Responses to a conjugate pneumococcal vaccine in preterm infants immunized at 2, 3, and 4 months of age.

Preterm infants are at an increased risk of invasive pneumococcal disease infection and, additionally, have a diminished response to Haemophilus influenzae type b (Hib) conjugate vaccines. There are little data examining the response of preterm infants to a seven-valent pneumococcal conjugate vaccine (PCV7). We examined the responses of preterm infants immunized at 2, 3, and 4 months of age to PCV7. A total of 133 preterm and 54 term infants were immunized with PCV7 and the Neisseria meningitidis group C (MCC), diphtheria, tetanus, pertussis, polio, and Hib vaccines. Pneumococcal serotype-specific IgG was measured by enzyme-linked immunosorbent assay (ELISA) pre- and postimmunization and at 12 months or following a booster of PCV7. Term and preterm responses were compared using linear and logistic regression analyses. Term infants had higher preimmunization geometric mean concentrations (GMCs) for all serotypes. Preterm infants had lower postimmunization GMCs for serotype 23F. Gestational age affected postimmunization GMCs for serotypes 4, 6B, and 23F. Preterm infants were as likely to have levels of ≥0.35 µg/ml as term infants for all serotypes except 23F. The proportions of infants with titers of ≥0.35 µg/ml for all 7 serotypes were comparable between groups. A total of 28 of 29 term infants who received a booster had levels of ≥0.35 µg/ml for all serotypes. One infant had undetectable levels for serotype 6B. Of the 32 preterm infants boosted, 9 had levels of <0.35 µg/ml for 1 serotype, and 1 had levels of <0.35 µg/ml for 2 serotypes. In nonboosted infants, GMCs for all serotypes except 6B had fallen by 12 months of age. These results support the need for a booster dose in the second year of life.

Functional analysis of the molecular factors controlling Qa1-mediated protection of target cells from NK lysis.

CD94/NKG2 receptors on mouse NK cells recognize the nonclassical class I molecule Qa1 and can deliver inhibitory signals that prevent NK cells from lysing Qa1-expressing cells. However, the exact circumstances under which Qa1 protects cells from NK lysis and, in particular, the role of the dominant Qa1-associated peptide, Qdm, are unclear. In this study, we examined in detail the lysis of Qa1-expressing cells by fetal NK cells that express CD94/NKG2 receptors for Qa1 but that lack receptors for classical class I molecules. Whereas mouse L cells and human C1R cells transfected with Qa1 were resistant to lysis by these effectors, Qa1-transfected TAP-deficient human T2 cells showed no resistance despite expressing high levels of surface Qa1. However, these cells could be efficiently protected by exposure to low concentrations of Qdm peptide or certain Qdm-related peptides. By contrast, even prolonged exposure of TAP-deficient RMA/S cells to high doses of Qdm peptide failed to induce levels of surface Qa1 detectable with a Qa1-specific mAb or to protect them from NK lysis, although such treatment induced sensitivity to lysis by Qa1-specific CTL. Collectively, these findings indicate that high surface expression of Qa1 is necessary but not sufficient for protection, and that effective protection requires the expression of sufficient levels of suitable Qa1-peptide complexes to overcome activatory signals. Results obtained with a series of substituted Qdm peptides suggest that residues at positions 3, 4, 5, and 8 of the Qdm sequence, AMAPRTLLL, are important for recognition of Qa1-Qdm complexes by inhibitory CD94/NKG2 receptors.

Stochastic acquisition of Qa1 receptors during the development of fetal NK cells in vitro accounts in part but not in whole for the ability of these cells to distinguish between class I-sufficient and class I-deficient targets.

Fetal mouse NK cells are grossly deficient in the expression of Ly49 molecules yet show a limited ability to distinguish between wild-type and MHC class I-deficient target cells. In this paper we report that during their development in vitro from immature thymic progenitors, a proportion of C57BL/6 fetal NK cells acquires receptors for a soluble form of the nonclassical class I molecule Qa1b associated with the Qdm peptide, but not for soluble forms of the classical class I molecules Kb and Db. The acquisition of these Qa1 receptors occurs in a stochastic manner that is strictly controlled by cytokines, and in particular is strongly inhibited by IL-4. All fetal NK clones tested, including those that lack detectable Qa1 receptors, express mRNA for CD94 and for both inhibitory and noninhibitory members of the NKG2 family. Fetal NK cells lacking receptors for Qa1 (and also for classical class I molecules) cannot distinguish between wild-type and class I-deficient blasts but, surprisingly, distinguish efficiently between certain wild-type and class I-deficient tumor cells. A variant line that lacks several members of the NKG2 family kills both types of tumor cell equally well, suggesting the existence of NKG2-containing inhibitory receptors that recognize as yet undefined nonclassical class I molecules of restricted distribution.

MHC class I expression protects target cells from lysis by Ly49-deficient fetal NK cells.

Using appropriate conditions natural killer (NK) cells can be cultured from the liver and thymus of day 14 fetal mice. These fetal NK cells are phenotypically and functionally indistinguishable from adult NK cells with the exception that they lack measurable expression of all of the Ly49 molecules that can currently be detected with antibodies. Despite this, they preferentially kill tumor cells and blast cells deficient in the expression of major histocompatibility complex class I molecules, although the degree of discrimination is usually weaker than that shown by adult NK cells and varies depending on the particular combination of effector and target cells used. Polymerase chain reaction analysis revealed that although fetal NK cells are severely deficient in the expression of mRNA for Ly49A, B, C, D, G, H, and I they express high levels of Ly49E mRNA, raising the possibility that Ly49E may have an important and special function in the early development of the NK lineage.

Fetal mouse NK cell clones are deficient in Ly49 expression, share a common broad lytic specificity, and undergo continuous and extensive diversification in vitro.

NK cells obtained by exposing mouse fetal thymocytes to appropriate combinations of IL-4, IL-2, and PMA are phenotypically indistinguishable from cultured adult splenic NK cells with the exception that they generally lack measurable expression of all of the inhibitory Ly49 molecules that can currently be detected with Abs (Ly49A, -C, -G, and -I) and of the activating molecule Ly49D. Despite this deficiency, fetal NK cells have a similar specificity to Ly49-expressing adult splenic NK cells. Individual fetal NK cell clones display an essentially invariant and broad specificity similar to that of polyclonal populations of fetal or adult NK cells, although significant differences in the fine specificity of clones can occasionally be detected. Most remarkably, cloned fetal NK cell lines display heterogeneous expression of a restricted set of surface molecules that includes 10A7, Ly6C, 3C2, CD8, certain isoforms of CD45, and also, occasionally, Ly49 molecules. This heterogeneity is not related to the cell cycle or activation status of the cells, and micromanipulation recloning demonstrates unambiguously that it is not due to a lack of a single cell origin. Diversity is generated rapidly and the capacity for diversification appears to persist indefinitely in vitro. The expression of individual variable Ags is independent and stochastic, resulting in fetal NK "clones" being potentially composed of hundreds of phenotypically distinct cells. We hypothesize that fetal NK cells behave as progenitor cells that are undergoing a process of rapid, extensive, and continuous diversification and that are individually capable of generating and regenerating a complex NK cell repertoire.

Phenotypic and functional characterization of long-lived NK cell lines of different maturational status obtained from mouse fetal liver.

Culture of day 14 mouse fetal liver (FL) cells in high dose IL-2, together with appropriate combinations of IL-4 and PMA, resulted in the generation of cell lines, termed FL-A lines, that were phenotypically and functionally indistinguishable from cultured adult splenic NK cell populations with the single important exception that no Ly49-expressing cells were present. By contrast, when FL cells were cultured in low-dose IL-2 alone, a second population of slow-growing NK-like cells, termed FL-B cells, emerged. These cells expressed the NK markers asialoGM1, 10A7, 2B4, and Fc gammaRII/III but differed from FL-A and splenic NK cells in expressing IL-2R alpha and stem cell factor receptor (SCFR) but no B220. Most lines derived in this manner had minimal or no cytolytic activity and only very low levels of NK1.1. However, they could secrete substantial quantities of several lymphokines including IL-3, granulocyte-macrophage (GM)-CSF, TNF-alpha, and, most surprisingly, IL-2. A minority of FL-B lines, typified by line 903, displayed marked cytolytic activity, moderate levels of NK1.1, reduced production of IL-2, and the capacity for accelerated growth in high-dose IL-2. FL-B lines generally expressed mRNA for CD3gamma but not for other CD3 chains, whereas FL-A and fetal thymic (FT) NK lines often expressed mRNA for all four CD3 chains. Despite many similarities to pro-T cells, FL-B cells showed no capacity to differentiate into mature T cells. Taken together, our results suggest that NK lines of different maturity can be obtained from fetal liver, with FL-B lines being the most immature, FL-A lines the most mature, and lines such as FL-B 903 representing an intermediate state of differentiation.