Germinal Center Formation Motif Signaling in B Cells and Reduced

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Altered Regulation of FcγRII on Aged Follicular Dendritic Cells Correlates with Immunoreceptor Tyrosine-Based Inhibition Motif Signaling in B Cells and Reduced This information is current as Germinal Center Formation of October 3, 2021. Yüksel Aydar, Péter Balogh, John G. Tew and Andras K. Szakal J Immunol 2003; 171:5975-5987; ;

doi: 10.4049/jimmunol.171.11.5975 Downloaded from http://www.jimmunol.org/content/171/11/5975

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Altered Regulation of Fc␥RII on Aged Follicular Dendritic Cells Correlates with Immunoreceptor Tyrosine-Based Inhibition Motif Signaling in B Cells and Reduced Germinal Center Formation1

Yu¨ksel Aydar,* Pe´ter Balogh,* John G. Tew,† and Andras K. Szakal2*

Aging is associated with reduced trapping of Ag in the form of in immune complexes (ICs) by follicular dendritic cells (FDCs). We postulated that this defect was due to altered regulation of IC trapping receptors. The level of FDC-M1, complement receptors 1 and 2, Fc␥RII, and FDC-M2 on FDCs was immunohistochemically quantitated in draining lymph nodes of actively immunized mice for 10 days after Ag challenge. Initially, FDC Fc␥RII levels were similar but by day 3 a drastic reduction in FDC-Fc␥RII expression was apparent in old mice. FDC-M2 labeling, reﬂecting IC trapping, was also reduced and correlated with a dramatic Downloaded from reduction in germinal center (GC) B cells as indicated by reduced GC size and number. Nevertheless, labeling of FDC reticula with FDC-M1 and anti-complement receptors 1 and 2 was preserved, indicating that FDCs were present. FDCs in active GCs normally express high levels of FcRs that are thought to bind Fc portions of Abs in ICs and minimize their binding to FcRs on B cells. Thus, cross-linking of B cell receptor and FcR via IC is minimized, thereby reducing signaling via the immunoreceptor tyrosine-based inhibition motif. Old FDCs taken at day 3, when they lack Fc␥RII, were incapable of preventing immunoreceptor tyrosine-based http://www.jimmunol.org/ inhibition motif signaling in wild-type B cells but old FDCs stimulated B cells from Fc␥RIIB؊/؊ mice to produce near normal levels of speciﬁc Ab. The present data support the concept that FcR are regulated abnormally on old FDCs. This abnormality correlates with a reduced IC retention and with a reduced capacity of FDCs to present ICs in a way that will activate GC B cells. The Journal of Immunology, 2003, 171: 5975–5987.

he follicular dendritic cells (FDCs)3 reside in the light a reduced number of germinal center (GC) B cells (19). Although zone of secondary lymphoid follicles, where their den- there is a marked age-related depression in GC development and T dritic processes interdigitate and form a three-dimen- specific Ab production, the ratio of the FDC reticulum to GC num- sional network, an FDC reticulum (1-4). FDCs function in capture bers remains 1:1 (18–21), emphasizing the dependence of GC de- by guest on October 3, 2021 and long-term retention of immune complexes (ICs) (5–7), the velopment on FDCs even in aging. blocking of apoptosis, the survival of specific B cells (8, 9), and the Fc␥RII and CR2 (CD21) exist on both FDCs and B cells and are promotion of the development of potent high-affinity (10) recall involved in important ligand-receptor interactions including FDC- responses (11, 12). The capture and retention of ICs are mediated Fc␥R-Fc in IC and FDC-CD21 ligand (CD21L)-B cell CD21. by an array of cell surface receptors that include Fc␥RII (13, 14) FDC-CD21L provides B cells with a critical costimulatory signal and complement receptors (complement receptors 1 and 2 (CR1/ and FDC-Fc␥RII minimizes coligation of B cell receptor (BCR) 2)) (15–17). and B cell-FcR (11, 13–17, 22). Fc␥RII is highly expressed on Senescence leads to a pronounced reduction in the secondary FDCs in secondary follicle GCs and the expression pattern of humoral immune response, the appearance of atrophic FDCs (18) Fc␥RII on FDC reticula supports the development of a potent Ab that trap and retain little ICs, produces few iccosomes, and induces response (11, 13, 23). The expression of Fc␥RII on FDCs in GCs is critical for FDCs to retain and mediate the conversion of ICs to a highly immunogenic form and for the generation of strong recall responses (11, 13). Moreover, Fc␥RII promotes the maturation of *Department of Anatomy and Neurobiology, The Immunology Group, and †Depart- the FDC reticulum (24); Fc␥RIIBϪ/Ϫ mice show a substantial de- ment of Microbiology and Immunology, Virginia Commonwealth University, Med- ical College of Virginia, Richmond, VA 23298 lay in the development of FDC reticula (24). The ability of FDCs ␥ Ϫ/Ϫ Received for publication October 24, 2002. Accepted for publication September from Fc RIIB mice to augment Ag-specific IgG production by 22, 2003. wild-type B cells (11, 13) or when incubated with the anti-Fc␥RII The costs of publication of this article were defrayed in part by the payment of page mAb 2.4G2 is markedly depressed. Furthermore, it appears that charges. This article must therefore be hereby marked advertisement in accordance engagement of Ig-Fc with numerous Fc␥RII on FDCs decreases with 18 U.S.C. Section 1734 solely to indicate this fact. the chances of signaling B cells via the immunoreceptor tyrosine- 1 This work was supported by National Institutes of Health Grant AG-17063. Y.A. is the recipient of a scholarship from the Ministry of National Education in Turkey. based inhibition motif (ITIM) induced by cross-linking BCR and B cell Fc␥RII via Ab-Ag complexes (J.G.T., unpublished data). 2 Address correspondence and reprint requests to Dr. Andras K. Szakal, Department of Anatomy and Neurobiology, MCV/VCU, 1101 East Marshall Street, Richmond, Thus, FDCs minimize a negative signal to B cells. VA 23298. E-mail address: [email protected] Although the phenomenon of age-associated reduction in IC 3 Abbreviations used in this paper: FDC, follicular dendritic cell; IC, immune com- trapping in old mice is well known (18, 19), the cellular and mo- plex; GC, germinal center; CD21L, CD21 ligand; CR1/2, complement receptors 1 and 2; ITIM, immunoreceptor tyrosine-based inhibition motif; PNA, peanut agglutinin; lecular bases for this defect have not been defined. Numerous stud- SHIP, Src homology 2-containing inositol phosphatase. ies indicate the importance of Fc␥RII and CR1/2 in IC trapping

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 5976 AGE-RELATED CHANGES IN MOLECULAR INTERACTIONS OF FDC AND B CELLS and mediation of costimulatory signals to B cells in humans as Materials and Methods well as mice (3, 7, 9, 11, 13). However, potential age-related Animals changes in the function and molecular interactions of the Fc␥RII BALB/c mice at 2–3 and 20–21 mo of age were purchased from the Na- and CR1/2 have not been determined. tional Institute on Aging Contract Facility (Harlan, Indianapolis, IN) and Age-related changes in IC trapping, reductions in the FDC re- maintained under speciﬁc pathogen-free conditions. The mice were housed ticulum, GC numbers, and size prompted the hypothesis that in old in standard plastic shoebox cages with ﬁlter tops. Food and water were mice reduced IC retention is due to a defect in expression of sur- supplied ad libitum. The young mice were used between 2 and 3 mo of age and old mice were used between 21 and 22 mo of age. The mice were face receptors that involve IC trapping. To test this hypothesis, handled in accordance with Virginia Commonwealth University Animal lymph node sections of actively immunized mice were immuno- Care and Use guidelines. histochemically quantitated for Fc␥RII and CR1/2 receptors, for Immunizations FDC reticulum size (anti-FDC-M1), for the capacity to trap ICs (anti-FDC-M2), and for GCs (peanut agglutinin (PNA)). The re- As described previously for active immunization (25, 26), mice received an sults showed that aging reduced the expression of Fc␥RII on FDCs initial 0.1-ml injection s.c. behind the nape of the neck consisting of 200 ␮g/ml aluminum potassium sulfate (A7167; Sigma-Aldrich, St. Louis, by day 3 after Ag challenge that correlated with the reduced trap- MO), precipitated OVA (A5503; Sigma-Aldrich), and 5 ϫ 108 heat-killed ping of ICs (FDC-M2 reactivity). In accordance with these results, Bordetella pertussis. Two weeks later, the mice were given a booster im- old FDCs taken at day 3 could not prevent ITIM signaling and Src munization of 50 ␮g of OVA i.p. and by s.c. injection 5 ␮g of OVA into homology 2-domain-containing inositol phosphatase (SHIP) phos- each of the front legs and hind feet. Finally, all mice were challenged at least 2 wk after the last immunization by s.c. injection of 5 ␮g of OVA into phorylation because of the dramatic age-related reduction of each of the front legs and hind feet and used at 0, 1, 3, 5, and 10 days after ␥ Fc RII expression. the challenge. Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 1. Representative light micrographs illustrating the kinetics of immunohistochemical labeling of FDC reticula in draining axillary lymph nodes of actively immunized young (6–8 wk) and old (22 mo) mice with FDC-M1. The same relationships were apparent in brachial and popliteal lymph nodes. Mice were primed and boosted with OVA and 3 wk after the ﬁnal booster the animals were challenged with 5 ␮gof OVA (day 0). D, Day. Magniﬁcation, ϫ120. The Journal of Immunology 5977

Abs and other reagents The rat mAbs against CR1/2 (CD21/35, clone 7G6), CD16/32 (clone 2.4G2), and biotinylated mouse anti-rat ␬-chain mAb (MRK-1) were obtained from BD PharMingen (San Diego, CA). Low-Tox rabbit complement was purchased from Cedarlane Laboratories (Westbury, NY). The normal rat serum, phenylhydrazine-HCl, the PNA lectin conjugated with peroxidase (PNA-HRP), sodium orthovanadate, sodium fluoride, sodium deoxycholic acid, Igepal CA-630, PMSF, and the streptavidin-peroxidase (streptavidin-HRP) were obtained from Sigma-Aldrich. Rabbit anti-mouse SHIP-145/135 (SHIP-1␣, 145 kDa; SHIP-1␤, 135 kDa), HRP-conjugated anti-phosphotyrosine (4G10), and protein A agarose were obtained from Upstate Biotechnology (Lake Placid, NY). Peroxidase-conjugated affi- ϩ Ј nipure goat anti-rabbit IgG (H L) and affinipure F(ab )2 goat anti-mouse IgG (H ϩ L) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). The FDC-M1 and FDC-M2 hybridoma cell lines were generously provided by Dr. M. Kosco-Vilbois (Serono, Switzerland), and their supernatant was used as primary Ab. FIGURE 2. Size of the average FDC reticulum in the draining lymph nodes of young and old mice as assessed by FDC-M1 labeling. Young (f) IC preparation and incubation of FDCs with IC and and old (Ⅺ) mice were actively immunized and challenged with OVA as complement described for the animals in Fig. 1. The averages were calculated using data The FDCs were prepared and incubated with ICs and complement as de- from three to six mice per day. Note that FDC-M1-positive FDC reticula scribed previously by Aydar et al. (27). In brief, OVA and affinity-purified Downloaded from were essentially identical in both young and old mice and showed about a anti-OVA were used to make ICs for use in cultures. A final ratio of 1 30% increase in size during the first 10 days of GC response. The data ng/ml OVA to 6 ng/ml anti-OVA was used to make up the ICs in Ag represent the mean in square micrometers Ϯ SEM. http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 3. Representative light micrographs illustrating the kinetics of immunohistochemical labeling of FDC reticula in draining axillary lymph nodes of actively immunized young (6–8 wk) and old (22 mo) mice with anti-CR1/2 (CD21/35, clone 7G6). The same relationships were apparent in brachial and popliteal lymph nodes. These sections are from the same mice described in Figs. 1 and 2. D, Day. Magniﬁcation, ϫ120. 5978 AGE-RELATED CHANGES IN MOLECULAR INTERACTIONS OF FDC AND B CELLS

IgG was measured by means of a solid-phase ELISA as previously described (13, 26, 29).

Immunohistochemistry Young and old mice were actively immunized with OVA and challenged 0, 1, 3, 5, and 10 days before use. After removal, the draining lymph nodes (axillary, brachial, and popliteal) were pooled and frozen in CryoForm- embedding medium (IEC, Needham Heights, MA) and frozen sections of 10-␮m thickness were cut on a Leitz (1720 Digital) cryostat and air dried. Five representative sections, three from the center and one from each side of the lymph nodes were collected and processed together for the quantitative analysis. Following acetone ﬁxation, the sections were rehydrated and the endogenous peroxidase activity was quenched with PBS containing 0.1% phenylhydrazine-HCl. After washing, the sections were saturated with 10% BSA, then incubated with unlabeled rat mAbs. The bound Abs were detected using biotinylated mouse anti-rat ␬-chain mAb followed by incubating the sections with streptavidin-HRP conjugate. The reaction was visualized using 3-amino-9-ethylcarbazole in a 0.1 M sodium acetate buffer

containing 0.03% H2O2 substrate. Downloaded from Analyses of immunohistochemistry results BIOQUANT NOVA Advanced Image Analysis software (R&M Biomet- rics, Nashville, TN) was used to analyze the immunohistochemistry studies. BIOQUANT NOVA is the core measurement software for image analysis applications. It measures area, length, number, position, density, and a

range of more specialized parameters. http://www.jimmunol.org/ FIGURE 4. The density and size of average FDC reticulum labeling in Setting the threshold. A threshold is a set for red, green, and blue inten- the draining axillary lymph nodes of young and old mice as assessed by sity values that are used to distinguish objects of interest in an image. The anti-CR1/2. Young (f) and old (Ⅺ) mice were actively immunized and word “threshold” also refers to the colorful highlighting on the screen that challenged with OVA. These are the same lymph nodes as those shown in results from setting a threshold range. Initially, we set an arbitrary thresh- the preceding figures from three to six mice per group. A, Note that the old that was the best threshold possible for the objects of interest. The same labeling densities of FDC reticula were essentially identical in young and threshold was used strictly throughout the measurements to enable a reli- ␥ old mice. The data represent the mean Ϯ SEM in color value of pixels. B, able comparison of average density for CR1/2, Fc RII, and FDC-M2 stain- ␥ Note that FDC-M1-positive FDC reticula were essentially identical in size ing, FDC reticulum size for FDC-M1, CR1/2, Fc RII, and FDC-M2, and PNA-positive GC size for PNA staining between young and old mice on in both young and old mice. The data represent the mean in square mi- successive days after Ag challenge. For each measurement, the objects of by guest on October 3, 2021 crometers Ϯ SEM. interest were selected by drawing an enclosed circle and filling it with the predefined threshold. Average density measurement. Density measurements in a color imaging excess. Young and old FDCs were incubated with ICs for2hinLow-Tox system (like BIOQUANT NOVA) are done by examining three channels rabbit complement to load FDCs with CD21L. (red, green, and blue), which comprise a color image. All three color channels may be used when setting a threshold to define the objects of interest Cell cultures and ELISA to be measured. But, the density measurement is made only on the channel designated in the density array’s comment. The default (and most com- Cell cultures were set up by adding enriched FDC preparations to B and T monly used) channel is the green channel since it most closely approxi- memory lymphocytes as described by Aydar et al. (27). In brief, FDCs mates the range of colors perceptible by the human eye. The average den- were isolated from lymph nodes (axillary, lateral axillary, inguinal, popli- ␥ teal, mesenteric, and para-aortic) of normal young adult mice by using a sity analyses for FDC-M1, CR1/2, Fc RII, and FDC-M2 were done using modification of a previously described procedure (26). Three days before average density. Average density looks at a group of pixels and measures isolation, the mice were exposed to whole body irradiation (600 rad, Ce137 their average color value. It is the most common way to compare the source). The irradiation dose does not interfere with FDC accessory func- amount of light-absorbing stain present in one object to others. Computers tions but eliminates many of the B and T cells, which otherwise form work on a color scale from 0 to 255. Zero is black and 255 is the brightest clusters with FDCs and decrease the FDC purity (28). Single-cell suspen- color. Note that this is backwards from conventional “absorption” scales sions were prepared from the lymph nodes by using enzymatic digestion. where darker regions have larger data values than lighter regions. To make Each lymph node capsule was opened using two 26-gauge needles, then the the data easily traceable for the readers, we reversed the relative density lymph nodes were placed in an enzyme mixture consisting of 1 ml of assignments by subtracting the measured values from the 255 so that 0 collagenase D (16 mg/ml, C-1088882; Roche, Basel, Switzerland), 0.5 ml corresponds to white and 255 corresponds to black (darker regions have of DNase I (5000 U/ml, D-4527; Sigma-Aldrich), and 0.5 ml of DMEM larger data values than lighter regions in our illustrations). ␮ supplemented with 20 mM HEPES, 2 mM glutamine, 50 g/ml gentami- Area measurement. The area array records the area of selected objects in cin, and MEM nonessential amino acids (Life Technologies, Rockville, a single field or across multiple fields. The system measures the selected MD). After 30 min at 37oCinaCO incubator, the medium and released 2 areas as square micrometers. When the density threshold settings and size cells were removed and transferred to a 15-ml conical centrifuge tube con- settings are satisfactory, the areas are counted using “Object Mode for all taining 5 ml of DMEM with 20% FCS and placed at 4oC. The remaining Objects” comment. For the present studies, we measured FDC reticulum tissue was subjected to a second 30-min digestion in a fresh aliquot of ␥ enzyme mixture and the cells were collected as before. Isolated cells were size for FDC-M1, CR1/2, Fc RII, and FDC-M2. We also measured PNA- gently layered on a continuous 50% Percoll gradient and centrifuged for 25 positive GC size for PNA. FDC reticulum size, labeled with FDC-M1, ␥ min at 700 ϫ g. The low-density (1.050–1.060 g/ml) fraction of cells CR1/2, Fc RII, or FDC-M2, was determined by using the automeasure- containing the FDCs was collected, washed by centrifugation at 400 ϫ g, ment mode of BIOQUANT NOVA, filling the FDC reticulum with pre- and resuspended in DMEM with 10% FCS, then incubated at 37oC for 1h defined threshold that most closely approximates the borders of FDC re- in a small tissue culture dish (Costar 3035; Costar, Cambridge, MA) to ticulum. PNA-positive GC size was determined by using the manual deplete adherent cells. After incubation, the nonadherent cells were col- measurement mode of BIOQUANT NOVA, which allows drawing of a lected, washed, and resuspended in DMEM with 10% FCS. OVA-specific circle around labeling the GC, then measures the inside of the circle. The Journal of Immunology 5979

FIGURE 5. Representative light micrographs illustrating the kinetics of immunohistochemical labeling of FDC reticula in draining axillary lymph nodes of actively immunized young and old mice with anti- Downloaded from Fc␥RII (CD16/32, clone 2.4G2). These are the same lymph nodes as those shown in the preceding ﬁgures. Note the striking decrease in FDC reticula after day 1 in the aged FDCs (D, day). The same relationships were apparent in brachial and popliteal lymph nodes. Magniﬁcation, ϫ120. http://www.jimmunol.org/ by guest on October 3, 2021

Determination of average density, average FDC reticulum size, young or old FDCs for 30 min at 37oC and then the cells were stimulated and average GC size with 10 ␮g/ml goat anti-mouse BCR Abs for 5 min at 37oC. Draining lymph nodes (two axillaries, two brachial, and two popliteal) Preparation of cell lysates were sectioned and used to quantitate average density, average FDC reticulum size, and average GC size per follicle. The average density for FDC- The cocultured cells were lysed in cold 10 mM Tris-HCl buffer (pH 7.5) M1, CR1/2, Fc␥RII, and FDC-M2 were done by adding up the densities of supplemented with 0.5% Nonidet P-40, 100 mM NaF, 1 mM Na3VO4, and follicles in the draining lymph nodes and dividing the sum by the number protease and phosphatase inhibitors (100 mM aprotinin, 5 ␮M leupeptin, of reticula. Similarly, the FDC reticulum sizes for FDC-M1, CR1/2, and 200 mM PMSF) for 30 min at 4oC, with agitation. The cell lysates were Fc␥RII, and FDC-M2 were measured by summing up the FDC reticulum centrifuged at 12,000 ϫ g for 30 min at 4oC, and the soluble fractions were size of follicles in the draining lymph nodes and dividing the sum by the collected for immunoprecipitation of SHIP. number of FDC reticula. The average PNA-positive GC size was determined by adding up PNA-positive GC sizes in follicles in all draining Immunoprecipitation of SHIP and Western blotting analysis lymph nodes and then dividing the sum by the number of GCs examined. The cell lysates were incubated with anti-SHIP (2 ␮g/ml) overnight at 4oC. Cell culture and stimulation To precipitate the Ab-associated proteins, the lysates were incubated with 100 ␮l of protein A-coated beads (protein A agarose) overnight at 4oC and A20 cells (mouse B lymphoma cell line) were obtained through American beads were recovered by centrifugation at 4000 ϫ g for 5 min at 4oC and Type Culture Collection (Manassas, VA) and were grown in DMEM sup- washed ﬁve times with ice-cold lysis buffer (radioimmunoprecipitation plemented with 10% (v/v) heat-inactivated FBS (HyClone, Logan, UT), 20 buffer). All washes were performed with protease and phosphatase inhib- mM HEPES, 2 mM glutamine, 50 ␮g/ml gentamicin, and MEM-nones- itors. Bound proteins were eluted from the beads by boiling for 5 min in 50 sential amino acids. Cells were harvested and washed with cold HBSS and ␮l of Laemmli sample buffer containing 50 ␮l of 2-ME/ml and centrifuged then the cells were equilibrated for 30 min at 37oC. To study the inhibition to remove the beads. The supernatants were collected and kept at Ϫ80oC of SHIP phosphorylation by FDCs, 2 ϫ 107 A20 cells were cocultured with until used. The eluted proteins were separated by SDS-PAGE (7% (w/v) 5980 AGE-RELATED CHANGES IN MOLECULAR INTERACTIONS OF FDC AND B CELLS

a quantitative assessment of FDC-associated molecules involved in FDC reticulum development in the draining lymph nodes of young and old mice. The average density of molecules in an FDC reticulum was determined for FDC-M1, CR1/2, Fc␥RII, and FDC-M2 staining during the GC reaction in response to Ag chal- lenges. The lymph nodes were evaluated at time 0 (pre-existing FDC reticula 3 wk after primary immunization) and then at days 1, 3, 5, and 10 after Ag injection. FDC reticulum size and PNA- positive GC size were expressed in square micrometers. Average density was expressed as average color value of the pixels in the region of the interest. Kinetics of FDC-M1-positive FDC reticulum development To determine the size of an average FDC reticulum, the FDC- specific mAb FDC-M1 was used. FDC-M1 is a murine follicular dendritic cell-reactive mAb that labels primarily the FDC reticula and some tingible body macrophages. Representative sections illustrating typical labeling patterns for FDC-M1 are presented in Fig. 1. The average reticulum size was quantitated using sections from three to six mice per group (Fig. 2). The pre-existing FDC- Downloaded from M1-positive FDC reticulum size was moderately large (ϳ40,000 ␮m2) at day 0 (3 wk after primary immunization) in these actively OVA-immunized young and old mice (Fig. 2). FDC-M1-positive FDC reticulum size enlarged gradually at days 1 and 3 and peaked at day 5 (ϳ70,000 ␮m2) in both young and old mice (Fig. 2). By day 10, FDC-M1-positive FDC reticulum size was reduced to pre- http://www.jimmunol.org/ FIGURE 6. The density and size of average FDC reticulum labeling in challenge level and became more compact both in young and old the draining axillary lymph nodes of young and old mice as assessed by anti-Fc␥RII (CD16/32, clone 2.4G2). Young (f) and old (Ⅺ) were actively mice (Fig. 2). No significant size differences were found between immunized and challenged with OVA. These are the same lymph nodes young and old mice, suggesting the number of FDCs in the reticula (three to six mice per group) as those shown in the preceding figures. A, of aged mice is not deficient. Note the striking decline in labeling intensity from day 1 in the reticula from aged FDCs. The data represent the mean Ϯ SEM in color value of Kinetics of CR1- and 2-positive FDC reticulum development pixels and significant age-related differences are indicated by the asterisk Since it is known that complement plays a significant role along p Ͻ 0.05).B,Note that Fc␥RII-positive FDC reticulum size was sig- with Fc␥RII in the trapping of ICs by FDCs, we examined the ,ء) by guest on October 3, 2021 nificantly reduced in old mice compared with young and remained very CR1/2 reactivity of FDCs in the reticula of draining lymph nodes small during the 3, 5, and, 10 days of the GC response. The data represent using a mAb reactive with CR1/2. FDCs labeled intensely when the mean in square micrometers Ϯ SEM and significant age-related dif- compared with B cells (Fig. 3). The average density of CR1/2 .(p Ͻ 0.0001 ,ء) ferences are indicated by the asterisk staining, and the average size of CR1/2-positive FDC reticula were determined and results for old and young mice at days 0, 1, 3, 5, gel) and electrotransferred to nitrocellulose membranes (Protran BA 79). and 10 were indistinguishable (Fig. 4). The size of the pre-existing The blots were blocked with 5% (w/v) BSA in a buffer containing Tween CR1/2- positive FDC reticula was moderately large (ϳ38,000 20 plus TBS overnight at 4oC and probed with HRP-conjugated anti- ␮m2) in both young and old mice at day 0 (Fig. 4B), but it in- phosphotyrosine (4G10) Ab. Signals were detected using the ECL kit (Am- creased and peaked at day 5 (ϳ68,000 ␮m2). By day 10, the CR1/ ersham Pharmacia Biotech, Piscataway, NJ). For loading controls, the membranes probed with HRP-conjugated anti-phosphotyrosine Ab were 2-positive FDC reticulum size slightly reduced and became more washed twice for 10 min in TBST after signal detection. Then the mem- compact in both the young and old lymph nodes (Fig. 4B). Overall,

branes were stripped with 0.1 M glycine, 50 mM KCl in distilled H2Oat there was no significant difference between the CR1/2-positive pH 2.2 for 15 min at room temperature to remove the anti-phosphotyrosine. FDC reticulum size of young and old mice during the GC response ␮ Stripped membranes were incubated with rabbit anti-mouse SHIP (2 g/ and results were consistent with FDC-M1. ml) for overnight at 4oC and labeled with HRP-conjugated secondary Abs. Signals were detected using the ECL kit. Kinetics of Fc␥RII-positive FDC reticulum development Western blot data quantitation We next examined whether age-related changes in the level of The ECL signal was quantitated using a scanner and a densitometry pro- Fc␥RII expression were detectable using anti-Fc␥R mAb (clone gram (Scion Image, Frederick, MD). The films were exposed to nitrocel- 2.4G2) (Fig. 5). The rat mAb 2.4G2 recognizes both mouse Fc␥RII lulose membrane for 20 min at ambient temperature in a dark room and and Fc␥RIII. However, histochemistry studies using Fc␥RIIBϪ/Ϫ were developed using a Kodak XOMAT developer (Kodak, Rochester, mice showed no labeling for the rat mAb 2.4G2 on FDCs, although NY). The films were scanned and the interest of protein bands was selected using the Scion Image software. The program measured the protein bands FDC reticula were present in active GCs as indicted by a variety of as mean density of all pixels. other FDC markers and Fc␥RIII was present on other cells (13). The conclusion of this previous study was that FDCs have little if Results any Fc␥RIII and, thus, we attribute labeling of FDC reticula with In old mice, FDC reticula develop poorly when assessed by 2.4G2 to be due to Fc␥RII-positive FDCs (13). Note the intense amount of Ag trapped, which is clearly reduced and contain re- labeling of FDC reticula compared with B cell areas and a remark- duced amount of immune complexes (18, 19). To understand the able loss of labeling of the reticula in old mice after day 1 (Fig. 5). aberrant development of FDC reticula and their reduced capacity Morphometry showed that at 0 time the average density for Fc␥RII to trap IC in old mice, the present studies were designed to obtain labeling was low for both young and old mice between (55 and 65 The Journal of Immunology 5981

FIGURE 7. Representative light micrographs illustrating the kinetics of immunohistochemical labeling of FDC reticula in draining axillary lymph nodes of Downloaded from actively immunized young and old mice with FDC- M2. These are the same lymph nodes as those shown in the preceding ﬁgures. Note the striking decrease in FDC reticula after day 3 in the aged FDCs (D, day). The same relationships were apparent in brachial and popliteal lymph nodes. Magniﬁcation, ϫ120. http://www.jimmunol.org/ by guest on October 3, 2021

pixels) (Fig. 6A). The average density for Fc␥RII labeling, which Kinetics of FDC-M2-positive FDC reticulum development ␥ reflects the amount of Fc RII on FDCs in the reticula, increased The reactivity of mAb FDC-M2 with FDCs is dependent on significantly by day 1 in both young and old mice and remained trapped ICs capable of fixing complement. FDC-M2 reacts specif- high in young mice between 194 and 173 pixels (Fig. 6A). In ically with the C4 fragment of complement associated with the marked contrast, in old mice the average density rapidly dropped FDC ICs (30). Thus, FDC-M2 labeling closely reflects the location off and stayed low (between 137 and 51 pixels) during the rest of of ICs on FDCs. the FDC reticulum reaction (Fig. 6A). The kinetics of the FDC-M2 labeling was studied to obtain a The size of pre-existing Fc␥RII-positive FDC reticulum 3 wk after priming was small (ϳ7,000 ␮m2) (Fig. 6B) in comparison to functional parameter that reflects Ag localization that can be cor- ␥ 30,000–40,000 ␮m2 for FDC-M1 and CR1/2 labeling, indicating related with Fc RII and CR1/2 labeling of FDCs, both of which that only a proportion of the FDC reticula labeled at day 0 both in are functional in IC trapping. Representative sections showing young and old mice. Between days 0 and 1, there was a substantial FDC-M2 labeling of FDC reticula are shown in Fig. 7. The aver- ϳ3- fold increase in Fc␥RII-positive FDC reticulum size in both age densities of pre-existing FDC-M2-positive FDC reticula were young and old mice. An additional ϳ2-fold increase occurred be- rather high (100–110 pixels) at day 0 in both young and old mice tween days 1 and 3 in young mice (Fig. 6B). In contrast, in old (Fig. 8A). The average density for FDC-M2 labeling increased in mice the FDC reticulum size increased by ϳ3-fold between days young mice significantly by day 1 and remained high (between 175 0 and 1, but the FDC reticulum size then dramatically decreased and 210 pixels) at days 5 and 10. In contrast, in old mice the from 20,000 ␮m2 and leveled off at 5,000 ␮m2. This represents average density after a brief increase at day 1 (ϳ150 pixels) rap- Ͼ10-fold deficit in Fc␥RII on FDCs in the reticula of aged mice at idly dropped off and stayed low (at 60 pixels) after day 5 of the GC days 3, 5, and 10. reaction (Fig. 8A). 5982 AGE-RELATED CHANGES IN MOLECULAR INTERACTIONS OF FDC AND B CELLS

the draining lymph nodes of young and old mice was determined and the values of responder draining lymph nodes were then plot- ted against time (Fig. 10). In young draining lymph nodes, the average pre-existing PNA-positive GC size at day 0 was small but much larger than an FDC reticulum. In young draining lymph nodes, there was a signiﬁcant increase in mean GC size from day 1 to day 5 (Fig. 10). The size of de novo OVA challenge-induced GCs increased through day 5 in the young lymph nodes, but in old mice the size was only 15% of normal at days 5 and 10. The results indicate a general failure of Ag on old FDCs to stimulate GC B cells and are consistent with the lack of Fc␥RII and FDC-M2 labeling in old mice.

Comparison of FDC reticula and GC numbers in the pool of draining lymph nodes at day 5 Kinetic analysis of the average FDC reticulum or average GC in the pool of draining lymph nodes of young and old mice revealed some remarkable deﬁcits in old mice. By day 5, the FDC reticula

and GCs were well developed and in some cases the number of Downloaded from FDC reticula and GCs appeared to be reduced in old mice. Anal- ysis of FDC reticula and GC number in the pool of draining lymph nodes (two axillaries, two brachial, and two popliteal) indicated between 55 and 65 FDC reticula and GCs in young mice (Fig. 11). Furthermore, the number of FDC-M1- and anti-CR1/2-positive FDC reticula were similar in young and old mice (Fig. 11). In http://www.jimmunol.org/ FIGURE 8. The density of average FDC reticulum labeling in the drain- marked contrast, the number of anti-Fc␥RII- and FDC-M2-posi- ing axillary lymph nodes of young and old mice as assessed by FDC-M2. tive FDC reticula was reduced by 70–80% in old mice and this Young (f) and old (Ⅺ) were actively immunized and challenged with OVA. These are the same lymph nodes (three to six mice per group) as correlated with a 70–80% reduction in PNA-positive GCs those shown in the preceding figures. A, Note that the average density for (Fig. 11). FDC-M2ϩ FDC reticula in old mice showed a moderate increase 1 day after challenge with the Ag but started to diminish at day 3 and remained Comparison of ITIM signaling in B cells in the presence of low for the rest of the GC response. In young mice, these values remained young and old FDC approximately twice as high as those of old mice. The data represent the Ϯ Recent results suggest that FDCs can block ITIM signaling in A20 mean SEM in color value of pixels and significant age-related differ- by guest on October 3, 2021 cells induced by co-cross-linking BCR and B cell Fc␥RII and that -p Ͻ 0.02).B,Note that the FDC ,ء) ences are indicated by the asterisk ␥ M2-positive FDC reticulum size was significantly reduced in old mice FDC Fc RII is critical to blocking this process (J.G.T., unpub- compared with young mice and remained very small during the 3, 5, and lished data). We reasoned that FDCs bind Ig-Fc and minimize 10 days of the GC response. The data represent the mean in square mi- co-cross-linking BCR to B cell Fc␥RII and subsequent activation crometers Ϯ SEM and significant age-related differences are indicated by of the ITIM. It followed, therefore, that the lack of Fc␥RII on old p Ͻ 0.0001). FDC would correlate with ITIM activation as indicated by SHIP ,ء) the asterisk phosphorylation. To test this, A20 cells were stimulated using anti- BCR, a well-known inducer of SHIP phosphorylation, as an analog The pre-existing FDC-M2-positive FDC reticulum size in young for ICs (Fig. 12). In contrast with anti-BCR (a positive control), the 2 Ј ␥ mice was significantly larger (45,285 ␮m ) than that of old mice F(ab )2 of anti-BCR cannot engage B cell Fc RII and represents a (8,194 ␮m2) and was comparable to FDC-M1 and CR1/2 labeling negative control. As expected, young FDCs blocked SHIP phos- (Fig. 8B). In young mice, the size of FDC-M2-positive FDC re- phorylation while old FDCs did not and were similar to control ␥ ␥ ticulum increased gradually and peaked at day 5 (72,623 ␮m2) FDCs that lacked Fc RII or with Fc RII blocked by specificAb comparable to M1 and CR1/2 labeling. At day 10, FDC-M2-pos- (Fig. 12). itive FDC reticulum size was slightly reduced and became more compact in young mice (Fig. 8B). In old mice, the size of FDC- Complement and IC-mediated FDC Ag stimulation M2-positive FDC reticulum increased from 8,194 ␮m2 at day 0 to In a recent study, old FDCs functioned as well as young FDCs 22,573 ␮m2 at day 1, but declined and leveled off at ϳ8,000 ␮m2, when the primary B cell signal was delivered by LPS and when representing greater than ϳ12-fold reduction in FDC reticulum FDCs were incubated with ICs and complement to provide old size at days 3, 5, and 10 (Fig. 8B). FDCs with adequate CD21L to engage the B cell coreceptor complex (27). However, BCR and Fc␥RII are not coligated and ITIM Kinetics of PNA-positive GC development signaling does not occur when LPS is the primary stimulator, thus In the preceding studies, the development of the FDC reticulum adeficit in Fc␥RII on old FDCs would not be a problem. This was monitored. We reasoned that the response of the GC B cells prompted us to reason that increasing the CD21L concentration on to Ag provided by the FDCs could be measured by monitoring the old FDCs would not restore immune responses when specificB development of PNA-positive GCs, since B cells labeling with cells were stimulated with ICs given that old FDCs lack normal PNA are activated and proliferating cells. Representative sections levels of Fc␥RII. Memory B and T lymphocytes from young mice (Fig. 9) illustrating the PNA labeling of developing GCs are shown were incubated with ICs and complement to keep these elements for the same group of young and old axillary lymph nodes from equivalent and to reduce the study to a comparison of old vs young day 0 through day 10 of the GC response. The average GC size in FDCs. As predicted, the old FDCs with additional CD21L failed to The Journal of Immunology 5983

FIGURE 9. Representative light micrographs illustrating the kinetics of immunohistochemical labeling of PNAϩ GCs in the draining axillary lymph nodes of ac-

tively immunized young and old mice with FDC-M2. Downloaded from These are the same lymph nodes as those shown in the preceding ﬁgures. Note the clear difference in labeling of the GCs in young and old mice. The same relationships were apparent in brachial and popliteal lymph nodes. D, day. Magniﬁcation, ϫ120. http://www.jimmunol.org/ by guest on October 3, 2021

induce a normal response with young memory B and T lympho- (31–35). In the present study, we compared the expression of sur- cytes (Fig. 13) and this result is consistent with the lack of Fc␥RII face receptors (i.e., Fc␥RII, and CR1/2) on young and old FDCs on old FDCs. that is critical for IC retention and is involved in FDC-B cell interactions (5–7, 10–17). The experiments were designed to study ␥ Ϫ/Ϫ Old FDCs and Fc RIIB B lymphocytes the kinetics of receptor expression during the GC reaction and Based on previous (27) and present observations, we reasoned that provided the first comparative analysis of the kinetics of expres- if a major problem with old FDCs relates to an inability to express sion of Fc␥RII, CR1/2, and the epitopes for FDC-M1 and FDC-M2 normal levels of Fc␥RII and bind Ig-Fc, then old FDCs should be during recall responses in young and old mice. The mAb FDC-M1 able to stimulate B cells lacking Fc␥RII quite normally. It is was used to monitor FDC presence (the FDC reticulum) and FDC- known that FDCs from Fc␥RIIBϪ/Ϫ mice fail to stimulate wild- M2, which reacts with the C4 fragment of complement associated type B cells where co-cross-linking of BCR and Fc␥RII can occur, with the FDC-ICs (30), was used to monitor Ag trapping/retention but that they stimulate B cells in the Fc␥RIIBϪ/Ϫ mice normally by FDCs in the FDC reticulum. Our goal was to understand why (13). Accordingly, we tested old FDCs for their capacity to stim- old FDCs trap less IC, exhibit depressed Ag-specific and costimu- ulate Ab production by Fc␥RIIBϪ/Ϫ lymphocytes in a system latory signals, and support only low level Ab production (3, 18, 20, where ITIM signaling via BCR and B cell Fc␥RII cannot occur. In 27). Results revealed that both the average density of FDC-M1 the absence of the IC-derived inhibitory B cell signaling, old FDCs labeling and the area of FDC reticulum labeled with FDC-M1 were were able to induce a much improved Ab response and when given similar in young and old mice. Thus, the data suggested that re- ICs with complement to provide CD21L, the responses were com- duced IC trapping in old mice was not due to a decrease in the parable to those of young FDCs (Fig. 14). number of FDCs or to reticulum development. However, Fc␥RII labeling on old FDCs was dramatically reduced in the 3- to 10-day Discussion period of the GC reaction. Not only was the average density of Humoral responses exhibiting high levels of high affinity Ab to Fc␥RII labeling on old FDCs diminished but FDC reticula number T-dependent Ags require not only T and B cells but also FDCs and sizes were dramatically reduced, indicating that only some 5984 AGE-RELATED CHANGES IN MOLECULAR INTERACTIONS OF FDC AND B CELLS

FIGURE 12. Old FDCs fail to inhibit SHIP phosphorylation in A20 cells stimulated with anti-BCR. A20 cells were cocultured with young and old FDCs for 30 min at 37oC to allow FDC-A20 cell interactions (for in FIGURE 10. Size of the average PNA-positive GCs in the draining lymph vitro GCs to form) and then stimulated with anti-mouse BCR, a well- nodes of young and old mice as assessed by labeling with PNA labeling. f Ⅺ known inducer of SHIP phosphorylation. Cells were then lysed, the lysate

Young ( ) and old ( ) mice were actively immunized and challenged with Downloaded from was immunoprecipitated with anti-SHIP, resolved using SDS-PAGE, OVA. These are the same lymph nodes as those shown in the preceding fig- transferred to a nitrocellulose membrane, and probed using anti- ures. The averages were calculated using data from three to six mice per day. phosphotyrosine Ab. Note that incubation of A20 cells with young FDCs Note that the GC size was significantly reduced in old mice compared with reduced ITIM signaling as indicated by reduced SHIP phosphorylation young mice and remained very small during the 3, 5, and 10 days of the GC response. The data represent the mean in square micrometers Ϯ SEM and compared with A20 cells stimulated with anti-BCR. On the other hand, ␥ Ϫ/Ϫ p Ͻ 0.003). incubation of A20 cells with old FDCs, Fc RIIB FDCs, or FDCs with ,ء) significant age-related differences are indicated by the asterisk Fc␥RII blocked with 2.4G2 failed to block SHIP phosphorylation in A20 cells stimulated with anti-BCR. This result is representative of three ex- http://www.jimmunol.org/ periments of this type. reticula have FDCs expressing Fc␥RII and not all of the FDCs in the positive reticula express substantial levels of Fc␥RII. This observation correlates with reduced IC trapping by old FDCs (18, 19) itive reticula and the average size of FDC-M1-positive reticula was and this was confirmed with a reduced FDC-M2 labeling that cor- similar in young and old mice, it appears that many old reticula related with the reduced Fc␥RII. Moreover, reduced PNA-positive that lacked Fc␥RII failed to trap ICs and were unable to support GC numbers in old mice correlated well with FDC-M2 staining. GC formation. Moreover, these results are consistent with studies This further supports our previous observation that the ratio of the showing that FDCs treated with anti- Fc␥RII (2.4G2) or from FDC reticula determined by Ag trapping to GC numbers is 1:1 by guest on October 3, 2021 Fc␥RIIϪ/Ϫ mice support only low level Ab production with wild- (18), emphasizing the dependence of GC development on Ag-bear- type B cells (11, 13). Consequently, the present data suggest that ing FDCs. Remarkably, even though the number of FDC-M1-pos- Fc␥RII expression on FDCs is not only critical for mediation of IC retention but for regulation of the B cell recall response. Given that old animals produce less specific Ab, it might be reasoned that the reduced Ab might yield less IC and altered FDC maturation as indicated by a lack of Fc␥RII. However, FDCs trap Ag-Ab complexes over a wide range of Ag:Ab ratios (36) and Ab levels of 2–300 ␮g are sufficient to trap ICs well in young mice (37), and these levels are achieved in aged mice (20). More significantly, when young and old animals are provided with identical amounts of specific Ab by passive transfer, ICs form in old animals normally and can be found in the afferent lymph and the subcapsular sinuses of lymph nodes as they are in the young. However, in young animals ICs are retained on FDCs and numerous GCs develop. In marked contrast, in old animals a much smaller amount of IC is retained on the FDC reticulum and GCs are reduced in number and are much smaller (18, 19). Similarly, when young and old mice are injected with young memory B and T cells, to be FIGURE 11. Correlation between the number of FDC reticula expressing Fc␥RII, IC trapping (indicated by FDC-M2), and a reduced number of PNA- certain memory B and T cells are in equal number, the recall re- positive GCs in old mice. Analysis of FDC reticula and GC number was done sponses in aged mice that depend on old FDCs are abnormally low using a pool of draining lymph nodes (two axillaries, two brachial, and two (20). Thus, the problem with FDC function in aged mice does not popliteal). Young (f) and old (Ⅺ) represent results from mice that were ac- appear to simply be a problem with Ab and IC formation or the tively immunized and challenged with OVA in each of the front legs and hind presence of memory cells. feet. The data represent the mean number of FDC reticula positive for FDC- FDC-Fc␥RII appears to be needed to trap optimal amounts of ␥ M1, anti-CR1/2, anti-Fc RII, or FDC-M2, or the mean number of GC positive ICs and then plays a role organizing these ICs in a stimulatory for PNA in pooled draining lymph nodes Ϯ SEM and significant age-related differences are indicated by the p value. Some GCs in old mice appeared to format for B cells (11, 13). Direct interaction of ICs with B cells fragment and closely associated fragments in a single follicle were counted as is known to lead to ITIM signaling and a lack of B cell respon- a single GC. These data were obtained from mice taken at day 5 but the same siveness (38–40). Recent data suggest that FDCs can block the relationship exists at days 3 and 10. Y, Young; O, old. ITIM signaling in A20 cells apparently by minimizing interaction The Journal of Immunology 5985

FIGURE 13. Incubation of old FDCs with ICs and complement from young mice did not restore stimulatory activity. Note that young FDCs (y) stimulate OVA-specific Ab production by memory lymphocytes (Ly) but old (o) FDCs could support only a lower anti- OVA response. Incubation of old FDCs with active C (aC) and IC did not restore the Ag-handling capacity of old FDCs and was not different from IC plus heat- inactivated complement (inaC). These data are typical of three experiments of this type and results are de- picted as the mean SEM of total IgG produced (p Ͻ 0.005). Downloaded from of Ig-Fc in ICs with A20 cell Fc␥RII and thus promoting B cell was delivered by LPS and costimulatory signals were provide by activation.4 Activation of the ITIM in cells results in phosphory- aged FDC (27). In this model, the critical FDC costimulatory sig- lation of SHIP (38, 41). SHIP is the main ligand for phosphory- nal was delivered by CD21L and additional CD21L was provided lated ITIM and that SHIP phosphorylation correlates with ITIM by incubating the aged FDCs with ICs and complement. The ad- signaling (42–46). Based on these observations, we hypothesized ditional CD21L dramatically improved the ability of old FDCs to http://www.jimmunol.org/ that old FDCs lacking Fc␥RII would not block SHIP phosphory- augment Ab production by LPS-stimulated B cells (27). We sought lation in cells stimulated by anti-BCR. The capacity of old FDCs, to determine whether an increased level of CD21L would remedy taken 3 days after Ag challenge, to block SHIP phosphorylation in the age-related deficit in handling specific Ag. Old FDCs were A20 cells induced by anti-BCR was shown to be impaired (Fig. treated with OVA-anti-OVA and complement and then tested in a 12). The data suggest that Ag handling and costimulatory activity recall response using memory T and B cells from OVA-immune of old FDCs become defective and their capacity to prevent ITIM mice. However, the treatment of old FDCs with complement and signaling deteriorates with aging. It appears that this is a conse- IC did not repair the depressed Ag-handling capacity of old FDCs quence of reduced expression of Fc␥RII on old FDCs that fails to and anti-OVA production remained depressed (Fig. 13). We rea- by guest on October 3, 2021 block the inhibitory effect of ICs by binding of Fc in the IC. This son that the lack of FDC-Fc␥RII on aged FDCs cannot prevent ultimately results in B cell inactivation and significantly depressed coligation of B cell Fc␥RII and BCR (13) and lack of Fc␥RII on secondary Ab responses. FDCs may also interfere with the ability of FDCs to arrange for the Recently, we reported that incubation of old FDCs with C and formation of iccosomes, which are the IC-coated small spherical ICs repaired the age-related defect when the primary B cell signal structures on which Ag is delivered from FDCs to B cells for endocytosis and processing (19, 20, 31, 47). Thus, it is not sur- prising that while additional CD21L may augment LPS-stimulated 4 Y. Aydar, J. Wu, J. Song, A. K. Szakal, and J. G. Tew. Fc␥RII expression on follicular dendritic cells and ITIM signaling in B cells. Submitted for publication. B cells it does not restore normal Ag handling and the suppression

FIGURE 14. Old FDCs stimulated Fc␥RIIBϪ/Ϫ B cells to produce near normal levels of specific Ab. Wild-type old FDCs were cocultured with young wild-type OVA-specific lymphocytes (Ly) or with young OVA-specificFc␥RIIBϪ/Ϫ lymphocytes in the presence of IC with or without complement. The depressed Ab response typical for old FDCs was not apparent with Fc␥RIIBϪ/Ϫ B lymphocytes because ITIM signaling induced by cross-linking of BCR and B cell Fc␥RII cannot occur. Addition of complement also improved the Ab response by providing more CD21L for costimulation. KO, Knockout. 5986 AGE-RELATED CHANGES IN MOLECULAR INTERACTIONS OF FDC AND B CELLS

A. K. Szakal, and J. G. Tew. 1999. Follicular dendritic cells protect malignant B cells from apoptosis induced by anti-Fas and antineoplastic agents. J. Immunol. 163:6442. 9. Qin, D., J. Wu, G. F. Burton, A. K. Szakal, and J. G. Tew. 1999. Follicular dendritic cells mediated maintenance of primary lymphocyte cultures for long- term analysis of a functional in vitro immune system. J. Immunol. Methods 226:19. 10. Wang, Y., G. Huang, J. Wang, H. Molina, D. D. Chaplin, and Y. X. Fu. 2000. Antigen persistence is required for somatic mutation and afﬁnity maturation of immunoglobulin. Eur. J. Immunol. 30:2226. 11. Tew, J. G., J. Wu, M. Fakher, A. K. Szakal, and D. Qin. 2001. Follicular dendritic cells: beyond the necessity of T-cell help. Trends Immunol. 22:361. FIGURE 15. Important differences in the molecular interactions in- 12. Fakher, M., J. Wu, D. Qin, A. Szakal, and J. Tew. 2001. Follicular dendritic cell volved in signaling B cells with FDCs from young or old mice. Based on accessory activity crosses MHC and species barriers. Eur. J. Immunol. 31:176. immunocytochemical data, there is an exceptionally higher density of 13. Qin, D., J. Wu, K. A. Vora, J. V. Ravetch, A. K. Szakal, T. Manser, and Fc␥RII on young FDCs than on B cells. The Fc␥RII on young FDCs J. G. Tew. 2000. Fc␥ receptor IIB on follicular dendritic cells regulates the B cell recall response. J. Immunol. 164:6268. apparently engages essentially all Ig-Fc on the Ag-Ab complexes. Thus, very little Ig-Fc is left that would be able to bind to B cell Fc␥RII receptors 14. Yoshida, K., T. K. van den Berg, and C. D. Dijkstra. 1993. Two functionally ␥ different follicular dendritic cells in secondary lymphoid follicles of mouse and activate ITIM signaling. By contrast, old FDCs are deﬁcient in Fc RII spleen, as revealed by CR1/2 and FcR␥II-mediated immune-complex trapping. and do not engage all (or most) Ig-Fc on the ICs. This would allow B Immunology 80:34. cell-BCR and Fc␥RII receptors to be cross-linked and ITIM activated. This 15. Papamichail, M., C. Gutierrez, P. Embling, P. Johnson, E. J. Holborow, and ultimately results in B cell inactivation and depressed Ab production. N, M. B. Pepys. 1975. Complement dependence of localisation of aggregated IgG in

Nucleus, ITAM, immunoreceptor tyrosine-based activation motif. germinal centres. Scand. J. Immunol. 4:343. Downloaded from 16. Yoshida, K., T. K. van den Berg, and C. D. Dijkstra. 1993. Two different mech- anisms of immune-complex trapping in the mouse spleen during immune responses. Adv. Exp. Med. Biol. 329:377. 17. Klaus, G. G., and J. H. Humphrey. 1977. The generation of memory cells. I. The induced by ITIM signaling in specific B cells. However, when we role of C3 in the generation of B memory cells. Immunology 33:31. ␥ Ϫ/Ϫ 18. Szakal, A. K., J. K. Taylor, J. P. Smith, M. H. Kosco, G. F. Burton, and J. G. Tew. used Fc RIIB lymphocytes where ITIM signaling via BCR 1988. Morphometry and kinetics of antigen transport and developing antigen- and B cell Fc␥RII cannot occur, old FDCs given ICs with com- retaining reticulum of follicular dendritic cells in lymph nodes of aging immune http://www.jimmunol.org/ plement were able to induce Ab responses that were comparable to mice. Aging Immunol. Infect. Dis. 1:7. those of young FDCs (Fig. 14). This experiment provides addi- 19. Szakal, A. K., Z. F. Kapasi, A. Masuda, and J. G. Tew. 1992. Follicular dendritic cells in the alternative antigen transport pathway: microenvironment, cellular tional independent support for the concept that old FDCs lacking events, age and retrovirus related alterations. Semin. Immunol. 4:257. normal levels of Fc␥RII do not inhibit ITIM signaling and fail to 20. Burton, G. F., M. H. Kosco, A. K. Szakal, and J. G. Tew. 1991. Iccosomes and stimulate wild-type B cells normally. the secondary antibody response. Immunology 73:271. In conclusion, our data may be summarized in a simple working 21. Kosco, M. H., G. F. Burton, Z. F. Kapasi, A. K. Szakal, and J. G. Tew. 1989. Antibody-forming cell induction during an early phase of germinal centre devel- model illustrated in Fig. 14. Based on immunocytochemical data, opment and its delay with ageing. Immunology 68:312. Fc␥RII receptors on the surface of young FDCs are markedly 22. Qin, D., J. Wu, M. C. Carroll, G. F. Burton, A. K. Szakal, and J. G. Tew. 1998. by guest on October 3, 2021 higher than on the surface of B cells. We reason that the Fc␥RII on Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG FDCs engage essentially all Ig-Fc in the Ag-Ab complexes. Thus, responses. J. Immunol. 161:4549. very little Ig-Fc is left that would be able to bind Fc␥RII on B cells 23. Maeda, K., G. F. Burton, D. A. Padgett, D. H. Conrad, T. F. Huff, A. Masuda, and activate ITIM signaling by cross-linking with BCR (Fig. 15). A. K. Szakal, and J. G. Tew. 1992. Murine follicular dendritic cells and low affinity Fc receptors for IgE (Fc⑀RII). J. Immunol. 148:2340. Fc␥RII levels on old FDCs were low, particularly in the 3- to 24. Rao, S. P., K. A. Vora, and T. Manser. 2002. Differential expression of the 10-day period after secondary immunization, and we reason that inhibitory IgG Fc receptor Fc␥IIB on germinal center cells: implications for old FDCs would not engage all (or most) Ig-Fc on the ICs (Fig. selection of high-affinity B cells. J. Immunol. 169:1859. 15). This would allow B cell BCR and Fc␥RII receptors to be 25. Kosco, M. H., E. Pflugfelder, and D. Gray. 1992. Follicular dendritic cell-dependent adhesion and proliferation of B cells in vitro. J. Immunol. 148:2331. cross-linked, ITIM signaling to be initiated, B cell maturation 26. Wu, J., D. Qin, G. F. Burton, A. K. Szakal, and J. G. Tew. 1996. Follicular would then be inhibited, and consequently aged mice would lack dendritic cell-derived antigen and accessory activity in initiation of memory IgG the ability to mount a vigorous recall response. responses in vitro. J. Immunol. 157:3404. 27. Aydar, Y., P. Balogh, J. G. Tew, and A. K. Szakal. 2002. 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