These findings may reflect an imbalance between thymic-dependent and independent pathways of T cell renewal. During thymic selection the proper rearrangement of the T cell receptor (TCR) genes could be affected by mutations in the NBS1 gene encoding a protein involved in repairing double-strand breaks of DNA [5]. The NBS1 protein and histone γ-H2AX, one of the primary DNA damage sensors, have been identified recently in normal, double-positive thymocytes at the sites of TCR gene-specific breaks introduced during V(D)J recombination. Thus, surveillance of TCR recombination intermediates by NBS1 protein might prevent oncogenic translocations and control the selection process of thymocytes [14].

The expression of CD45RA and CD45RO isoforms of the CD45 common leucocyte antigen is regulated strictly during intrathymic T cell selection. Normally, CD3-negative, double-positive (DP) cortical thymocytes express CD45RO. Positively selected DP thymocytes become single CD4⁺ or CD8⁺ switch their CD45 isotype from CD45RO to CD45RA and leave the thymus as newly generated post-thymic lymphocytes. In contrast, the CD45RO⁺ thymocytes are eliminated by apoptosis [15]. Thus, CD45RO-bearing memory CD4⁺ and CD8⁺ cells could represent either immature lymphocytes that escaped apoptosis, or activated peripheral T cells (transition of RA to RO isoform). On the other hand, expression of the CD45RA isoform is not a unique marker of thymic origin because the T cell subset of putative thymic progeny may still contain the CD45RA ‘revertants’ (transition of CD4RO to CD45RA after antigen elimination), which are identical phenotypically to the naive T cell population with thymic progeny [16,17].

Selective deficiency of the naive CD4⁺ T cell subsets in NBS patients (CD56⁺ and CD8⁺ cells expressed CD45RA) might not imply altered expression or splicing of the CD45 gene, but suggests a failure in intrathymic CD4⁺ T cell selection and/or prevalence of extrathymic pathways in T cell generation. Children normally generate new T cells via thymic output of CD45RA⁺ naive T cell subsets, with only a minimal contribution of antigen-driven peripheral expansion of the CD45RO⁺ memory T cell pool [17]. Thus, the diminished naive CD45RA/CD4⁺ T cell subset with CD4^low in both lymphopenic and non-lymphopenic NBS children possibly represents a restricted thymic output of immature naive CD4⁺ T cells rather than ‘revertants’ from the periphery, which should possess CD4 receptors of higher density [17]. The naive CD4⁺ cells proliferate well in response to mitogens [phytohaemagglutinin (PHA), concanavalin-A (ConA)][18], autologous and allogeneic cells [19] and act as a suppressor–inducer T cell subset that activates suppressor CD8⁺ T cells which in turn decrease IgG synthesis by B cells [20,21]. Thus, deficiency of the naive CD4⁺ T cell subset might contribute to the defects of cellular and humoral immune response described previously in NBS patients [2–4,6–9,22,23].

Thymus-independent T cell generation is due to peripheral expansion of mature T cells and/or extrathymic differentiation of bone marrow progenitors. These pathways predominate in advancing age as well as in impaired thymic function, as in NBS. Extrathymic expansion or differentiation takes place in lymph nodes, gut, bone marrow and liver, often leading to an excess of non-classical lymphocyte subsets such as TCR-γδ⁺ T cells or marrow-derived NK cells. The newly generated T cells express CD45RO^high isoforms and have TCRs of restricted diversity. Their absolute number remains either reduced throughout life (possibly as in group A) or is elevated [15,16], which results probably from either a response to infectious agents or expansion of malignant lymphocyte clones (possibly as in group B). Other characteristics of T lymphocytes generated by peripheral expansion fit well with our results: first, the density of CD45RO isoforms in CD4⁺ and CD8⁺ memory cell subsets was high. Had they have survived intrathymic apoptosis and left the thymus as CD45RO⁺, they should have resembled the cells of immature phenotype-expressing CD4, CD8 receptors of low density. However, the density of these receptors and others (CD3, αβ-TCR, γδ-TCR) did not decrease. Secondly, double-positive T cells should be present in the periphery but they were absent (data not shown). Thirdly, the T cell population bearing the memory phenotype is possibly not fully active in the NBS patients. Normal memory CD4⁺ T cells exert a helper effect on B cells and participate in the induction of class I-restricted cytotoxic T cells protecting against infection. However, despite a relative excess of the memory CD4⁺ T cell population, which should enhance IgG synthesis, NBS patients had hypo- or dysgammaglobulinaemia [1–4,6–9] and were negative for the presence of specific serum IgG antibodies against Streptococcus pneumoniae and HBs-Ag vaccination [9], and did not respond to diphtheria and tetanus vaccination [23]. A deficiency in naive CD4⁺ T cells, a shift towards a memory phenotype in this T cell subset and functional T cell defects resembling those found in NBS have also been described in ataxia–telangiectasia (A–T) [24]. Therefore, it would be difficult at present to suggest some specific role for NBS1 protein in the development of specific T cell defects in NBS which would be different to those found in A–T.

The NK lymphocytes contain CD3^–/CD8^low cells [13]. Due to an excess of NK cells the CD8^low subset of NBS patients represented about 50% of the whole CD8⁺ population versus 5% in controls. This might account for the decline in the value of the mean fluorescence intensity for CD8 receptors expressed in the total CD8⁺ population and in the naive CD8/CD45RA⁺ cell subset (both contain NK cells).

The proportion of the whole CD8⁺ population and the naive CD8⁺ cell subset were unchanged, regardless of the total lymphocyte count. In contrast, the absolute number of total CD8⁺ cells as well as of naive CD8/CD45RA⁺ and CD3/CD8⁺ subsets, was decreased more than twofold in group A compared to the control or group B. Thus, lymphopenia was caused by the loss of naive CD4⁺ and CD8⁺ T cells, but not of CD45RA/CD56⁺ or CD45RA/CD8^low non-T cell subsets (part of CD56⁺ subpopulation).

The deletional mechanism which preferentially induces the loss of naive CD4⁺ and CD8⁺ T cell subsets (group A) or only the naive CD4⁺ T cell subset (group B) is not known, but may involve apoptosis. Fas-independent apoptosis has been described in naive CD4⁺ and CD8⁺ lymphocytes of lymphopenic CVID patients [25] and in cultured human T cell blasts following activation in interleukin (IL)-2-deprived medium [26]. The T cells of NBS patients are poor producers of IL-2, thus an increase in their susceptibility to apoptosis may be due to similar mechanisms. Preferential loss of the naive CD4⁺ cell subset (group B) may indicate a prevelance of Fas-dependent apoptosis of CD4⁺ T cells, because CD8⁺ T cells (relatively less affected) are eliminated mainly by TNF receptor triggering [27].

Impaired renewal of CD4⁺ and CD8⁺ T cell subsets could be compensated partially by an excess of NK and TcR-γδ T cell subsets. NK cells do not express a rearranged TcR and the NBS1 gene seems not to interfere with the maturation of NK cells. The ratio of CD45RA/CD56⁺ to CD45RO/CD56⁺ was decreased significantly due to over-expression of CD45RO isoform, thus at least some of the NK cells were activated in vivo. The CD45 isoforms in the NK population are critical in providing signals necessary for optimal cytotoxic activity [28]. Density and distribution of the CD16 receptor within the CD56⁺ NK population were similar to controls (data not shown). The CD16 receptor is engaged in cytokine synthesis and ADCC reaction [29]. As the distribution of the CD8 receptor in the NK cell subpopulation was also unaffected, NK lymphocytes in NBS patients are possibly functionally active. Elevation of NK cells in single cases of NBS patients have been reported by us [5] and others [30].

An increase in γδ-TCR-bearing T cells results possibly from extensive extrathymic T cell expansion or differentiation and reflects further the failure of T cell regeneration in thymic-dependent pathways [31]. Increased numbers of γδ-TCR T cells have been described in patients with A–T as an inherited defect of genetic recombination, in SCID patients, in the elderly, in the response to mycobacterial and other infectious antigens [32–34].

Thus, in NBS patients γδ-TCR T cells together with NK lymphocytes probably form a significant line of defence against infectious agents. These cells are highly active, poorly specific cytotoxic effectors capable of recognizing antigens in the context of relatively non-polymorphic antigen-presenting molecules not encoded in the MHC [35].