thus, our results indicate that, when evaluated in a limiting dilution condition, NIMA-reactive cord blood cells are detectable and a preferential expansion of NK cells is observed.

High frequency of Epstein-Barr virus (EBV) lymphoblastoid cell line-reactive lymphocytes in cord blood: evaluation of cytolytic activity and IL-2 production.

We investigated natural immunity towards autologous EBV lymphoblastoid cell lines (EBV-LCL) in the cord blood. Cord blood lymphocytes (CBL) from 20 healthy neonates were examined together with three EBV+ and one EBV- adult donors. We found that high frequencies of EBV-LCL-reactive cytotoxic lymphocytes, ranging from 1/190 to 1/12,205 were detectable in EBV- and EBV+ donors, as well as in the cord blood of 15 out of 20 neonates. Surface phenotype analysis, depletion experiments with MoAbs specific for T and natural killer (NK) lymphocyte subsets, and T lymphocyte cloning procedures strongly indicate virus-specific cytotoxic T lymphocytes (CTL) as the major population responsible for the lysis of autologous EBV-LCL in EBV+ donors. Conversely, a high frequency of NK cells seems to be involved in the killing activity observed in neonates and in the EBV- donor. Frequencies of EBV-LCL-induced IL-2-producing lymphocytes were high in EBV+ donors (range 1/2247-1/6633) and heterogeneous, but consistent, in cord blood (range 1/5072-1/57,819) and in the EBV- adult (1/17,148). CD8+ lymphocytes were responsible for IL-2 production in EBV+ individuals, while CD4+ T cells were charged with this role in cord blood and in the EBV- donor. These data demonstrate that CBL are able to develop a strong innate immunity, directed against autologous EBV-infected cells, mediated by both NK cells and CD4+ T lymphocytes. This characteristic may be relevant for protection against viral infections in both neonates and patients given cord blood transplantation (CBT).

Expression of CD45R0 antigen on the surface of resting and activated neonatal T lymphocyte subsets.

Expression of CD45R0 antigen has been evaluated on the surface of T lymphocyte subsets obtained from cord blood and on peripheral blood lymphocytes (PBL) from both term and preterm neonates. In some experiments, expression of the same antigen has also been evaluated after in vitro activation of cord blood lymphocytes (CBL) with phytohemagglutinin (PHA) or in mixed lymphocyte culture. The CD45R0 molecule was found to be present on a significantly lower percentage of CD3+, CD4+ and CD8+ CBL as compared with the same subsets evaluated in adult individuals. Moreover, at variance with adult PBL, the great majority of CBL displayed a low level of fluorescence when stained with UCHL1 (CD45R0-specific monoclonal antibody), and both PHA and allogeneic stimuli were able to strongly increase the expression of the CD45R0 molecule on CBL. The percentage of CD45R0+ PBL obtained from both term and preterm neonates on the 1st day of life was comparable to that of CBL, however the expression of this molecule increases remarkably within a few days in the majority of term neonates, while it seems to take more time for the antigen to be fully expressed in preterm infants.

In vivo activated cord blood lymphocytes express high affinity interleukin-2 receptor: evaluation of their responsiveness to in vitro stimulation with recombinant interleukin-2.

We report the phenotypical and functional characterization of a cord blood mononuclear lymphocyte subset (CBMC) which expresses the p55 chain of interleukin-2 receptor (IL-2R, CD25). The great majority of CD25-positive CBMC are activated CD3-positive T lymphocytes in that most of them are HLA-DR-positive, coexpress both CD4 and CD8 antigens and at least a part of them display high affinity IL-2R. In keeping with the expression of both chains of IL-2R (p55 and p75), enriched CD25-positive CBMC are able to display strong IL-2-induced proliferation and lymphokine-activated killing activity. The percentage of CD25-positive cells is higher in cord blood than in adult peripheral blood and their presence could be peculiarly important in the neonatal period, when specific T-cell-mediated immune responses are still on their way to full maturation.

Neonatal B lymphocyte subpopulations and method of delivery.

We studied four groups of healthy term newborn infants: (1) 11 infants born by vaginal delivery; (2) 11 infants born by elective cesarean section; (3) 10 infants born by emergency cesarean section with labor, and (4) 10 infants born by complicated vaginal delivery. Total and differential leukocyte counts, cortisol blood level, and B lymphocyte subpopulations (SmIg, sIgD, sIgM, CD19, CD20, CD21, CD23) were evaluated in cord blood samples from the four infant groups. Furthermore, the Pentothal blood level was measured in infants born by elective cesarean section and in their mothers at delivery. Higher total and differential leukocyte counts and cortisol blood levels were observed in group 1 and 4 infants as compared with group 2 and 3 infants. A significant correlation was observed between cortisol blood level and leukocyte counts. The percentages of positivity to cell surface markers of B lymphocyte subpopulations were significantly higher in infants born by elective cesarean section. A negative significant correlation of thiopentone with sIgM and CD21 was observed. These data indicate a significant influence of method of delivery and of thiopentone on B lymphocyte subpopulations.

Neutrophil chemotaxis in infants delivered by caesarean section.

We evaluated polymorphonuclear leucocyte (PMN) chemotaxis and cortisol levels in cord blood from 15 healthy term infants delivered by caesarean section and from 15 healthy vaginally delivered term infants. Mean neutrophil chemotaxis was significantly higher in infants delivered by caesarean section (78.3 +/- 23.4 microns) than in vaginally delivered infants (57.8 +/- 16.6 microns; P = 0.01). Mean blood cortisol level was significantly lower in infants delivered by caesarean section (9.14 +/- 2.76 micrograms/dl) than in infants born by vaginal delivery (20.71 +/- 6.98 P = 0.0001). No relationship was found between PMN chemotaxis and blood cortisol level. The higher neutrophil chemotactic activity observed in infants delivered by caesarean section could be related to general maternal anaesthesia.

[Anesthesia and the chemotaxis of neutrophilic granulocytes in newborn infants]. / Anestesia e chemiotassi dei granulociti neutrofili nel neonato.

The authors investigated the modifications of neutrophil chemotaxis and cortisol plasma levels in the newborn delivered by cesarean section or vaginal delivery. Chemotaxis turned out to be higher in the group undergoing cesarean section, while cortisol was lower in this group. The increased activity of neutrophils can be explained with the lessened inhibitory effect of endogenous steroids and with the stimulating action of nitrous oxide.

Natural cytotoxicity in the neonate: high levels of lymphokine activated killer (LAK) activity.

In 28 healthy full-term newborns the percentage of circulating cells expressing the Leu7 antigen, the marker of natural killer (NK) cells, was significantly lower than in healthy adults. However, newborns and adults did not differ with regard to the percentage of cells reacting with the Leulla, Leullc and TEC NK-1, monoclonal antibodies directed against the IgG Fc receptor of killer cells. Spontaneous NK activity of neonatal cells was profoundly reduced compared to the adult. In contrast, antibody dependent cellular cytotoxicity and NK-like activity generated in mixed lymphocyte cultures were similar in the two groups and lymphokine-activated killer cell (LAK) activity was high in the neonate. Natural killing is thought to play an important role in antiviral immunity since the neonate has a deficient capacity to deal with viral infections. Consequently, the present data indicate either that spontaneous NK is the most informative in vitro measure of newborn natural cytotoxicity in vivo, or, alternatively, that natural killing is not as important in antiviral immunity as previously suggested.

Serum growth-promoting activity measured as [3H]thymidine incorporation into human activated lymphocytes and serum transferrin levels in newborns and mothers.

Serum growth-promoting activity measured as [3H]thymidine incorporation into human activated lymphocytes and serum transferrin levels were measured during the perinatal period in newborns and mothers. Both thymidine activity (TA) and transferrin levels were significantly increased at the time of delivery in mothers compared to control women, and there was a progressive return to control levels in the first 5 postpartum days. A significant correlation was found between TA and placental weight. In the newborns, TA was low in cord blood after vaginal delivery but not in the cord blood from babies born by cesarean section. In premature newborns, TA was lower than in full term newborns. In all newborns during the first 24 postnatal hours, there was an increase in TA with levels rising above adult control values: levels in cord blood were positively correlated with birth weight but not with thymidine activity. These data afford complementary insights into the humoral controls of growth in newborn infants.

Deficiency of neutrophil bactericidal activity in term and preterm infants. A longitudinal study.

The present longitudinal study of 40 healthy term infants and 20 preterm infants shows that neutrophil bactericidal activity against Staphylococcus aureus and Streptococcus faecalis is transiently but significantly impaired in term infants during the 1st week of life. The deficiency is more severe and prolonged in preterm infants, and may contribute to the neonate's increased susceptibility to infections.

Lymphocyte subpopulations in the neonate: a subset of HNK-1-, OKT3-, OKT8+ lymphocytes displays natural killer activity.

It has been recently reported that cord blood lymphocytes (CBL) contain a subpopulation of OKT8+, sheep erythrocyte-rosetting negative (E-) cells not detectable in adult peripheral blood lymphocytes (a-PBL). The present studies were undertaken to characterize this subset of lymphocytes functionally and phenotypically. OKT8+ cells were purified from E-depleted CBL by negative selection on nylon-wool columns as well as by positive selection on plates coated with rabbit antibody to murine IgG (panning). The purified CBL displayed natural killer (NK) activity against K562 erythroleukemic cells. Although most of these CBL were large granular lymphocytes, they lacked typical NK markers such as HNK-1 and OKM1 surface antigens. Most OKT8+, OKT3- cells were also OKT10+, Ia+ and had the receptor for peanut agglutinin. These CBL may represent a stage along the differentiation pathway leading to mature NK or T cells.

Lymphocyte subpopulations in the neonate: identification of an immature subset of OKT8-positive, OKT3-negative cells.

T cell subpopulations of lymphocytes from cord blood (CBL) of 24 newborns and from peripheral blood (a-PBL) of 24 healthy adult volunteers were assessed in T cell-enriched, T cell depleted and unseparated lymphocyte fractions by using OKT3, 4, 6, and 8 monoclonal antibodies. The results show that T cell-enriched CBL include adult numbers of OKT3+, OKT4+, OKT6+ and OKT8+ lymphocytes whereas the T cell-depleted fraction consists of a high percentage of OKT8+, OKT3-, non-E rosette-forming cells bearing a PNA receptor. The presence of the PNA receptor and the lack of the OKT3+ antigen strongly support the hypothesis that the subset of OKT8+ cells in cord blood includes immature T lymphocytes that may represent an intermediate stage between thymocytes and mature peripheral T cells.

Age-related variation in growth-promoting activity human plasma measured in human lymphocytes.

The age-related variations in the growth-promoting activity of human plasma have been studied from birth (cord blood) to adulthood using a bioassay which measures the incorporation of tritiated thymidine into lectin-activated human lymphocytes. Cord blood values were low (0.69 +/- 0.004 U/ml). A definite increase was found at 5 days of age, correlating with the level at birth. Higher levels were attained after 1 month of age, with a 2-fold increase during the first months of life. Lower values were found in children 1-10 yr old, and high levels were found during puberty. This pattern, different from those of sulfation activity and plasma somatomedins suggests that factors other than somatomedins may be involved in growth stimulation during the first year of life in humans.

Colostral T lymphocytes detected by intracytoplasmic and membrane markers.

Colostral lymphocytes were studied using two established T-cell markers: intracytoplasmic alpha-naphtyl-acetate esterase (ANAE) staining and membrane receptors for sheep erythrocytes (E rosettes). ANAE staining allowed counting and identification of T-cell subsets independently of the status of membrane structures and receptors frequently altered in colostral cells. The fact that a sizeable number of colostral lymphocytes had the same phenotype as the majority of mature circulating peripheral blood lymphocytes supports the hypothesis that colostral lymphocytes may play a role in protecting neonates against infections, in transferring immune information to the newborn, or in modulating the immune response via release of soluble factors. A considerable percentage of colostral T lymphocytes are ANAE-negative. This phenotype is similar to that observed among thymocytes.

Peripheral blood T lymphocyte subpopulations in newborns.

Monocyte-depleted lymphocyte suspensions from the peripheral blood of 36 healthy newborns and 23 adults were compared as to percent of cells forming rosettes with sheep erythrocytes (T lymphocytes) and proliferative responses after in vitro culture with various doses of Phytohemagglutinin, Concanavalin A and Pokeweed mitogen. No significant difference was found between the proportions of T lymphocytes of newborns and adults. Mitogen responsiveness of the newborn lymphocytes showed dramatic between-subject variability completely unrelated to the proportion of T lymphocytes. It is suggested that during neonatal life there is massive variable recirculation of T lymphocytes subsets (and/or stages of maturation) characterized by differential degrees of responsiveness to the mitogens tested.