Impaired germinal center reaction in mice with short telomeres

Abstract

Reduction of germinal center reactivity is a landmark of immunosenescence and contributes to immunological dysfunction in the elderly. Germinal centers (GC) are characterized by extensive clonal expansion and selection of B lymphocytes to generate the pool of memory B cells. Telomere maintenance by telomerase has been proposed to allow the extensive proliferation undergone by B lymphocytes in the GC during the immune response. We show here that late generation mTR(-/-) mice, which lack the mouse telomerase RNA (mTR) and have short telomeres, present a dramatic reduction in GC number following antigen immunization. Upon immunization with an antigen, wild-type splenocyte telomeres are elongated and this is accompanied by a high expression of the telomerase catalytic subunit in the spleen GC. In contrast, telomerase-deficient mTR(-/-) splenocytes show telomere shortening after immunization, presumably due to cell proliferation in the absence of telomerase. All together, these results demonstrate the importance of telomere maintenance for antibody-mediated immune responses and support the notion that telomere elongation detected in wild-type spleens following immunization is mediated by telomerase.

Fig. 1. mTERT is expressed in the spleen follicles and GC. Representative images of spleen follicles/GC stained with K-370 anti-telomerase antibody (brown). All spleen sections were processed in parallel. (A) A spleen follicle from a non-immunized mouse pre-incubated with the peptide that was used to derive the K-370 antibody prior to K-370 incubation shows no specific staining. (B) Typical staining with K-370 antibody of a spleen follicle from a non-immunized wild-type mouse. (C) Strong K-370 staining of a spleen GC 14 days after second immunization with KLH. All images shown in this figure are at the same magnification (40× objective). We confirmed that follicles and GC were correctly identified by subsequent staining of the sections with markers B220 and PNA (the panels in this figure do not show these additional stains for purposes of clarity, but see for example Figure 3E).

Fig. 2. Reduced numbers of spleen follicles in late generation mTR^–/– mice. (A and B) Representative images of spleen sections from age-matched non-immunized wild-type and G6 mTR^–/– mice. All images are shown at the same magnification (20× objective); the fact that the follicles in (B) are smaller is coincidental, as follicles do not have an invariable size and in a given section there are larger and smaller follicles. Sections were stained with naive B cell-specific antibody B220 (blue) and with GC marker PNA (brown). The figures show mostly spleen follicles and not GC, as there was no immunization. Non-immunized mice show a weak, spatially restricted area of PNA-positive cells (brown) inside the follicle, which is unambiguously identified by B220 staining (blue). The images shown are not the full spleen section and are shown for illustrative purposes. For quantification of follicle numbers see (C). (C) The average number of follicles visualized per spleen section from several wild-type and G6 mTR^–/– mice is shown with bars. The total number of spleen sections from each mouse used to calculate follicle number per section is indicated at the bottom of the graph. In all cases, half of the spleen was used to estimate the number of follicles. All spleens had a similar size, therefore similar volumes of the spleen were scanned for follicles. The follicles were counted along the full longitudinal sections of the spleen, and an average of 20–30 follicles per section in the wild-type and 10–15 follicles per section in the G6 mTR^–/– (KO-G6) mice were counted. Standard deviation bars are shown. (D) Relative activation with respect to wild-type mice of splenocytes from G6 mTR^–/– mice following mitogen stimulation (see Materials and methods for details). α–CD3 and Con–A, T cell-specific mitogens; LPS, B cell-specific mitogen; Ion-PMA, B + T cell mitogens. Total numbers of mice used for each experiment are indicated below the graph. Standard deviation bars are also shown. See Materials and methods for detailed description of the experiment.

Fig. 3. Reduced numbers of GC in late generation mTR^–/– mice after immunization with KLH. (A) Representative images of spleen sections from non-immunized and immunized wild-type mice (14 days after the second immunization with KLH). Sections were stained with naive B cell marker B220 (blue) and with GC marker PNA (brown). All parts of this figure are the same magnification (20× objective). Non-immunized mice show a weak and spatially restricted area of PNA-positive cells (brown) inside the follicle. In contrast, immunized mice mostly present GC, which are characterized by a strong and wide area of PNA-positive cells [compare the two panels in (A)]. (B) Representative images of spleen sections from non-immunized and immunized G6 mTR^–/– mice (14 days after the second immunization with KLH). Sections were stained with naive B cell marker B220 (blue) and with GC marker PNA (brown). All parts of this figure are at the same magnification (20× objective). Non-immunized mice show a weak and spatially restricted area of PNA-positive cells (brown) inside the follicle. In contrast, immunized mice mostly present GC, which are characterized by a strong, wide area of PNA-positive cells [compare the two panels in (B)]. (C) The average number of GC visualized per spleen section from wild-type, G5 and G6 mTR^–/– mice is shown with bars. The total number of spleen sections used for the analysis is indicated at the bottom of the graph. GC were counted as described for follicles in legend to Figure 2. Standard deviation bars are shown. (D) Analysis of the in vivo immune response to KLH in wild-type and G6 mTR^–/– mice. Sera were collected 14 days after the primary immunization and the IgM and IgG anti-KLH titers were calculated by serially diluting the serum samples. The three dilutions used for each serum are represented. Results are given as the serum dilution giving half-maximal binding to Ag as measured at 492 nm that was 1873 and 1110 for total IgM and 1.6 × 10⁴ and 1.08 × 10⁴ for total IgG in two different wild-type mice, WT1 and WT2, respectively (▪ and ♦); 301 and 249 for total IgM and 1.0 × 10⁴ and 1.0 × 10⁴ for total IgG in two different G6 mTR^–/– mice, KO1-G6 and KO2-G6, respectively (○ and ▵). (E) Representative images of wild-type and G1 mTR^–/– spleens 14 days after second immunization with KLH (immunized). Sections were stained with B cell-specific antibody B220 (blue) and with GC marker PNA (brown). Both images are at the same magnification (20× objective). (F) The average number of GC visualized in spleen sections from wild-type and G1 mTR^–/– mice is shown with bars. The total number of spleen sections used for the analysis is indicated at the bottom of the graph. Standard deviation bars are shown.

Fig. 3. Reduced numbers of GC in late generation mTR^–/– mice after immunization with KLH. (A) Representative images of spleen sections from non-immunized and immunized wild-type mice (14 days after the second immunization with KLH). Sections were stained with naive B cell marker B220 (blue) and with GC marker PNA (brown). All parts of this figure are the same magnification (20× objective). Non-immunized mice show a weak and spatially restricted area of PNA-positive cells (brown) inside the follicle. In contrast, immunized mice mostly present GC, which are characterized by a strong and wide area of PNA-positive cells [compare the two panels in (A)]. (B) Representative images of spleen sections from non-immunized and immunized G6 mTR^–/– mice (14 days after the second immunization with KLH). Sections were stained with naive B cell marker B220 (blue) and with GC marker PNA (brown). All parts of this figure are at the same magnification (20× objective). Non-immunized mice show a weak and spatially restricted area of PNA-positive cells (brown) inside the follicle. In contrast, immunized mice mostly present GC, which are characterized by a strong, wide area of PNA-positive cells [compare the two panels in (B)]. (C) The average number of GC visualized per spleen section from wild-type, G5 and G6 mTR^–/– mice is shown with bars. The total number of spleen sections used for the analysis is indicated at the bottom of the graph. GC were counted as described for follicles in legend to Figure 2. Standard deviation bars are shown. (D) Analysis of the in vivo immune response to KLH in wild-type and G6 mTR^–/– mice. Sera were collected 14 days after the primary immunization and the IgM and IgG anti-KLH titers were calculated by serially diluting the serum samples. The three dilutions used for each serum are represented. Results are given as the serum dilution giving half-maximal binding to Ag as measured at 492 nm that was 1873 and 1110 for total IgM and 1.6 × 10⁴ and 1.08 × 10⁴ for total IgG in two different wild-type mice, WT1 and WT2, respectively (▪ and ♦); 301 and 249 for total IgM and 1.0 × 10⁴ and 1.0 × 10⁴ for total IgG in two different G6 mTR^–/– mice, KO1-G6 and KO2-G6, respectively (○ and ▵). (E) Representative images of wild-type and G1 mTR^–/– spleens 14 days after second immunization with KLH (immunized). Sections were stained with B cell-specific antibody B220 (blue) and with GC marker PNA (brown). Both images are at the same magnification (20× objective). (F) The average number of GC visualized in spleen sections from wild-type and G1 mTR^–/– mice is shown with bars. The total number of spleen sections used for the analysis is indicated at the bottom of the graph. Standard deviation bars are shown.

Fig. 3. Reduced numbers of GC in late generation mTR^–/– mice after immunization with KLH. (A) Representative images of spleen sections from non-immunized and immunized wild-type mice (14 days after the second immunization with KLH). Sections were stained with naive B cell marker B220 (blue) and with GC marker PNA (brown). All parts of this figure are the same magnification (20× objective). Non-immunized mice show a weak and spatially restricted area of PNA-positive cells (brown) inside the follicle. In contrast, immunized mice mostly present GC, which are characterized by a strong and wide area of PNA-positive cells [compare the two panels in (A)]. (B) Representative images of spleen sections from non-immunized and immunized G6 mTR^–/– mice (14 days after the second immunization with KLH). Sections were stained with naive B cell marker B220 (blue) and with GC marker PNA (brown). All parts of this figure are at the same magnification (20× objective). Non-immunized mice show a weak and spatially restricted area of PNA-positive cells (brown) inside the follicle. In contrast, immunized mice mostly present GC, which are characterized by a strong, wide area of PNA-positive cells [compare the two panels in (B)]. (C) The average number of GC visualized per spleen section from wild-type, G5 and G6 mTR^–/– mice is shown with bars. The total number of spleen sections used for the analysis is indicated at the bottom of the graph. GC were counted as described for follicles in legend to Figure 2. Standard deviation bars are shown. (D) Analysis of the in vivo immune response to KLH in wild-type and G6 mTR^–/– mice. Sera were collected 14 days after the primary immunization and the IgM and IgG anti-KLH titers were calculated by serially diluting the serum samples. The three dilutions used for each serum are represented. Results are given as the serum dilution giving half-maximal binding to Ag as measured at 492 nm that was 1873 and 1110 for total IgM and 1.6 × 10⁴ and 1.08 × 10⁴ for total IgG in two different wild-type mice, WT1 and WT2, respectively (▪ and ♦); 301 and 249 for total IgM and 1.0 × 10⁴ and 1.0 × 10⁴ for total IgG in two different G6 mTR^–/– mice, KO1-G6 and KO2-G6, respectively (○ and ▵). (E) Representative images of wild-type and G1 mTR^–/– spleens 14 days after second immunization with KLH (immunized). Sections were stained with B cell-specific antibody B220 (blue) and with GC marker PNA (brown). Both images are at the same magnification (20× objective). (F) The average number of GC visualized in spleen sections from wild-type and G1 mTR^–/– mice is shown with bars. The total number of spleen sections used for the analysis is indicated at the bottom of the graph. Standard deviation bars are shown.

Fig. 3. Reduced numbers of GC in late generation mTR^–/– mice after immunization with KLH. (A) Representative images of spleen sections from non-immunized and immunized wild-type mice (14 days after the second immunization with KLH). Sections were stained with naive B cell marker B220 (blue) and with GC marker PNA (brown). All parts of this figure are the same magnification (20× objective). Non-immunized mice show a weak and spatially restricted area of PNA-positive cells (brown) inside the follicle. In contrast, immunized mice mostly present GC, which are characterized by a strong and wide area of PNA-positive cells [compare the two panels in (A)]. (B) Representative images of spleen sections from non-immunized and immunized G6 mTR^–/– mice (14 days after the second immunization with KLH). Sections were stained with naive B cell marker B220 (blue) and with GC marker PNA (brown). All parts of this figure are at the same magnification (20× objective). Non-immunized mice show a weak and spatially restricted area of PNA-positive cells (brown) inside the follicle. In contrast, immunized mice mostly present GC, which are characterized by a strong, wide area of PNA-positive cells [compare the two panels in (B)]. (C) The average number of GC visualized per spleen section from wild-type, G5 and G6 mTR^–/– mice is shown with bars. The total number of spleen sections used for the analysis is indicated at the bottom of the graph. GC were counted as described for follicles in legend to Figure 2. Standard deviation bars are shown. (D) Analysis of the in vivo immune response to KLH in wild-type and G6 mTR^–/– mice. Sera were collected 14 days after the primary immunization and the IgM and IgG anti-KLH titers were calculated by serially diluting the serum samples. The three dilutions used for each serum are represented. Results are given as the serum dilution giving half-maximal binding to Ag as measured at 492 nm that was 1873 and 1110 for total IgM and 1.6 × 10⁴ and 1.08 × 10⁴ for total IgG in two different wild-type mice, WT1 and WT2, respectively (▪ and ♦); 301 and 249 for total IgM and 1.0 × 10⁴ and 1.0 × 10⁴ for total IgG in two different G6 mTR^–/– mice, KO1-G6 and KO2-G6, respectively (○ and ▵). (E) Representative images of wild-type and G1 mTR^–/– spleens 14 days after second immunization with KLH (immunized). Sections were stained with B cell-specific antibody B220 (blue) and with GC marker PNA (brown). Both images are at the same magnification (20× objective). (F) The average number of GC visualized in spleen sections from wild-type and G1 mTR^–/– mice is shown with bars. The total number of spleen sections used for the analysis is indicated at the bottom of the graph. Standard deviation bars are shown.

Fig. 4. Mitogen-induced proliferation in splenocytes from non-immunized and KLH-immunized wild-type and G6 mTR^–/– mice. Activation following mitogen stimulation of splenocytes from immunized wild-type and G6 mTR^–/– mice with respect to that of non-immunized mice (see Materials and methods). For simplicity, activation of non-immunized wild-type splenocytes is considered to be 100% for all mitogens used, and the remainder of the values are expressed as a percentage relative to wild-type activation. α–CD3 and Con–A, T cell-specific mitogens; LPS, B cell-specific mitogen; Ion-PMA, B + T cell mitogens. Non-immunized and 14 days after second immunization with KLH. Total numbers of mice used in each experiment are indicated above the graph. Standard deviation bars are also shown.

Fig. 5. Apoptosis following mitogen stimulation in splenocytes and thymocytes from wild-type, G5 and G6 mTR^–/– mice. Cell cycle distribution of total splenocytes after LPS treatment. LPS, B cell-specific mitogen. White, apoptosis; light grey, cells in G₁; dark grey, cells in S–phase; black, cells in G₂. The total number of mice used for the analysis is indicated at the bottom of the graph. Standard deviation bars are shown.

Fig. 6. Metaphase chromosomes from wild-type and G5 mTR^–/– splenocytes from mice prior to and after KLH immunization. (A and B) Metaphases from wild-type splenocytes. (A) Metaphase corresponding to splenocytes from a non-immunized mouse. (B) Metaphase of splenocytes from a KLH-immunized mouse. Quantification of telomere length of these cells is shown in Table I. Images are shown for illustrative purposes only. (C and D) Metaphases from fifth generation mTR^–/– splenocytes. (C) Metaphase corresponding to splenocytes from a non-immunized mouse. (D) Metaphase of splenocytes from a KLH-immunized mouse. Quantification of telomere length of these cells is shown in Table I. Images are shown for illustrative purposes only. (E) Distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocytes from control wild-type mice (Wt-1, Wt-2, Wt-3) or wild-type mice that have been immunized with KLH (Wt-5, Wt-6 and Wt-7). The frequency of chromosomes with long telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized. (F) The distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocyte-derived control G1 mTR^–/– mice (KOG1-1 and KOG1-2) or G1 mTR^–/– mice that have been immunized with KLH (KOG1-3 and KOG1-4). The frequency of chromosomes with short telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized. (G) The distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocyte-derived control G5 mTR^–/– mice (KOG5-1 and KOG5-2) or G5 mTR^–/– mice that have been immunized with KLH (KOG5-3, KOG5-4 and KOG5-5). The frequency of chromosomes with long telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized.

Fig. 6. Metaphase chromosomes from wild-type and G5 mTR^–/– splenocytes from mice prior to and after KLH immunization. (A and B) Metaphases from wild-type splenocytes. (A) Metaphase corresponding to splenocytes from a non-immunized mouse. (B) Metaphase of splenocytes from a KLH-immunized mouse. Quantification of telomere length of these cells is shown in Table I. Images are shown for illustrative purposes only. (C and D) Metaphases from fifth generation mTR^–/– splenocytes. (C) Metaphase corresponding to splenocytes from a non-immunized mouse. (D) Metaphase of splenocytes from a KLH-immunized mouse. Quantification of telomere length of these cells is shown in Table I. Images are shown for illustrative purposes only. (E) Distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocytes from control wild-type mice (Wt-1, Wt-2, Wt-3) or wild-type mice that have been immunized with KLH (Wt-5, Wt-6 and Wt-7). The frequency of chromosomes with long telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized. (F) The distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocyte-derived control G1 mTR^–/– mice (KOG1-1 and KOG1-2) or G1 mTR^–/– mice that have been immunized with KLH (KOG1-3 and KOG1-4). The frequency of chromosomes with short telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized. (G) The distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocyte-derived control G5 mTR^–/– mice (KOG5-1 and KOG5-2) or G5 mTR^–/– mice that have been immunized with KLH (KOG5-3, KOG5-4 and KOG5-5). The frequency of chromosomes with long telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized.

Fig. 6. Metaphase chromosomes from wild-type and G5 mTR^–/– splenocytes from mice prior to and after KLH immunization. (A and B) Metaphases from wild-type splenocytes. (A) Metaphase corresponding to splenocytes from a non-immunized mouse. (B) Metaphase of splenocytes from a KLH-immunized mouse. Quantification of telomere length of these cells is shown in Table I. Images are shown for illustrative purposes only. (C and D) Metaphases from fifth generation mTR^–/– splenocytes. (C) Metaphase corresponding to splenocytes from a non-immunized mouse. (D) Metaphase of splenocytes from a KLH-immunized mouse. Quantification of telomere length of these cells is shown in Table I. Images are shown for illustrative purposes only. (E) Distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocytes from control wild-type mice (Wt-1, Wt-2, Wt-3) or wild-type mice that have been immunized with KLH (Wt-5, Wt-6 and Wt-7). The frequency of chromosomes with long telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized. (F) The distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocyte-derived control G1 mTR^–/– mice (KOG1-1 and KOG1-2) or G1 mTR^–/– mice that have been immunized with KLH (KOG1-3 and KOG1-4). The frequency of chromosomes with short telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized. (G) The distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocyte-derived control G5 mTR^–/– mice (KOG5-1 and KOG5-2) or G5 mTR^–/– mice that have been immunized with KLH (KOG5-3, KOG5-4 and KOG5-5). The frequency of chromosomes with long telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized.

Fig. 6. Metaphase chromosomes from wild-type and G5 mTR^–/– splenocytes from mice prior to and after KLH immunization. (A and B) Metaphases from wild-type splenocytes. (A) Metaphase corresponding to splenocytes from a non-immunized mouse. (B) Metaphase of splenocytes from a KLH-immunized mouse. Quantification of telomere length of these cells is shown in Table I. Images are shown for illustrative purposes only. (C and D) Metaphases from fifth generation mTR^–/– splenocytes. (C) Metaphase corresponding to splenocytes from a non-immunized mouse. (D) Metaphase of splenocytes from a KLH-immunized mouse. Quantification of telomere length of these cells is shown in Table I. Images are shown for illustrative purposes only. (E) Distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocytes from control wild-type mice (Wt-1, Wt-2, Wt-3) or wild-type mice that have been immunized with KLH (Wt-5, Wt-6 and Wt-7). The frequency of chromosomes with long telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized. (F) The distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocyte-derived control G1 mTR^–/– mice (KOG1-1 and KOG1-2) or G1 mTR^–/– mice that have been immunized with KLH (KOG1-3 and KOG1-4). The frequency of chromosomes with short telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized. (G) The distribution of telomere fluorescence intensity values of the average of p- and q-telomeres (black bars) in splenocyte-derived control G5 mTR^–/– mice (KOG5-1 and KOG5-2) or G5 mTR^–/– mice that have been immunized with KLH (KOG5-3, KOG5-4 and KOG5-5). The frequency of chromosomes with long telomeres increases in the splenocytes from immunized mice. N, non-immunized; I, immunized.