Single and combined deletions of the NTAL/LAB and LAT adaptors minimally affect B-cell development and function

Abstract

NTAL (non-T-cell activation linker, also called LAB) and LAT (linker for activation of T cells) are evolutionarily related transmembrane adaptor proteins that are phosphorylated upon immunoreceptor engagement. Using quantitative reverse transcription-PCR, both NTAL and LAT were found to be expressed in B cells. However, LAT expression was limited to early B cells, whereas NTAL expression typified mature B cells. To delineate their roles in B-cell development and function, Ntal-deficient mice were generated and crossed with Lat-deficient mice. B cells developed in Lat(-/-) Ntal(-/-) double-deficient mice and in mice lacking either of the two adaptors with the same efficiency as in wild-type mice. Upon B-cell antigen receptor cross-linking, Ntal(-/-) B cells exhibited slightly increased Ca(2+) mobilization and proliferation. In addition, Ntal-deficient mice had increased levels of natural antibodies and slightly increased humoral response to a T-dependent antigen. Normal titers of serum-specific immunoglobulins were produced in response to a T-cell-independent antigen. Although NTAL is also expressed in plasma cells, its absence did not affect the hypergammaglobulinemia E and G1 that developed in mice with a mutation in tyrosine 136 of LAT. Therefore, NTAL does not play a role in B cells symmetric to the role played by LAT in T cells.

FIG. 1.

Comparative expression of Ntal and Lat transcripts throughout mouse B-cell development. (A) Diagram of mouse B-cell development showing anatomic localizations and cell surface markers. Cell populations corresponding to the specified stages of B-cell development were sorted from either bone marrow or spleen (see Materials and Methods). RNA samples were prepared and the relative levels of Ntal and Lat mRNAs analyzed by quantitative RT-PCR. (B) Results are expressed as relative units of Ntal and Lat mRNA normalized using Hprt transcripts. Positive control samples (+) correspond to mature B cells (NTAL panel) or mature T cells (LAT panel) isolated from wild-type spleen, whereas negative control samples (−) corresponds to total spleen cells isolated from Ntal^−/− × Lat^−/− double-deficient mice. Data shown are representative of three independent experiments.

FIG. 2.

Lack of NTAL expression in Ntal^−/− splenocytes as detected by Western blotting. The arrow indicates the position of the band corresponding to NTAL (29 kDa). The upper band corresponds to an unidentified nonspecifically reactive protein. The intensity of this band demonstrates that each lane was loaded with comparable amounts of total proteins. The reduced amounts of NTAL found in Ntal^+/− splenocytes compared to Ntal^+/+ (WT) splenocytes denote the occurrence of a gene dosage effect.

FIG. 3.

Flow cytometric analysis of B-cell subpopulations in Ntal^−/− bone marrow. Single-cell suspensions from bone marrow were stained with the indicated antibodies and analyzed by flow cytometry. Numbers indicate the percentages of cells in the specified quadrants or gates. The total number of cells found within each organ is shown for wild-type (WT) and Ntal^−/− mice (averaged from six experiments).

FIG. 4.

Flow cytometric and quantitative RT-PCR analyses of B-cell subpopulations in Ntal^−/− peritoneum and spleen. (A and C). Single-cell suspensions from spleen (A) and peritoneum (C) were stained with the indicated antibodies and analyzed by flow cytometry. Numbers indicate the percentages of cells in the specified quadrants or gates. The total number of cells found within each organ is shown for wild-type (WT) and Ntal^−/− mice (averaged from 20 (spleen) and six (peritoneum) experiments). In the case of CD21/35 and CD23 profiles, windows indicate T1 (CD21/35^lo CD23^lo), follicular (CD21/35^int CD23^hi), and marginal zone (CD21/35^hi, CD23^lo) B cells. (B) Cell populations corresponding to marginal zone and follicular B cells were sorted from the spleen on the basis of CD21/35 and CD23 profiles, and RNA samples were prepared and the relative levels of Ntal and Lat mRNAs were analyzed by quantitative RT-PCR.

FIG. 5.

Proliferative and Ca²⁺ responses in splenic B cells from Ntal^−/− mice and Lat^−/− Ntal^−/− double-deficient mice. (A) Spleen B cells purified from wild-type (WT) or Ntal^−/− mice were stimulated with increasing concentration of goat F(ab)′₂ anti-mouse IgM antibody (0-40 μg/ml), with lipopolysaccharide (1 μg/ml), or with phorbol myristate acetate and ionomycin (P/I). After 40 h of culture, the ATP content of each culture, a value proportional to the extent of cell proliferation, was measured by luminescence. (B) Calcium flux analysis in response to BCR stimulation in wild-type and Ntal^−/− mature B cells. The diagram depicts calcium flux (y axis) as a function of time (x axis). Arrow below the x axis indicates the time point of stimulation with F(ab)′₂ goat anti-mouse IgM antibody. Data shown are representative of four independent experiments. (C) Intra- and extracellular Ca2⁺ mobilization was recorded (see Materials and Methods) in mature B cells from wild-type (WT) or Ntal^−/− spleens. Same symbols as in panel B. The time point at which the extracellular Ca2⁺ concentration was restored to 1.3 mM is indicated by an arrow. (D) Calcium flux analysis in response to BCR stimulation in wild-type and Ntal^−/− B220⁺ IgD⁻, immature B cells. Same symbols as in panel B. (E) Calcium flux analysis in response to BCR stimulation in wild-type, Ntal^−/−, and Lat^−/− Ntal^−/− mature B cells. Same symbols as in panel B. (D) Calcium flux analysis in response to BCR stimulation in wild-type and Lat^−/− mature B cells. Same symbols as in panel B.

FIG. 6.

Analysis of protein tyrosine phosphorylation and of mitogen-activated protein kinase phosphorylation in splenic B cells from Ntal^−/− mice. Purified B cells of Ntal^−/− (lanes 1-5) and wild-type mice (lanes 6-10) were either left untreated or activated with affinity purified F(ab)′₂ fragments of an anti-IgM monoclonal antibody for the indicated periods of time. Postnuclear lysates were subsequently separated by 12% SDS-PAGE and the activation of the individual members of the mitogen-activated protein kinase family (ERK, JNK, p38) assessed using phosphospecific anti-JNK, anti-p38, and anti-ERK antibodies. For analysis of global tyrosine phosphorylation, the antiphosphotyrosine monoclonal 4G10 antibody was employed. To verify equal loading, the blots were stripped and reincubated with an anti-ERK1/2 polyclonal rabbit antiserum. The data shown are representative of seven independently performed experiments.

FIG. 7.

Humoral responses in Ntal^−/− mice. (A) For measuring B-cell responses to T-independent antigen, group of 8-10 mice (wild-type, open circles; Ntal^−/−, solid circles) were immunized with TNP-Ficoll. Mice were bled before (T0) and 7 days (T7) after immunization. TNP-specific immunoglobulins of the indicated isotypes (IgM and IgG3) were assessed in individual mice and plotted on a logarithmic scale. Values represent relative binding units obtained within the linear range of the titration curves compared with a standard nonimmunized control. (B) For measuring B-cell responses to the T-dependent antigen TNP-OVA, groups of 8-10 mice (wild-type, open circles; Ntal^−/−, solid circles) were immunized with TNP-OVA and bled before (T0) and 14 days (T14) after immunization. Relative amounts of TNP-specific IgM and IgG1 were analyzed as described in A. Data shown are representative of two independent experiments. (C) Humoral immune response following suboptimal immunization against OVA. Wild-type (n = 6), Ntal^+/− (n = 6), and Nta^−/− (n = 6) mice were bled 7 days after the second immunization. Total Ig as well as Ig of the indicated isotypes and reactive with OVA were determined by ELISA. (D) Natural antibodies to DNA, DNP, and gelatin were assayed in preimmune sera from wild-type (n = 6) and Ntal^−/− (n = 6) mice by ELISA. The concentration of total IgG reactive with the indicated antigens was measured. Differences in both C and D are significant at P < 0.01. (E) Ntal deficiency does not prevent the hypergammaglobulinemia E and G1 that develops in LAT^Y136F mice. The concentrations of IgG1 and IgE in individual mice are plotted on a logarithmic scale. The mean of each distribution is indicated by an horizontal bar.

FIG. 8.

Normal B-cell development in Lat^−/− Ntal^−/− double-deficient mice. Single-cell suspensions from bone marrow (A), spleen (B), and peritoneum (C) were stained with the indicated antibodies and analyzed by flow cytometry. The genotypes are specified above the dot plots. Numbers indicate the percentages of cells in the specified quadrants or gates. In the case of the peritoneum, windows indicate T (CD5^hi B220⁻), B-1 (CD5^int B220^int), and follicular B (CD5⁻ B220^hi) cells. As expected, both Lat^−/− and Lat^−/− Ntal^−/− mice are deprived of mature T cells.