Type II membrane CD69 regulates the formation of resting T-helper memory

Kenta Shinodaa,1, Koji Tokoyodaa,b,1,2, Asami Hanazawaa, Koji Hayashizakia, Sandra Zehentmeierb, Hiroyuki Hosokawaa, Chiaki Iwamuraa, Haruhiko Kosekic, Damon J. Tumesa, Andreas Radbruchb, and Toshinori Nakayamaa,2

aDepartment of Immunology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan; bGerman Rheumatism Research Center, 10117 Berlin, Germany; and cLaboratory for Developmental Genetics, RIKEN Research Center for Allergy and Immunology, Yokohama 230-0045, Japan

Edited by Max D. Cooper, Emory University, Atlanta, GA, and approved March 12, 2012 (received for review November 10, 2011)

Memory T-helper (Th) are crucial for the maintenance lived plasma cells. Thus, we have clarified the role of CD69 in Th of acquired immunity to eliminate infectious pathogens. We have memory and further defined the role of Th memory in humoral previously demonstrated that most memory Th lymphocytes reside immunity, thus providing strategies for modulating T-B inter- and rest on stromal niches of the bone marrow (BM). Little is actions in immunological memory. known, however, regarding the molecular basis for the generation Results and maintenance of BM memory Th lymphocytes. Here we show that CD69-deficient effector CD4 T lymphocytes fail to relocate into CD69 Is Required for Generation of Memory Th Cells. In steady state, CD69 was expressed on, at most, 2% of CD44lo, naive CD4 and persist in the BM and therefore to differentiate into memory hi cells. Consequently, CD69-deficient CD4 T cells fail to facilitate the T cells and 20% to 30% of CD44 CD4 T cells in the spleen and fi BM, whereas it was expressed on approximately 70% of BM production of high-af nity antibodies and the generation of BM Ly-6ChiCD44hi CD4 T cells, which have been identified as a long-lived plasma cells in the late phase of immune responses. population including professional resting memory Th cells (Fig. Thus, CD69 is critical for the generation and maintenance of S1A). Despite the constitutive expression of CD69, BM professional memory Th lymphocytes, which can efficiently help CD69+CD44hi CD4 T cells were not activated in terms of DNA humoral immunity in the late phase. The deficit of immunological synthesis (Fig. S1 B and C), nor overall transcription, as we have memory in CD69-deficient mice also highlights the essential role of shown previously (11). BM for the establishment of Th memory. To investigate the relevance of CD69 expression and function in memory Th cells, we monitored antigen-specific CD4 T cells T-B interaction | homing | trafficking | plasmablast | microenvironment after immunization in an adoptive transfer model (22). Naive CD4 T cells are activated in the secondary lymphoid organs immedi- fi ately after immunization and, within 8 wk, some of the activated mmunological memory is a de ning characteristic of the adap- cells migrate to the BM via blood flow and persist there as memory Itive immune response and can be manifested by CD4 and CD8 cells for a long time (11). Naive normal mice were transferred with T cells and B cells. The generation and maintenance of memory CD69-deficient or WT DO11.10 transgenic (Tg) naive CD4 T-helper (Th) lymphocytes is crucial for acquired immunity to T cells, immunized with ovalbumin (OVA) plus lipopolysaccha- eliminate various infectious pathogens (1–3). In their absence, the fi ride (LPS) and then analyzed for the transferred CD4 T cells. Four generation of high-af nity memory B cells and long-lived plasma days after immunization, activated CD69-deficient or WT cells (4–6) and the maintenance and secondary expansion of – DO11.10 Tg CD4 T cells were similarly detectable in the spleen, memory CD8 T cells (7 10) are impaired. We have previously whereas none were detected in the BM of either group (Fig. 1 A shown that Ly-6C is a specific marker of memory Th cells in bone hi and B and Fig. S2A). In the memory phase, CD69-deficient marrow (BM) (11). Upon challenge with antigen, Ly-6C memory DO11.10 Tg CD4 T cells did not accumulate in the BM compared Th cells rapidly express and CD154 (CD40L) and effi- fl A fi with WT cells as detected by ow cytometry (Fig. 1 )andby ciently induce the production of high-af nity antibodies by B cells. histological analysis (Fig. 1B), although most WT and CD69-de- Despite their eminent importance for the regulation of immune ficient DO11.10 Tg cells disappeared from the spleen (Fig. 1A). reactions and immunological memory, the molecular mechanisms Most WT DO11.10 Tg CD4 T cells of the BM in the memory regulating relocation of Th memory precursors to the BM have not phase express CD69, as do BM Ly-6ChiCD44hi CD4 T cells in been investigated. steady status (Fig. 1C and Fig. S2A). To confirm the defect of BM CD69 is a type II membrane protein expressed as a homodimer memory Th cells in CD69-deficient mice, we alternatively analyzed composed of heavily glycosylated subunits (12). T cells express the immune response of WT and CD69-deficient mice to 4-(hy- CD69 rapidly upon stimulation of the T-cell receptor (TCR) (13, droxy-3-nitrophenyl) acetyl-coupled (NP)-OVA plus incomplete 14), which is why CD69 has been mostly regarded as an activation Freund adjuvant (IFA), by monitoring OVA-specific CD4 T cells marker (15, 16). The precise role of CD69 in immunity has not based on their expression of IFN-γ. In the memory phase of the been determined because its ligand is unknown. Freshly prepared immune response, significantly fewer OVA-reactive IFN-γ–pro- undergoing selection events express CD69, and reg- ducing CD4 T cells were detected in BM of CD69-deficient mice ulatory roles for CD69 expression in T-cell development in the compared with WT mice (Fig. 1D). Thus, CD69 is required for the thymus have been suggested (17, 18). However, phenotypical establishment of professional resting Th memory in the BM. analysis in previous studies using CD69-deficient mice has re- vealed that CD69 does not appear to be required for the de- velopment of CD4 T cells (19, 20). Although multiple target processes of CD69 have been suggested in the effector phase of Author contributions: K.S., K.T., A.R., and T.N. designed research; K.S., K.T., A.H., K.H., and S.Z. performed research; H.H., C.I., and H.K. contributed new reagents/analytic tools; and an immune response, such as in an anticollagen antibody-induced K.S., K.T., D.J.T., A.R., and T.N. wrote the paper. arthritis model (21) and allergic airway inflammation model (20), fl its function in immunological memory has not been elucidated. The authors declare no con ict of interest. Here we show the role of CD69 in the generation of memory This article is a PNAS Direct Submission. Th cells and investigate the ability of CD69-deficient Th cells to 1K.S. and K.T. contributed equally to this work. facilitate humoral immunity. CD69-deficient CD4 T cells failed 2To whom correspondence may be addressed. E-mail: [email protected] or tnakayama@ to form memory cells because of defective relocation into, and faculty.chiba-u.jp. persistence in, the BM. Moreover, the Th memory-deficient mice This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. IMMUNOLOGY could not produce high-affinity antibodies and generate long- 1073/pnas.1118539109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1118539109 PNAS | May 8, 2012 | vol. 109 | no. 19 | 7409–7414 Downloaded by guest on October 6, 2021 Fig. 1. CD69 is required for the generation of memory Th cells. (A– C)CD4Tcells(3× 105) from CD69+/+ − − (open column) and CD69 / (filled column) DO11.10 Tg mice were transferred into BALB/c mice, and the mice were then injected with 100 μg of OVA plus 10 μgLPS.(A)Acti- − − vated CD69 / OVA-specificCD4 T cells do not accumulate in BM. The number of OVA-TCR+ donor T cells was determined by flow cytome- try. Representative OVA-TCR/CD44 − staining profiles of B220 CD4 T cells in spleen and BM of host mice at days 4 and 48 after immunization are shown (Left). The cell numbers (mean ± SD) of donor T cells was quantified as OVA-TCR+B220−CD44hi CD4 T cells (Right). (B)Fewactivated − − CD69 / OVA-specificCD4Tcellsare observed in BM. Representative his- tological section of OVA-TCR (green) vs. laminin (red) labeling in BM of the host mice at day 56 after transfer are shown (Left). Outlined area at the left is enlarged (Center). The absolute number of OVA-TCR+ CD4 TcellspersquaremillimeterofBM was quantified (Right; n =5).(C) Most antigen-specificmemoryTh cells of BM express CD69. OVA-TCR+ CD4 T cells in the spleen and BM of the host mice at day 48 after immunization were stained with anti-CD69 antibodies (background staining with an isotype control shown in gray) and analyzed by flow cytometry. The inserted numbers indicate the mean fluorescence intensity (n =3).(D) Antigen-specific functional memory Th cells are impaired in CD69- − − deficient mice. At day 90 after immunization with 100 μg NP-OVA plus IFA, OVA-specific CD4 T cells of spleen and BM of CD69+/+ (open column) and CD69 / (filled column) mice were quantified by flow cytometry. For detecting OVA-reactive CD4 T cells, IFN-γ+CD44hi CD4 T cells were counted after ex vivo stimulation with NP-OVA for 5 h. The frequencies of IFN-γ+ cells among CD44hi CD4 T cells of the spleen and BM are shown (mean ± SD; n = 5). All data are representative of two or more independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001).

CD69 inhibits sphingosine-1-phosphate (S1P)/S1P receptor 1 Affinity maturation of antibodies in B cells occurs on antigen- (S1P1)-mediated cell egress from lymphoid organs after exposure bearing follicular dendritic cells of germinal centers (GCs) of the to IFN-α/β (23). Accordingly, inhibition or deficiency of CD69 secondary lymphoid organs and is assisted by T follicular helper can be expected to enhance the movement of activated CD4 (TFH) cells (24–27). To evaluate the generation of TFH cells and + + T cells to the periphery. This, however, was not observed in the GC formation in CD69-deficient mice, CXCR5 PD-1 TFH cells present study; rather, the number of antigen-specific CD4 T cells and GL-7+PNAhiIgDlo GC B cells were counted by flow cytom- in the peripheral blood was comparable on days 4, 15, and 48 etry, and expression of transcriptional repressor Bcl6, which fi + after immunization between WT and CD69-de cient CD4 is a critical regulator of TFH cell differentiation, on CXCR5 PD- + T-cell-transferred mice (Fig. S3). 1 TFH cells were evaluated (Fig. S5). TFH and GC B cells of the spleen were normally generated in CD69-deficient mice (Fig. S5). CD69-Deficient CD4 T Cells Fail to Provide Efficient Help for B Cells. To To examine where and when CD69-deficient CD4 T cells ex- investigate the function of CD69 in Th cells, we tested whether hibit the defects in the ability to help production of high-affinity CD69-deficient CD4 T cells can help B cells for antibody pro- antibodies, we enumerated antibody-secreting cells (ASCs) in duction in vivo. In immune responses to NP-coupled chicken spleen and BM of mice transferred with CD69-deficient or WT γ-globulin (NP-CGG) plus LPS or NP-coupled keyhole limpet DO11.10 Tg CD4 T cells (Fig. 2B and Fig. S2A). At days 14 and hemocyanin (NP-KLH) plus alum, CD69-deficient mice pro- 28 after immunization, CD69-deficient CD4 T cells could induce duced significantly less NP-specific high-affinity, but not total, splenic ASCs but not BM ASCs, which include long-lived plasma antibodies compared with WT mice, on days 28 and 90 after cells (Fig. 2B). To analyze whether homing of plasmablasts to the immunization of NP-CGG (Fig. S4A) or on day 28 after immu- BM indeed requires the help of BM memory CD4 T cells, splenic nization of NP-KLH (Fig. S4B), whereas, on day 14, the levels of plasmablasts of WT mice, on day 14 after immunization of NP- anti–NP-CGG (Fig. S4A) and total serum IgG and IgG1 (Fig. KLH, were transferred into KLH-immunized or intact WT or S4C) were comparable. Next, CD4 T cells were sorted from CD69-deficient mice, and analyzed for the homing of NP-specific spleens of CD69-deficient or WT DO11.10 Tg mice and trans- ASCs to the BM (Fig. 2C). In immunized hosts, the WT plas- ferred into normal mice, and then the transferred mice were mablasts failed to efficiently home to the BM in the absence of immunized with NP-OVA and analyzed for NP-specific anti- CD69, suggesting that antigen-specific CD4 T cells of BM con- bodies (Fig. 2A and Fig. S2A). CD69-deficient CD4 T cells failed trol the establishment of BM plasma cells (Fig. 2C). Interest- to induce the production of high-affinity antibodies, especially ingly, also in nonimmunized mice, CD69 significantly enhanced in the late phase (Fig. 2A). In contrast, CD69-deficient mice the efficient homing of plasma blasts to the BM (Fig. 2C), sug- transferred with WT DO11.10 Tg CD4 T cells could normally gesting that BM CD4 T cells support the establishment of BM produce NP-specific high-affinity antibodies (Fig. S4D). Thus, the plasma cells independent of antigen specificity. Taken together, defective production of high-affinity antibodies in CD69-deficient our results indicate that CD69-deficient CD4 T cells fail to help mice appeared to be caused by the lack of CD69 expression on B cells in the late phase of an immune response because of the CD4 T cells. defective control of homing of long-lived plasma cell precursors,

7410 | www.pnas.org/cgi/doi/10.1073/pnas.1118539109 Shinoda et al. Downloaded by guest on October 6, 2021 Fig. 2. Impaired help for B cells by CD69-de- ficient CD4 T cells in the late phase. (A) CD69- deficient CD4 T cells fail to induce the production of high-affinity antibodies in vivo. CD4 T cells (3 × 105) from CD69+/+ and CD69−/− DO11.10 Tg mice were transferred into BALB/c mice, and the mice were then immunized i.p. with 100 μg NP-OVA plus 10 μg LPS. Blood taken at each time point

was analyzed for anti–NP36-IgG1 and anti–NP4- −/− IgG1 by ELISA (n =5–7). (B) CD69 CD4 T cells fail to provide efficient help for the generation of long-lived plasma cells in vivo. CD4 T cells (3 × 105) from CD69+/+ and CD69−/− DO11.10 Tg mice were transferred into BALB/c mice, and the mice were then immunized i.p. with 100 μg NP-OVA

plus 10 μg LPS. NP-specificIgG1 ASCs in spleen and BM were detected by enzyme-linked immu- nosorbent spot assay at days 14 and 28 after immunization (n = 7). (C) Splenic plasmablasts fail to home to the BM of CD69-deficient mice. − Thy1.2 splenocytes from BALB/c mice at day 14 after immunization with 100 μg NP-KLH in alum were transferred into KLH-immunized (14 d before) or − − unimmunized CD69+/+ or CD69 / mice. One week after the cell transfer, NP-specific IgG ASCs in BM were detected by enzyme-linked immunosorbent spot assay (n =4;*P < 0.05 and **P < 0.01).

although they can normally form GCs and generate splenic the BM, CD4 T cells from spleen of WT or CD69-deficient plasmablasts. DO11.10 Tg mice at day 4 after immunization were labeled with different fluorescent dyes and transferred into one normal BM Functional Th Cells Were Markedly Decreased in CD69-Deficient mouse, and, 2 h later, the transferred cells in the spleen and BM Mice. To identify the functional defect of CD4 T cells in CD69- were counted (Fig. 4A and Fig. S2B). The transferred CD69- deficient mice, we first analyzed their proliferation, apoptosis, deficient effector CD4 T cells could migrate to the spleen effi- and ability to produce cytokines. Splenic naive CD4 T cells from ciently compared with WT, but not into the BM (Fig. 4A). The CD69-deficient or WT mice were stimulated in vitro with anti- genetic loss of CD69 did not affect the differentiation of naive CD3 and anti-CD28. Clonal expansion (Fig. S6A) and pro- CD4 T cells into effector cells, nor did it indirectly affect cell duction of the cytokines IFN-γ and IL-2 (Fig. S6B) in CD69- trafficking, because the relocation of WT effector CD4 T cells deficient and CD69-sufficient CD4 T cells were comparable. To into the BM was significantly inhibited by treatment with Fab assess the regulation of survival and apoptosis in CD4 T cells, fragments of anti-CD69 antibodies 2 h (Fig. 4B)or24h(Fig. S7) effector CD4 T cells from CD69-deficient or WT mice were after cell transfer. Injection of CD69-specific antibodies into cultured with the survival factor IL-7. The rates of survival and immunized mice also inhibited the relocation of antigen-specific apoptosis in WT and CD69-deficient effector CD4 T cells were effector CD4 T cells into the BM (Fig. 4C). In addition, anti- indistinguishable (Fig. S6C). CD154, which is essential for help CD69–treated BM CD44hi CD4 T cells did not rehome to the to B cells (28–30), was expressed equivalently in WT and CD69- BM efficiently (Fig. 4D). These results indicate that CD69 reg- hi + + deficient splenic naive, CD44 , and CXCR5 PD-1 TFH cells ulates the homing of effector and memory Th cells to BM. (Fig. 3A). BM CD44hi CD4 T cells of CD69-deficient mice, however, did express less CD154 and also IFN-γ and TNF-α (Fig. CD69 Regulates Persistence of Th Cells on BM Stromal Cells. Al- 3 A and B), whereas they normally expressed the other immu- though anti-CD69–treated effector cells could not efficiently noregulatory molecules such as inducible T-cell costimulator, migrate to the BM (Fig. 4B), the few BM-resident cells of mice PD-1, and OX40 (Fig. S6D). This corresponded to a drastic re- transferred with anti-CD69–treated cells could not also efficiently duction of Ly-6ChiCD44hi CD4 T cells, which were absent as contact laminin+ cells, i.e., stromal cells and endothelial cells a distinct population (Fig. 3C), and functional CD154+ Ly- (Fig. 5A). Thus, the absolute numbers of laminin+-interacting 6ChiCD44hi CD4 T cells were decreased in the BM of CD69- effector cells in the BM were markedly reduced by inhibition of deficient mice (Fig. 3D). Deficiency of CD69 itself does not af- CD69 (Fig. 5B). These data indicate that memory Th cells persist fect the expression of Ly-6C, because the absolute numbers and on BM stromal cells by using CD69. To confirm the residence of the frequency of Ly-6ChiCD44hi CD8 T cells in the BM of CD69- memory Th cells on the stroma, BM Ly-6Chi or CD69+ CD44hi deficient and WT mice were equivalent (Fig. S1A). The expres- CD4 T cells in steady status could also be analyzed histologically. sion levels of CD49b, CD119 (IFN-γR1), CD127 (IL-7Rα), More than 80% of CD69+CD4+ cells in the BM expressed Ly-6C CD186 (CXCR6), and CD197 (CCR7) on BM CD44hi CD69- highly as detected by flow cytometry (Fig. 5C). Therefore, we deficient CD4 T cells were normal (Fig. S6E). As BM CD44hi generated CD69:GFP knock-in mice (Fig. S8), and investigated CD4 T cells are potent in giving help for antibody production of the localization of BM CD69+CD4+ cells, which include most fi memory Th cells in vivo. More than 80% of GFP+CD4+ cells B cells (11), we examined the function of CD69-de cient BM − CD44hi CD4 T cells. BM CD44hi CD4 T cells of CD69-deficient compared with approximately 30% of GFP CD4+ cells contacted mice provide less help for the production of high-affinity anti- laminin+ stromal cells (Fig. 5D). Taken together, these data in- bodies in vivo (Fig. 3E). Thus, CD69-deficient mice have few dicate that CD69 regulates the persistence of memory Th cells on functional memory Th cells with Ly-6ChiCD44hi phenotype. survival niches organized by stromal cells.

CD69 Regulates Relocation of Effector Th Cells to BM. How does Discussion CD69 regulate the generation of memory Th cells in BM? In an We herein demonstrate that CD69 functions as a homing receptor immune response to OVA, antigen-experienced DO11.10 Tg on CD4 T cells and is required for relocation to, and persistence CD4 T cells of the spleen and peripheral blood but not BM were in, the BM, and that its function is essential for the generation of normally present at days 4 and 48 after immunization (Fig. 1A memory Th cells. In fact, CD69-deficient mice have few pro- and Fig. S3). The biased distribution indicated that CD69 works fessional resting memory Ly-6ChiCD44hi, functional CD154+,and in the relocation of activated CD4 T cells from blood to BM. To also antigen-specific memory Th cells in the BM. We have pre- IMMUNOLOGY analyze the migration ability of CD69-deficient CD4 T cells to viously reported that memory Th cells are maintained on their

Shinoda et al. PNAS | May 8, 2012 | vol. 109 | no. 19 | 7411 Downloaded by guest on October 6, 2021 Fig. 3. Defects of functional memory Th cells in the BM of CD69-deficient mice. (A)Ex- pression of CD154 is impaired selectively in CD4 T cells from BM of CD69−/− mice. Naive (CD44loCD62L+), splenic CD44hi

CD4Tcells,TFH cells, and BM CD44hi CD4 T cells were isolated and stimulated with immobilized anti-CD3 antibodies for 4 h in the

presence of brefeldin A. TFH cells were sorted as splenic CXCR5+ PD-1+ CD4 T cells from C57BL/6 mice at day 7 after immunization with NP-CGG plus IFA. Cells were fixed and stained with anti-CD154 antibodies (n =3–6). (B)Impaired production of BM CD4 T cells from CD69-deficient mice. BM CD44hi CD4 T cells were iso- lated and stimulated with immo- bilized anti-CD3 antibodies for 4 h in the presence of brefeldin A. Cells were fixed and stained with antibodies against IFN-γ and TNF- α (n =6).(C)Ly-6ChiCD44hi CD4 T cells are significantly reduced − − in the BM of CD69 / mice. The histograms show Ly-6C expression on CD44hiCD62L−CD25−B220− CD4 T cells of spleen and BM in CD69+/+ − − and CD69 / mice. The isotype control is shown in gray. Right: − − − Frequencies of Ly-6Chi cells among CD44hiCD62L CD25 B220 CD4 T cells of spleen and BM (n = 7). (D) CD69+/+ Ly-6ChiCD44hi CD4 T cells of BM rapidly express CD154 − − − − after stimulation with anti-CD3 antibodies ex vivo. CD44hiCD62L B220 CD4 T cells from BM of CD69+/+ and CD69 / mice were isolated, stained with anti–Ly-6C antibodies, and then stimulated for 4 h with immobilized anti-CD3 antibodies in the presence of brefeldin A. Harvested cells were fixed and stained with anti-CD154 antibodies (n =5).(E) Antigen-specific memory CD4 T cells from BM induce efficient production of high-affinity antibodies upon stimulation. A total of 105 CD44hiCD62−CD25− CD4 T cells from BM of NP-OVA–immunized mice at day 25 were transferred together with 2 × 106 splenocytes depleting Thy1.2+,CD11c+, CD11b+,Gr-1+,CD49b(DX5)+,andCD138+ cells by MACS (including approximately 92% of CD19+ cells) from the immunized mice into naive C.B-17/scid mice, followed by injection of NP-OVA. At day 7 after transfer and secondary immunization, the blood of the recipient mice was analyzed by ELISA for the presence of high-affinity antibodies (n =3).Alldata(means± SD) are representative of two or more independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001).

stromal niches in the BM (31) and now find that antigen-specific the blood is dependent on the S1P/S1P1 pathway (33, 34). The and CD69(GFP)+ CD4 T cells of the BM contact stromal cells in constitutive expression of CD69 on resting memory Th cells may a CD69-dependent fashion. Moreover, we show a role for memory inhibit their egress from the BM. Th cells in the BM: the defect of BM memory Th cells impairs the CD69-deficient effector CD4 T cells have a defect in the ability production of high-affinity antibodies because of the defective to enter into the BM (Fig. 4A). In addition, most CD69+CD4+ generation of long-lived plasma cells in the BM. memory Th cells of the BM contact stromal cells in vivo, whereas − The molecular mechanisms regulating Th cell trafficking to most CD69 CD4+ cells do not (Fig. 5D). The treatment of ef- the BM were unclear. CD69 is required for the trafficking of fector or memory cells with Fab fragments of anti-CD69 anti- effector CD4 T cells to the BM, especially for their relocation bodies markedly inhibited not only relocation to the BM but also and persistence on the BM stromal cells. Although most splenic the interaction with BM stromal cells after adoptive transfer antigen-specific CD4 T cells express CD69 at days 1 and 2 after (Figs. 4B and 5 A and B). These data suggest that CD69 of Th cells immunization, 80% to 90% have lost the expression by day 4 works as an adhesion molecule in a two-step process: in the (Fig. S9). This suggests that splenic CD69-expressing effector transmigration via BM sinusoidal endothelial cells and in the CD4 T cells detected at day 4 may preferentially migrate to the adhesion to the stromal niches of the BM. Although the existence BM and transit to memory cells in the BM. The S1P/S1P1 of a ligand for CD69 is expected based on crystal structure pathway is another regulator of cell trafficking. CD69 is physi- analysis (35), it is still unknown but tempting to speculate that the cally associated with S1P1 and directly inhibits S1P/S1P1-medi- ligand is expressed on BM stromal cells. Some of the C-type ated cell egress from lymphoid organs after exposure to IFN-α/β family molecules, e.g., dendritic cell-specific ICAM-3 grabbing (23). Both CD69-overexpressing and S1P1-deficient thymocytes nonintegrin and macrophage act as adhesion accumulate in the thymus and do not enter the periphery (17, molecules that regulate functions and are involved in 32). From these studies, CD69 is presumed to inhibit the egress establishing adaptive immunity (36). Thus, CD69 may also func- of mature thymocytes through blockage of S1P1. However, thy- tion as an adhesion molecule to BM stromal cells as well as other mocyte development and the generation of peripheral CD4 and members of the C-type lectin family. CD8 T cells were normal in CD69-deficient mice (21). Also in an Our previous data indicates that Ly-6ChiCD44hi CD4 T cells in immune response, antigen-experienced CD69-deficient splenic the BM reflect memory Th cells which are reactive, resting, and CD4 T cells normally egress from the spleen to the peripheral long-lived. CD69-deficient CD4 T cells are normally developed, blood (20) (Fig. S3). Therefore, the expression of CD69 itself on proliferate, and differentiate into effector cells. Although the thymocytes and splenocytes may not be physiologically involved numbers of CD44hi memory-phenotype CD4 T cells in spleen, in their egress from thymus and spleen, respectively. It had been lymph nodes, BM, and blood of CD69-deficient mice are also reported that the egress of CD4 T cells and B cells from BM to normal, only BM Ly-6ChiCD44hi CD4 T cells are selectively

7412 | www.pnas.org/cgi/doi/10.1073/pnas.1118539109 Shinoda et al. Downloaded by guest on October 6, 2021 − − Fig. 4. CD69 regulates the relocation of Th cells to the BM. (A) Relocation of CD69 / antigen-specific effector CD4 T cells to BM is impaired. A total of 1 × 107 − − splenic CD4 T cells from CD69+/+ (open column) and CD69 / (filled column) DO11.10 Tg mice 4 d after immunization with 100 μg OVA plus 10 μg LPS were labeled with CMFDA or CMTMR, respectively, mixed, and transferred into C.B-17/scid mice. Two hours later, CMFDA+ or CMTMR+ OVA-TCR+CD44hi CD4 T cells in spleen and BM of the host mice were assessed by flow cytometry (n = 5). (B) Relocation of antigen-specific CD4 T cells to the BM can be blocked with the Fab fragment of anti-CD69 antibody. Splenic CD4 T cells from DO11.10 Tg mice, 4 d after immunization with 100 μg OVA plus 10 μg LPS, were pretreated with the Fab fragment of anti-CD69 antibody (filled column) or control (open column) before labeling with CMFDA or CMTMR. These cells were mixed and transferred into C.B-17/scid mice. Two hours later, CMFDA+ or CMTMR+ OVA-TCR+CD44hi CD4 T cells in spleen and BM of the host mice were assessed by flow cytometry (n = 6). (C) Relocation of antigen-specific memory Th cells to the BM is dependent on CD69. CD4 T cells (3 × 105) from DO11.10 Tg mice were transferred into BALB/c mice, and the mice were then injected with 100 μg of OVA plus 10 μg LPS. At days 4, 6, and 8 after immunization, mice were injected with 100 μgof Fab fragments of anti-CD69 antibodies (filled column) or isotype control (open column), and analyzed for OVA-TCR+CD44hi CD4 T cells in spleen and BM at day − − 12 after immunization. (D) CD69-dependent rebound of BM-derived CD44hi CD4 T cells to the BM. A total of 2 × 106 B220 MHC class II cells from BM of BALB/c mice were treated with the Fab fragment of anti-CD69 antibodies (filled column) or isotype control (open column), labeled, transferred, and quantified as shown in B (n = 5). All data (mean ± SD) are representative of two or more independent experiments (*P < 0.05 and **P < 0.01).

lacking (Fig. 3 C and D). In an immune response to OVA, splenic CD69-deficient mice provides the opportunity to analyze BM OVA-specific CD69-deficient CD4 T cells at day 48 after im- memory cells for their unique roles in humoral immunity. The munization were detectable and similar in number to WT cells lack of CD69 in CD4 T cells induced the defective production of (Fig. 1 A and B). The selective loss of BM memory Th cells in high-affinity antibodies (Fig. 2A and Fig. S4A). To clarify the

Fig. 5. CD69 regulates the persistence of Th cells on BM stromal cells. (A) Anti-CD69– treated antigen-specific effector CD4 T cells of BM do not contact laminin+ stromal cells. As described in Fig. 4B, antigen-spe- cific effector CD4 T cells were treated with the Fab fragment of anti-CD69 antibodies or isotype control, labeled with CMFDA or CMTMR, respectively, and transferred into C.B-17/scid mice. Representative histologi- cal section of CMFDA (green), CMTMR (red), and laminin (gray) is shown (Left). A percentage of CMFDA+ or CMTMR+ cells contacting laminin+ cells in the BM of the host mice were enumerated by immuno- histological analysis (Right). (B) CD69- blocked effector Th cells significantly failed to persist on the stromal cells. Absolute numbers of CMFDA+ or CMTMR+ OVA- TCR+CD44hi CD4 T cells contacting laminin+ cells in the BM were calculated from data by flow cytometry in Fig. 4B and immuno- histological analysis in Fig. 5A (n = 3). (C) CD69+CD4+ cells in BM expressed Ly-6C highly. A representative CD69/CD4 profile of BM cells and a histogram of Ly-6C among CD69+CD4+ cells are shown. The isotype control is shown in gray (n = 3). (D) CD69-expressing memory Th cells contact with laminin+ cells. Representative histological section of GFP (green), CD4 (red), and laminin (gray) labeling in BM − of CD69gfp/+ mice is shown (Upper). A percentage of GFP+CD4+ or GFP CD4+ cells contacting laminin+ cells in the BM of CD69gfp/+ mice were enumerated by

− IMMUNOLOGY immunohistological analysis (Lower). Thirty-six of 43 GFP+CD4+ cells were in contact with laminin+ cells, compared with 24 in 82 GFP CD4+ cells. All data (mean ± SD) are representative of two or more independent experiments (**P < 0.01).

Shinoda et al. PNAS | May 8, 2012 | vol. 109 | no. 19 | 7413 Downloaded by guest on October 6, 2021 rationale of the defect, GC B cells and splenic and BM plasma approved by the Chiba University Review Board for Animal Care. For im- cells were enumerated, and CD154 expression and cytokine munization, mice were injected with OVA (Sigma), NP29-KLH, NP29-OVA, or production, which are essential for help to B cells, were measured NP36-CGG (Biosearch Technologies) with LPS (Invivogen), alum (Imject Alum; hi in TFH cells and splenic and BM CD44 CD4 T cells. Splenic B/ Pierce), or IFA (Sigma). plasma and T cells were numerically and functionally normal. In contrast, BM plasma cells and T-cell numbers were decreased. Cell Labeling and Adoptive Transfer. For adoptive transfer, CD4 T cells from These data indicate that the defect of BM plasma cells was caused BALB/c or DO11.10 Tg mice were sorted by magnetic-activated cell sorting by the absence of BM antigen-specific Th cells. BM Th cells may (MACS) and transferred i.v. into BALB/c or C.B-17/scid mice. For positive se- play a unique and essential role in the transmigration of plasma lection and neutralization by antibodies, we used the Fab fragment of anti- cells to the BM and/or their persistence in the BM. CD4 or anti-CD69 antibodies and streptavidin-MACS microbeads (Miltenyi Injection of Fab fragments of anti-CD69 antibodies blocked Biotec). For induction of OVA-TCR+ T cells, mice were immunized i.p. with C FH the relocation of effector CD4 T cells to the BM (Fig. 4 ). This 100 μgNP29-OVA plus LPS after adoptively transferred CD4 T cells from may be used as a possible therapeutic for prevention of the DO11.10 Tg mice. OVA-TCR+ cells were phenotyped by staining with anti- persistence of harmful memory cells, i.e., those responsible for bodies against PD-1 and CXCR5. To monitor donor cells in host mice, cells allergy and autoimmune diseases. To date the precise roles of were labeled with the cytoplasmic probes CellTracker Green 5-chlor- memory Th cells in acquired immunity have remained unclear. fl fi omethyl uorescein diacetate (CMFDA) and CellTracker Orange (5-(and-6)- However, this study and the use of Th memory-de cient mice (((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) (CMTMR; Invitrogen) will contribute to the clarification of their roles in the primary 7 before transfer. Briefly, cells (1 × 10 cells/mL) were incubated with 0.1 μMof and secondary immune responses. CMFDA or 5 μM of CMTMR in PBS solution for 15 min at 37 °C, washed, and ’ Materials and Methods incubated for another 30 min at 37 °C, according to the manufacturer sin- struction. Flow cytometric data were analyzed with FlowJo software Detailed descriptions of all materials and methods are provided in SI (Tree Star). Materials and Methods.

fi ACKNOWLEDGMENTS. We thank K. Katakura, K. Sugaya, T. Fukasawa, Mice. CD69-de cient mice were backcrossed more than 13 times onto C57BL/6 T. Geske, and H. Hecker-Kia for expert technical help. This work was or BALB/c backgrounds (21). DO11.10 Tg mice were provided by D. Loh supported by Global Center for Education and Research in Immune System (Washington University School of Medicine, St. Louis, MO) (37). For all Regulation and Treatment (Ministry of Education, Culture, Sports, Science, experiments, mice were used at 6 to 16 wk of age and were maintained and Technology), Grant-in-Aid for Scientific Research on Priority Areas under specific pathogen-free conditions. BALB/c, C57BL/6, and C.B-17/scid 22021011, Scientific Research (B) Grant 21390147, Young Scientists (A) Grant mice were purchased from Clea. CD69:GFP knock-in mice (CD69gfp/+) were 22689014, Research Activity Start-Up Grant 23890030, and Japan Society generated by homologous recombination in ES cells. Transfection of ES cells for the Promotion of Science Fellowship 22.56132; the Uehara Memorial Foundation; Takeda Science Foundation; Naito Foundation; Astellas and selection of clones were performed essentially as described for CD69 KO Foundation for Research on Metabolic Disorders (Japan); Deutsche For- mice (21). To introduce cDNA-encoding egfp into the cd69 locus, we gen- schungsgemeinschaft Grant SFB 650; and the Federal Ministry of Education fi erated a replacement vector to remove the rst exon of the cd69 gene and Research (Germany) for support through Forschungseinheiten der encompassing the initiation codon (Fig. S8A). CD69gfp/+ mice were back- Systembiologie. K.T. was a Research Fellow of the Alexander von Hum- crossed 11 times to the C57BL/6 background. All animal experiments were boldt Foundation.

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