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Vaccination with protein induces high-affinity memory B cells and persistent circulating antigen-specific antibodies for long-lasting humoral immunity1,2. Follicular helper T cells (TFH cells) have emerged as another class of immunoregulatory cells3–5 specialized to control the stepwise development of antigen-specific B cell immunity. In the first week after priming, antigen-specific TFH cells emerge6–8 to initiate antibody secretion, isotype switching and the germinal center (GC) reaction9. In the GC, TFH cells regulate the develop-ment of high-affinity memory B cells10–12 and the production of long-lived plasma cells13. After rechallenge with antigen, memory TFH cells promote the population expansion of antigen-specific mem-ory B cells and the rapid induction of high-affinity plasma cells7,14. Thus, antigen-specific TFH cell function is central to multiple facets of B cell immunity, but how this cognate regulatory activity is controlled remains poorly understood.

The development and function of antigen-specific TFH cells emerges progressively with separable requirements for cognate con-trol. The initial programming of TFH cells occurs after the first contact with dendritic cells (DCs) expressing complexes of peptide and major histocompatibility complex (MHC) class II (pMHCII) in the T cell zones of draining lymphoid tissue15. Loss of the chemokine receptor CCR7 and expression of the chemokine receptor CXCR5 relocates TFH cells to B cell zones6 and facilitates contact with antigen-primed pMHCII-expressing B cells16–18. The transcriptional repressor Bcl-6 (A000369) is expressed by the early pre-GC TFH cells8 and is sufficient and necessary to induce this program in vivo19–21. Intercellular contact of long duration17 with signals promoted by signaling lymphocytic- activation molecule receptors17 and engagement of inducible costimu-lator (ICOS) through its ligand ICOSL on B cells is required for the

entry of pre-GC TFH cells into the GC environment22,23. Ectopic ICOS expression by naive helper T cells promotes excessive TFH population expansion, enlarged GCs and autoantibody production9. Interactions between interleukin 21 (IL-21; A001258) and its receptor IL-21R (A001259) are central to effective B cell immunity24,25. Although TFH cells can develop in the absence of IL-21R (refs. 10,11), the lack of IL-21 signaling in B cells substantially compromises the GC reaction and the production of post-GC plasma cells10–12. The immunoregu-latory receptor PD-1 expressed by TFH cells has also been linked to the control of GC B cell survival and post-GC plasma cell production mediated by the PD-1 ligand PD-L2 (ref. 13). Hence, clonal expansion and the cognate delivery of specific TFH cell functions also require a highly regulated program of progressive development in vivo.

Antibody-secreting cells constitute the critical effector B cell com-partment produced through regulation by antigen-specific TFH cells in vivo. Plasma cells are defined as postmitotic antibody-secreting cells and represent the terminally differentiated component of the anti-body-secreting cell compartment1,2. Outside the GC, the development of plasma cells requires regulation by TFH cells and occurs rapidly during the first week after vaccination with proteins. These non-GC plasma cells do not somatically hypermutate26,27 but can switch anti-body classes and typically have short half-lives of 3–5 d (ref. 28). In contrast, the development of GC plasma cells involves affinity matura-tion and antibody class switching that results in long-lived compart-ments of high-affinity plasma cells1,2,29. The transcriptional repressor Blimp-1 (A003268) initiates the development of plasma cells in both pathways30, exerting its control largely through repression of Bcl-6 and the transcription factor Pax5 (ref. 31), which allows derepression of the transcription factor XBP-1 (refs. 32,33). Moreover, IL-21, which

1Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, USA. 2Present address: Roche, Basel, Switzerland (E.U.), and Institut National de la Santé et de la Recherche Médicale U563, Toulouse, France (N.F.). Correspondence should be addressed to M.G.M.-W. (mcheyzer@scripps.edu).

Received 2 July; accepted 29 September; published online 31 October 2010; addendum published after print 8 March 2011; doi:10.1038/ni.1954

Plasma cells negatively regulate the follicular helper T cell programNadége Pelletier1, Louise J McHeyzer-Williams1, Kurt A Wong1, Eduard Urich1,2, Nicolas Fazilleau1,2 & Michael G McHeyzer-Williams1

B lymphocytes differentiate into antibody-secreting cells under the antigen-specific control of follicular helper T cells (TFH cells). Here we demonstrate that isotype-switched plasma cells expressed major histocompatibility complex (MHC) class II, the costimulatory molecules CD80 and CD86, and the intracellular machinery required for antigen presentation. Antigen-specific plasma cells accessed, processed and presented sufficient antigen in vivo to induce multiple helper T cell functions. Notably, antigen-primed plasma cells failed to induce interleukin 21 (IL-21) or the transcriptional repressor Bcl-6 in naive helper T cells and actively decreased these key molecules in antigen-activated TFH cells. Mice lacking plasma cells showed altered TFH cell activity, which provided evidence of this negative feedback loop. Hence, antigen presentation by plasma cells defines a previously unknown layer of cognate regulation that limits the antigen-specific TFH cell program that controls ongoing B cell immunity.

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is abundantly produced by TFH cells8,10–12, potently induces Blimp-1 expression34 and the development of plasma cells in a manner dependent on the transcription factors STAT3 and IRF4 (ref. 35). XBP-1 controls the response of unfolded proteins and supports many facets of the cellular secretory apparatus32. Lower surface expres-sion of the B cell antigen receptor (BCR) and downregulation of the MHCII transactivator CIITA36 suggest that plasma cells remain highly specialized for antibody secretion in vivo and have a lower capacity for immunoregulation and responsiveness.

Contrary to those expectations, we demonstrate here continued high expression of MHCII, the costimulatory molecules CD80 and CD86 and the intracellular machinery for antigen presentation in antigen-specific isotype-switched plasma cells in vivo. Notably, after being primed in vivo, antigen-specific plasma cells expressed pMHCII complexes and were able to activate antigen-specific helper T cells. Antigen-pulsed plasma cells induced the proliferation and differentia-tion of effector cells in vitro from naive antigen-specific helper T cells but promoted Blimp-1 in favor of the induction of Bcl-6 and IL-21. Furthermore, plasma cells shut down IL-21 production and decreased Bcl-6 expression in activated helper T cells in an antigen-specific manner. In support of the idea of that negative regulatory function, CXCR5+PD-1+ TFH cells accumulated to large numbers in draining and distal lymphoid tissues after protein immunization of mice lack-ing B cell–expressed Blimp-1, which do not produce plasma cells in vivo. Large numbers of plasma cells seemed to be interspersed with helper T cells at the T cell–B cell borders and throughout the lym-phoid tissue during the second week after antigen priming, with evi-dence of contact of helper T cells with plasma cells in situ. Finally, we diminished the expression of Bcl-6 and IL-21 in antigen-specific TFH cells in vivo through adoptive transfer of antigen-pulsed plasma cells. Our data demonstrate an antigen-presentation function for plasma cells during adaptive immunity that serves to limit ongoing antigen-specific TFH cell function. Hence, we propose an additional layer of negative regulation during adaptive immunity that is a functional sensor of plasma cell production that can refine the development of antigen-specific B cell memory.

RESULTSAntigen-specific plasma cells express MHCII, CD80 and CD86The antigen-specific B cell response to nitrophenylacetyl (NP) coupled to keyhole limpet hemocyanin (KLH) as a protein carrier is regulated by TFH cells and can be assessed directly by flow cytometry14,37. After immunizing mice with NP-KLH, we were able to quantify antibody-secreting cells by intracellular labeling with antigen, the binding of antigen to the cell surface and secretion of antigen-specific antibod-ies by enzyme-linked immunospot assay (Supplementary Fig. 1). Therefore, we were able to isolate antigen-specific antibody-secreting cells (immunoglobulin M negative (IgM−) and CD138+) with dis-tinct developmental histories for subsequent analysis of function (Fig. 1a). We found that by day 5 after secondary immunization with the Toll-like receptor 4 agonist–based Ribi adjuvant system, >90% of isotype-switched antibody-secreting cells did not incorporate the thymidine analog BrdU over the preceding 24 h in vivo (Fig. 1b). Thus, most antibody-secreting cells (specific and nonspecific) used in this study could be considered noncycling, terminally differenti-ated plasma cells.

In the antigen-specific compartment, IgM−CD138+ plasma cells had high surface expression of MHCII protein (Fig. 1c). All recently formed plasma cells (non-GC at 7 d after initial priming; post-GC at 14 d after priming; and memory-response at 5 d after secondary chal-lenge) had expression of MHCII equivalent to that of naive B cells and CD11c+ DCs. Long-lived plasma cells that persisted in the spleen (>14 d after secondary challenge) or bone marrow (>14 d after secondary challenge; data not shown) had lower but readily demonstrable expression of MHCII. Isotype-switched plasma cells also upregulated expression of CD80 and CD86 and retained abundant expression at all developmental stages after antigen experience (Fig. 1d). These data suggested that antigen-experienced plasma cells retained cognate intercellular interaction with pMHCII-specific helper T cells.

The same expression pattern for MHCII, CD80 and CD86 was evident in switched plasma cells regardless of antigen specificity and across different mouse strains (Supplementary Fig. 2). In contrast, many IgM+ plasma cells had lost MHCII expression, with most also

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Figure 1 Isotype-switched plasma cells retain expression of MHCII and costimulatory molecules. (a) NP-specific plasma cell (PC) numbers over time (negative for propidium iodide (PI−), the dump channel (Dump−) and IgD−, and NP+CD138+) in the spleens of C57BL/6 mice after primary and secondary immunization with NP-KLH (open square, adjuvant-only for memory at day 5). (b) Expression of CD138 versus IgM on NP-specific cells (gated as PI−Dump−IgD− and NP+) at day 5 after secondary challenge (left); BrdU expression on NP-specific IgM−CD138+ plasma cells on day 5 after rechallenge (middle); and frequency of BrdU+ NP-specific IgM−CD138+ plasma cells on days 3 and 5 after rechallenge (Memory), relative to that of the total IgM−CD138+ plasma cell compartment (right), 20 h after a single intraperitoneal injection of BrdU. Number in outlined area (left) indicates percent CD138+IgM− cells. (c,d) Expression of I-Ab (MHCII; c) and CD80 and CD86 (d) on NP-specific IgM−CD138+ plasma cells, CD4+ helper T cells (T), CD11c+ DCs and naive B cells (B), presented as summary mean fluorescence intensity (MFI). *P < 0.05 and **P < 0.01 (Student’s t-test). Data are representative of four or more (a) or three or more (b–d) experiments with one mouse per experiment (mean and s.e.m.).

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being negative for CD80 and CD86 (Supplementary Fig. 3). The switched plasma cell compartment had very low but detectable expres-sion of mRNA encoding MHCII, its transactivator CIITA, and CD80 and CD86 (Supplementary Fig. 4), as expected given the negative effect of Blimp-1 on CIITA during the development of plasma cells36. Thus, isotype-switched plasma cells express key surface proteins asso-ciated with MHCII-restricted antigen presentation even in the pres-ence of low expression of mRNA encoding these proteins in vivo.

Plasma cells retain antigen-presentation machineryNext we investigated the mechanism used by plasma cells to control MHCII-restricted antigen processing and presentation. We readily detected invariant chain protein (Ii; also called CD74) and com-plexes of the MHCII–associated Ii peptide CLIP and MHCII mol-ecule I-Ab intracellularly in antigen-specific IgM−CD138+ plasma cells in quantities similar to those in resting B cells (Fig. 2a). We also found the peptide-editing complexes H-2M and H-2O by direct intracellular labeling for these proteins (Fig. 2b). We detected CD74, H-2M and H-2O proteins at similar abundance in nonspecific switched MHCIIhi and MHCIIint plasma cells that rep-resented recently formed and long-lived plasma cell compartments, respectively (Supplementary Fig. 5). The abundance of mRNA encoding CD74, H-2M and H-2O was low but detectable in this switched plasma cell compartment (Supplementary Fig. 5b–d). We found mRNA for the carboxypeptidases cathepsin B (CtsB) and CtsZ, associated with early endosomes, and the aminopeptidases AEP and CtsH, associated with lysosomes, in switched plasma cells (Fig. 2c). We also found mRNA for other cathepsin proteases (GILT, CtsS, CtsL, CtsC and CtsD), present in low abundance (Supplementary Fig. 6). Therefore, in addition to evidence of cell surface MHCII, CD80 and CD86, there is abundant evidence of intracellular machinery involved in antigen processing and presen-tation in the specialized MHCII peptide-loading pathway in vivo.

Antigen-specific plasma cells present antigen in vivoAlthough the machinery for antigen processing was in place, it remained important to address the ability of plasma cells to present specific antigenic peptides in vivo. Using hen egg lysozyme (HEL) as the immunogen, we directly visualized HEL-binding plasma cells in polyclonal B10.BR (H-2k-restricted) mice 5 d after a secondary boost with soluble antigen (Fig. 3a). Direct labeling with Aw3.18 (ref. 38), an antibody specific for the dominant helper T cell epitope of HEL amino acids 48–61 in I-Ak, showed considerable specific pMHCII on HEL-binding plasma cells (Fig. 3b). There was negligible back-ground antigen binding or expression of pMHCII in mice primed with irrelevant pigeon cytochrome c (PCC) antigen before the boost with soluble HEL (Fig. 3b). Thus, antigen-specific memory-response plasma cells present detectable amounts of the dominant pMHCII 5 d after soluble protein rechallenge in vivo.

Plasma cells drive proliferation of specific helper T cellsAt 10 d after primary adjuvant-driven immunization, most anti-gen-specific plasma cells express isotype-switched BCRs with point mutations and evidence of antigen-driven selection during the GC reaction27. To assess the ability of post-GC plasma cells to present antigen, we isolated IgM−CD138+ plasma cells 10 d after immuniz-ing mice with PCC and cultured the cells together with naive PCC-specific helper T cells with transgenic expression of the 5C.C7 αβ T cell antigen receptor (TCR; Fig. 3c). The switched plasma cells from PCC-immunized mice, but not those from control mice immunized with adjuvant only, promoted substantial proliferation of naive anti-gen-specific helper T cells without the addition of exogenous antigen. The addition of exogenous peptide promoted no further upregula-tion of the activation and memory marker CD44 or dilution of the cytosolic dye CFSE in vitro. Thus, antigen-specific post-GC plasma cells express sufficient pMHCII and provide adequate costimula-tion in vivo to drive the proliferation of naive antigen-specific helper T cells in vitro.

Next, to assess whether plasma cells can activate naive helper T cells in vivo, we pulsed plasma cells with HEL amino acids 48–61 and adoptively transferred the cells into mice containing CFSE-labeled, HEL-specific, naive helper T cells with transgenic expression of the 3A9 αβTCR (Fig. 3d). In this transfer, HEL peptide–pulsed plasma cells promoted the activation and proliferation of helper T cells, but the same dose of soluble HEL peptide did not. Thus, plasma cells probably gain physical access to naive helper T cells in vivo by migrat-ing through lymphatics to the T cell zones of the draining lymph nodes. In this manner, pMHCII-expressing plasma cells can activate antigen-specific helper T cells in situ.

Plasma cells cannot induce the development of pre-GC TFH cellsGiven the ability of switched plasma cells to act as antigen-present-ing cells (APCs), it became important to compare and contrast this activity with that of conventional APCs. We found that the same number of switched plasma cells and CD11c+ DCs pulsed with either peptide or protein antigen induced similar activation and proliferation in naive antigen-specific helper T cells (Fig. 4a and

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Figure 2 Plasma cells express the intracellular machinery for MHCII antigen processing and presentation. (a,b) Intracellular expression of CD74 (Ii) and I-Ab–CLIP (a) and of H-2M and H-2O (b) on NP-specific IgM−CD138+ plasma cells in C57BL/6 spleens on day 5 after secondary immunization with NP-KLH, and in naive B cells and CD4+ helper T cells, presented as summary MFI. (c) Quantitative PCR analysis of the expression of mRNA for various proteases (vertical axes) in CD4+ helper T cells, CD11c+ DCs, naive B cells and IgM−CD138+ plasma cells, presented in arbitrary units (AU) relative to expression in naive T cells, set as 1. *P < 0.01 and **P < 0.001 (Student’s t-test). Data are representative of three experiments (a,b) or three or more experiments (c) with one mouse each (mean and s.e.m.).

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Supplementary Fig. 7). We obtained similar results with two anti-gen systems (PCC and HEL) using different naive antigen-specific helper T cell populations (Supplementary Fig. 8). Even substan-tially decreasing the antigen dose or the ratio of APC to helper T cell resulted in very little difference between the various types of APC (Supplementary Fig. 9). Furthermore, analysis with acti-vated antigen-specific helper T cells purified by flow cytometry for quantitative PCR after culture (Supplementary Fig. 10) indicated that both types of APC induced robust and equivalent expression of

mRNA (Fig. 4b) and protein (Supplementary Fig. 11) for a series of known helper T cell–derived cytokines. However, in contrast, antigen-pulsed DCs induced high expression of IL-21 and Bcl-6 in activated helper T cells that was notably absent from the helper T cells in antigen-pulsed plasma cell cultures (Fig. 4c). Switched plasma cells uniquely induced Blimp-1, the transcriptional repres-sor of Bcl-6 and the TFH program. Thus, unlike DCs, switched plasma cells were unable to drive naive antigen-specific helper T cells toward the TFH fate in vitro.

Plasma cells decrease IL-21 and Bcl-6 in TFH cellsAntigen-specific TFH cells are required for the generation of isotype-switched plasma cells in vivo. Thus, we considered it unlikely that naive helper T cells would be the main target of the APC activity of switched plasma cells in vivo. Therefore, we assessed whether antigen-primed plasma cells had an effect on the development or

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Figure 3 Antigen-specific plasma cells process and present antigen in vivo. (a) Isotype-switched B cell compartment (Dump−IgD−IgM− and CD19+CD138+; far left), HEL binding versus CD138 expression (middle) and total number of switched HEL-specific plasma cells (far right) in B10.BR mice given primary immunization with HEL or PCC (above plots or below graph), assessed on day 5 after secondary challenge with soluble HEL. Numbers adjacent to outlined areas indicate percent CD138+CD19+ cells (far left) or HEL+CD138+ cells (middle). (b) Isotype-switched B cells (Dump−IgD−IgM−; far left), HEL binding versus pMHCII expression (middle) and summary MFI for pMHCII (far right), for cells from mice treated as in a. (c) Expression of CD44 and CFSE on CFSE-labeled 5C.C7 αβTCR–transgenic helper T cells cultured for 4 d with isotype-switched plasma cells isolated 10 d after vaccination of mice with adjuvant only (far left) or with PCC protein (middle), either alone (PCC) or with the addition of MCC peptide (+ peptide); numbers in outlined areas indicate frequency of responders. Far right, total CD44hiCFSElo helper T cells. (d) Expression of CD44 and CFSE (left and middle) on CFSE-labeled 3A9 αβTCR–transgenic HEL-specific helper T cells 24 h after transfer into B10.BR mice immunized with peptide alone (HEL) amino acids 48–61 (HEL(48–61) only), given transfer of IgM−CD138+ plasma cells pulsed with 10 μM HEL peptide (Peptide-pulsed PCs), or immunized with HEL in adjuvant (HEL + adjuvant; n = 1 mouse); numbers in outlined areas indicate frequency of responders. Far right, total CD44hiCFSElo helper T cells. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are representative of three experiments with one mouse each (mean and s.e.m.).

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Figure 4 Plasma cells induce proliferation, many helper T cell functions and Blimp-1 expression but not expression of Bcl-6 or IL-21. (a) Expression of CD44 and CFSE (left and middle) on CFSE-labeled 5C.C7 αβTCR–transgenic helper T cells activated for 4 d in vitro with IgM−CD138+ plasma cells or CD11c+ DCs alone (0), MCC (Peptide (MCC)) or PCC (Protein (PCC)); numbers in outlined areas indicate frequency of responder. Far right, total activated CD44hiCFSElo helper T cells. (b,c) Quantitative RT-PCR analysis of the expression of mRNA for various molecules (horizontal axes) in CD4+ helper T cells left unactivated (Naive) or activated in cultures with CD11c+ DCs (DC-activated) or IgM−CD138+ plasma cells (PC-activated) as APCs in the presence of PCC protein; results are normalized to β2-microglobulin and are presented relative to those of naive CD4+ helper T cells assessed ex vivo immediately after isolation, set as 1. TNF, tumor necrosis factor; IFN, interferon. *P < 0.05 (Mann-Whitney test). Data are representative of ten or more (a) or three or more (b,c) experiments with one mouse each (mean and s.e.m.).

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function of previously differentiated antigen-specific TFH cells. For this, we established primary helper T cell cultures with DCs and protein to induce TFH cells (as described above) and then subse-quently divided the activated antigen-specific helper T cells across secondary cultures with isotype-switched plasma cells or CD11c+ DCs, with or without antigen. CD11c+ DCs supported the further accumulation of antigen-specific helper T cells regardless of anti-gen addition (Fig. 5a). In contrast, plasma cells supported no fur-ther accumulation of helper T cells in the absence of antigen and supported the accumulation of significantly fewer helper T cells in the presence of antigen (Fig. 5a). The antigen-dependent decrease occurred without changes in the abundance of mRNA for the antiap-optotic factors Bcl-2 and Bcl-XL or the apoptosis promoters Bax and Bak in the helper T cells (Supplementary Fig. 12a), which suggested no substantial changes via apoptosis in the culture. Thus, in contrast to their effect on the activation of naive helper T cells, switched plasma cells had a broad negative effect on previously activated effector helper T cells.

Although IL-21 is produced by many effector helper T cell sub-types8, it has been a useful indicator of selective TFH differentiation in vitro20–23. Notably, we found an antigen-dependent decrease in IL-21 controlled by switched plasma cells (Fig. 5b). This functional deviation was selective for IL-21 without an effect on the produc-tion of other cytokines such as tumor necrosis factor or IL-10 (Supplementary Fig. 12b), which demonstrated antigen-specific downregulation of a major TFH cell function. Furthermore, Bcl-6 is a principal inducer of the TFH developmental program in vivo and in vitro19–21. In the secondary cultures, plasma cells resulted in significantly lower Bcl-6 expression in the antigen-specific helper T cells in an antigen-specific manner (Fig. 5b). We extended those results to in vivo antigen-activated 5C.C7 αβTCR–transgenic helper T cells that produced demonstrable amounts of IL-21 after prim-ing with antigen. In 24 h of short-term coculture, switched plasma cells resulted in lower expression of IL-21 and Bcl-6 in the helper T cells, in contrast to coculture with antigen-pulsed CD11c+ DCs (Fig. 5c). This effect was selective, as IL-4 and the transcription factor GATA-3 were unaffected and expression of the transcrip-tion factor T-bet was similarly affected by DC and plasma cell presentation (Supplementary Fig. 12c). Those trends were reca-pitulated with nontransgenic polyclonal antigen-specific helper T cells positive for the TCR α-chain variable region 11 and β-chain variable region 3 (Vα11+Vβ3+) at day 7 after priming (Fig. 5d and

Supplementary Fig. 12d). Therefore, antigen-specific plasma cells negatively regulate the function and developmental programming of antigen-specific TFH cells in vitro.

TFH cells accumulate in the absence of plasma cellsTo understand the role of plasma cell presentation in vivo, we immu-nized C56BL/6 mice conditionally lacking Blimp-1 in the B cell compartment (called ‘Blimp-1-cKO mice’ here) with NP-KLH. An antigen-specific primary and memory B cell response develops in these mice without antibody-secreting cells from all stages of the immune response30. Notably, the primary GC reaction was also sig-nificantly larger in these mice, for reasons that remain unresolved30 (P < 0.05; Supplementary Fig. 13a). We quantified CXCR5+PD-1+ TFH cells in the activated CD44hiCD62LloICOShi subset of CD4+ T cells that accumulated after primary immunization (Fig. 6a and Supplementary Fig. 13b). In the absence of plasma cells, CXCR5+PD-1+ TFH cells accumulated to significantly greater num-bers in the draining lymph nodes and spleen at all time points tested (days 9, 11 and 15; P < 0.05). In the TFH compartment, the brightest TFH cells (CXCR5++PD-1++) were significantly greater in abundance at day 11 in the absence of plasma cells (P < 0.01), which suggested ‘preferential’ accumulation of GC TFH cells in these mice.

After local development in the GC, high-affinity post-GC plasma cells migrate to distal tissues that favor longevity and/or func-tion of the secreting compartment. In the absence of plasma cells, CXCR5+PD-1+ TFH cells accumulated to significantly greater num-bers in the mesenteric lymph nodes, consistent with the loss of a local regulatory function for plasma cells in this region (P < 0.05; Supplementary Fig. 13c). More CXCR5−PD-1+ cells accumulated in the CD44hiCD62Llo compartment of CD4+ helper T cells in the bone marrow without plasma cells (Supplementary Fig. 13d). No change occurred in the Ly6C+ memory CD4+ helper T cell compart-ment, which suggested a selective effect on PD-1+ helper T cells in this typically plasma cell–rich environment. These data suggested an additional function for plasma cells in favored survival niches and perhaps for extended periods after initial development in local GC environments.

Next we attempted to reverse the accumulation of TFH cells in the immunized Blimp-1-cKO mice at day 11. We transferred

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Figure 5 Switched plasma cells selectively inhibit the expression of IL-21 and Bcl-6 in TFH cells. (a) Total CD44hiCD62Llo helper T cells after secondary culture of in vitro–activated CFSE-labeled 5C.C7 αβTCR–transgenic helper T cells in medium alone (0) or with CD11c+ DCs or IgM−CD138+ plasma cells as APCs with (Ag) or without (No) PCC antigen. (b) Quantitative RT-PCR analysis of the expression of IL-21 mRNA and Bcl-6 mRNA by in vitro–activated helper T cells reactivated in various secondary culture conditions (horizontal axes); results are normalized to β2-microglobulin and presented relative to those of naive helper T cells, set as 1. (c) Quantitative RT-PCR analysis of the expression of mRNA for various molecules (vertical axes) in 5C.C7 αβTCR–transgenic helper T cells activated in vivo, then sorted and reactivated in various culture conditions (horizontal axes); results presented as in b. Open bars, in vivo–activated helper T cells before culture. (d) Quantitative RT-PCR analysis of the expression of mRNA for various molecules (vertical axes) in nontransgenic, in vivo antigen–activated Vα11+Vβ3+ helper T cells on day 7 after immunization of mice (n = 4) with PCC and reactivated in various culture conditions (horizontal axes); results presented as in b. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with medium alone or compared according to brackets (Student’s t-test). Data are representative of three or more experiments (a–c) or four experiments (d) with one mouse each (mean and s.e.m.).

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unfractionated lymph node and spleen cells from C57BL/6 mice 4 d after secondary immunization with the same antigen-adjuvant com-bination. Transferring this source of antigen-specific plasma cells restored TFH cell numbers in the spleen of immunized Blimp-1-cKO mice without affecting cell numbers in littermate controls (Fig. 6b) and decreased the greater TFH cell numbers in the lymph nodes to the numbers in littermate controls (Supplementary Fig. 14a). At day 7 after priming, most antigen-specific helper T cells were in the follicular B cell areas but not in the GCs. At this early time point, there were significantly more CXCR5+PD-1+ TFH cells in both the spleen and lymph nodes of Blimp-1-cKO mice (P < 0.05; Fig. 6c and Supplementary Fig. 14b,c). Thus, in the absence of the development of plasma cells, the accumulation of greater numbers of TFH cells is consistent with the loss of a plasma cell–controlled local negative regulatory loop in vivo.

Plasma cells negatively affect TFH cells in vivoWhen we assessed cells in situ after immunization of mice with PCC, we found large numbers of IgD−CD138+ plasma cells interspersed

with T cells outside GCs at T cell–B cell borders and throughout T cell zones and the medullary regions (Fig. 7a–e). Furthermore, we found evidence of colocalization of CD138+ plasma cells with Vβ3+ T cells that included the dominant antigen-specific responder helper T cell compartment7,8, with evidence of cell contact at mul-tiple points between Vβ3+ T cells and CD138+ plasma cells at the T cell–B cell regions of these lymph nodes (Fig. 7f–h). Adoptive transfer of plasma cells pulsed with moth cytochrome c (MCC) pep-tide 2 d before the peak of the local antigen-specific TFH response induced selectively lower expression of Bcl-6 and IL-21 and not of IL-4 or GATA-3 (Fig. 7i), as assessed on a per-cell basis in puri-fied CD44hiCD62LloCXCR5+ Vα11+Vβ3+ TFH cells as described7,8. There was no influence on the splenic TFH cell response in the lym-phoid tissues that did not directly drain the site of adoptive transfer

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Figure 7 Plasma cells localize together with CD4+ T cells and negatively affect the antigen-specific TFH program in vivo. (a–h) Confocal microscopy of draining lymph nodes from PCC-immunized B10.BR mice at day 10 after priming. Top left, approximate location of medulla (M) relative to positions of images in a–f; below, colors of labeled antibodies for antigens. B, B cell follicle; T, T cell area. Areas outlined in a and in f,g are enlarged at right in b and in h, respectively. White arrowheads (h) indicate contact of CD138+ plasma cells with Vβ3+ T cells. Scale bars, 100 μm. (i) Quantitative RT-PCR analysis of the expression of mRNA for various molecules (vertical axes) in TFH cells sorted as Vα11+Vβ3+CD44hiCD62Llo and CXCR5+ from lymph nodes of B10.BR mice on day 7 after PCC vaccination; mice received transfer of no cells (0) or plasma cells (PC) 2 d before analysis. Results are normalized to β2-microglobulin and presented relative to those of naive helper T cells, set as 1. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are representative of three experiments with one mouse each (mean and s.e.m.).

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Figure 6 More accumulation of TFH cells in the absence of plasma cells in vivo. (a) Expression of CXCR5 versus PD-1 (left) on activated CD4 helper T cells (gated as CD16-CD32−B220−CD8−CD4+CD62LloCD44hi) from lymph nodes (LN) and spleens (SP) of Blimp-1-cKO mice (cKO; right) and littermates (wild-type (WT); left) 11 d after vaccination with NP-KLH in adjuvant. (b) Expression of CXCR5 versus PD-1 on activated spleen CD4+ helper T cells from Blimp-1-cKO mice (right) and littermates (left) 11 d after vaccination with NP-KLH in adjuvant; 2 days before analysis, mice received mixtures of 5.0 × 107 unfractionated lymph node and spleen cells from C57BL/6 mice intraperitoneally and at the base of the tail from day 4 after secondary NP-KLH immunization. (c) Expression of CXCR5 versus PD-1 on activated splenic CD4 helper T cells (gated as in a) from Blimp-1-cKO mice (right) and littermates (left) 7 d after vaccination with NP-KLH in adjuvant. Numbers adjacent to outlined areas indicate percent CXCR5+PD-1+ cells (left; outer outlined areas) or CXCR5++PD-1++ cells (right; inset outlined areas). Right, total PD-1+CXCR5+ or PD-1++CXCR5++ activated helper T cells. NS, not significant; *P < 0.05 (Student’s t-test). Data are representative of three experiments with one mouse each (mean and s.e.m.).

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(Supplementary Fig. 15). Thus, excessive numbers of plasma cells can negatively affect the local TFH program in antigen-responsive TFH cells in vivo.

DISCUSSIONOur findings challenge the existing paradigm that plasma cells are specialized only for antibody secretion. We have demonstrated that plasma cells participated in the cognate regulation of ongoing adap-tive immunity. Isotype-switched plasma cells processed and presented antigen in vivo and functioned as highly competent APCs for naive helper T cells. However, we propose that antigen-specific plasma cells act at a later phase in the developing immune response to negatively regulate antigen-specific TFH cell function. The effect of negative reg-ulation may extend into the GC reaction with an influence on distal immune-response sites and plasma cell–survival niches such as the bone marrow. We propose that there is an additional layer of negative regulation during adaptive immunity that senses the production of antigen-specific plasma cells to refine the ongoing development of high-affinity B cell memory.

Plasma cells are clearly specialized to secrete antigen-specific antibody. Blimp-1 orchestrates the development of plasma cells by repressing existing B cell programs (AID, Bcl-6 and Pax5), block-ing cell cycle entry (c-Myc and E2f1) and initiating substantial changes in the secretory apparatus (J-chain) and unfolded protein response (XBP-1)31. In our study here, we have provided evidence of the maintenance of proteins associated with MHCII antigen processing and presentation, despite a low abundance of mRNA. How the MHCII machinery is maintained in the presence of low mRNA abundance and beyond the acute phase of the initial pri-mary response remains unclear39,40. Long-term protein stability is possible, but it is more likely that the small amount of mRNA is sufficient for the production of protein in plasma cells highly spe-cialized for protein secretion31. There have been reports of MHCII and costimulatory molecules on plasma cells41,42 and transformed myelomas43. However, the ability of plasma cells to function as competent APCs was unexpected.

Post-mitotic cells that are highly specialized for secretion still need to sense and respond to environmental cues to exert effec-tive regulatory function in vivo. We have provided evidence here of continued expression of BCRs on newly formed and long-lived plasma cells. The TFH compartment is already subspecialized for cognate pairing with antigen-experienced B cells3–5,44. Multiple costimulatory and adhesion molecule pairs are expressed by these two cell types to create strong interactions of long duration after cognate contact17,18. First, contact between TFH cells and B cells leads to higher expression of IL-21 and Bcl-6 (refs. 10–12). There is also evidence of cognate contact between GC TFH cells and GC B cells by real-time imaging45 and the sorting of TFH cell–GC B cell conjugates46. However, the developmental consequences of these cognate interactions for the GC TFH cells remain unclear. IL-21 production by TFH cells is central to affinity maturation, GC persistence, function and B cell fate determination10,11. In our study here, we have demonstrated the ability of plasma cells to dampen the production of IL-21 by effector TFH cells in a nega-tive feedback loop. We propose that these actions constitute an acute regulatory activity to contract TFH cell function, thereby constraining ongoing B cell immunity. Furthermore, given the evidence of continued expression of BCRs and MHCII presen-tation machinery in long-lived plasma cells, we speculate that there is an additional role for these interactions in the long-term survival of plasma cells.

Commitment to the plasma cell fate requires contact with TFH cells. In the spleen, non-GC plasma cells first appear in T cell areas and then migrate out through the bridging channels into the red pulp1,2. In the lymph nodes, plasma cells can accumulate at the T cell–B cell borders and the medullary cords47. Post-GC plasma cell commitment is also considered to be dependent on GC TFH cell signals. Evidence of PD-1 involvement13, a direct role for IL-21 (refs. 10,11) and expression of Blimp-1 in the GC itself 48 further supports this position. Under normal physiological conditions, it is difficult to see the differentiation of plasma cells in the GC compartment. However, in the absence of the chemokine EBI-2, some GC B cells are unable to leave the GC and produce more discernible compartments of putative plasma cells in situ49. The rapid production of plasma cells after antigen rechallenge is dependent on CD4+ TFH cells; however, the distribution of memory response plasma cells in lymph nodes has not been well characterized. Nevertheless, these trends indicate that plasma cells or their immediate precursors reside in the same microenvironments as TFH subtypes to enable their selective control of the TFH program in vivo.

The finding of more TFH cells in the absence of plasma cells pro-vides support for the idea of the existence of this negative regula-tory loop in vivo. There were greater numbers of all CXCR5+PD-1+ TFH cells in these mice at many time points after antigen priming. The GC TFH cell compartment has the highest expression of PD-1 and CXCR5 (ref. 5), and these cells accumulate to greater numbers in the absence of plasma cells in vivo. It has also been suggested that these cells have the highest expression of ICOS and of GL-7 (ref. 50), as a GC subset–specific marker. Although B cell memory develops in the absence of plasma cells30, its capacity for long-term immunoprotection is limited in the absence of plasma cells. The Blimp-1-cKO mice are also devoid of secreted antibodies that may influence the contraction of adaptive immunity. However, in the absence of the FcγR Fc receptor for IgG on helper T cells, these effects probably do not directly influence the TFH program. Furthermore, a greater number of GC TFH cells correlates with dysregulated GC selection and autoantibody production5. Thus, in the absence of plasma cells, we would predict that a greater number of GC TFH cells results in more IL-21 production that could alter normal GC physiology and the development and function of memory B cells.

Our studies of the mechanism of negative regulation by plasma cells indicate a requirement for antigen to dampen antigen-specific TFH cell activity. Our in vitro studies indicated that cognate contact is necessary but did not report on the effector mechanism. PD-1 expression by TFH cells and PD-L2 expression on GC B cells have been shown to have a role in regulating selection in the GC and the number of long-lived plasma cells13. IL-21 itself has an important role in these dynamic activities and the generation of high-affinity memory B cells10,11. Thus, after cognate contact, the delivery of secondary modifying signals may be cell associated and secreted. It is also plausible that different sets of GC TFH cells may affect different types of GC B cells. For example, the pairing of IL-4-secreting GC TFH cells with IgG1-expressing GC B cells46 has introduced the possibility of TFH cell–B cell subset dedication. Similarly, the rules that govern antigen-specific regulation by plasma cells in vivo focus on specific cognate TCR-pMHCII interactions and may be subsequently modified by cell-associated and secreted molecular determinants of cell fate.

Under the normal dynamics of protein vaccination, constraining TFH cell numbers and lower IL-21 production may progressively increase competitive pressure critical for GC survival signals. Thus, we predict that the negative regulation of GC TFH cell function

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could enhance affinity maturation in the GC and promote memory B cells with higher overall affinities. This cellular ‘product-to-inducer’ negative regulatory mechanism may also increase GC selection thresholds to help guard against potential self-reactivity in the GC. In the memory response, a negative regulatory loop would act as an antigen-specific plasma cell–sensing mechanism to limit the accelerated and explosive production of plasma cells that accompanies antigen rechallenge. We suggest that sensing the antigen-specific plasma cell product of the TFH cell regula-tory pathway may also provide an efficient means of constrain-ing autoreactivity and fine tuning ongoing antigen-specific B cell immunity in vivo.

The cognate control of B cells by TFH cells is an important and active area of research. We believe that the effect of B cell–controlled antigen presentation on the development and function of antigen- specific TFH cells remains poorly understood. This cascade of bidirectional regulation is central to effective adaptive immunity and regulates the emergence of antigen-specific immune memory. The previously unknown mechanism of antigen-specific plasma cell con-trol that we have identified here is one facet of this largely unexplored cognate immune regulatory mechanism.

METHODSMethods and any associated references are available in the online version of the paper at http://www.nature.com/natureimmunology/.

Accession codes. UCSD-Nature Signaling Gateway (http://www.signaling-gateway.org): A000369, A001258, A001259 and A003268.

Note: Supplementary information is available on the Nature Immunology website.

AcKNoWLEdGMENtsWe thank L. Denzin (Memorial Sloan Kettering Cancer Center) for antibodies 2C3A and Ob1; A. Rudensky (Memorial Sloan Kettering Cancer Center) for monoclonal antibody 15G4; E. Unanue (Washington University) for the Aw3.18 cell line; K. Calame (Columbia University) for mice with loxP-flanked Prdm1 alleles excised by Cre recombinase expressed from a CD19 promoter; and K. Spencer for help with confocal imaging. Supported by the German Academic Exchange Service (E.U.), the German Research Foundation (E.U.), the Canadian Institutes of Health Research (MFE-98574 to K.A.W.) and the US National Institutes of Health (AI047231, AI040215 and AI071182 to M.G.M.-W.). This is The Scripps Research Institute manuscript 20113.

AUtHoR coNtRIBUtIoNsN.P., L.J.M.-W. and M.G.M.-W. conceived of and designed the project; N.P. provided and analyzed the data for all experiments except Figures 3a,b and 6; K.A.W. designed and completed the experiments and analyzed the data for pMHCII HEL-specific plasma cells (Fig. 3a,b); E.U. laid the experimental foundation for staining HEL-specific B cells; N.F. prepared the activated helper T cell samples for quantitative PCR analysis (Fig. 4b) and provided expertise in setting up the T cell in vitro experiments; L.J.M.-W. and M.G.M.-W. did the experiments for and analyzed the data from the Blimp-1-cKO mice (Fig. 6); K.A.W. and N.F. contributed ideas and participated in the manuscript preparation; and N.P., L.J.M.-W. and M.G.M.-W. wrote the manuscript.

coMPEtING FINANcIAL INtEREstsThe authors declare no competing financial interests.

Published online at http://www.nature.com/natureimmunology/. reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.

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48. Angelin-Duclos, C., Cattoretti, G., Lin, K.I. & Calame, K. Commitment of B lymphocytes to a plasma cell fate is associated with Blimp-1 expression in vivo. J. Immunol. 165, 5462–5471 (2000).

49. Gatto, D., Paus, D., Basten, A., Mackay, C.R. & Brink, R. Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses. Immunity 31, 259–269 (2009).

50. Yusuf, I. et al. Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150). J. Immunol. 185, 190–202 (2010).

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nature immunologydoi:10.1038/ni.1954

ONLINE METHODSMice. C57BL/6, B10.BR, B10.BR-Thy1.1, 5C.C7 αβTCR–transgenic and 3A9 αβTCR–transgenic mice were maintained in specific pathogen–free condi-tions at The Scripps Research Institute. Mice with loxP-flanked Prdm1 alleles (encoding Blimp-1) excised by Cre recombinase expressed from a CD19 pro-moter were obtained from K. Calame and backcrossed nine generations onto the C57BL/6 strain and were maintained in specific pathogen–free conditions at The Scripps Research Institute. The Scripps Research Institutional Animal Care and Use Committee approved all experiments.

Immunization. Mice were immunized subcutaneously with 400 μg NP-KLH, PCC protein (Sigma) or HEL protein (Sigma) in monophosphoryl lipid A–based adjuvant7 for the primary immunization. Secondary challenge was done 6 weeks or more after primary immunization and was either identical to the primary immunization or 40 μg soluble HEL in PBS.

Flow cytometry. Cell suspensions were made as described7. For surface staining, 4 × 108 cells per ml were incubated for 45 min at 4 °C with the following labeled monoclonal antibodies conjugated in the laboratory: anti-CD3 (145.2C11), anti-CD8 (53-6.7), anti-CD11c (HL3), anti-CD62L (MEL-14), anti-CD80 (16-10A1), anti-CD86 (GL1), anti-CD138 (281-2), anti-B220 (RA3-6B2), anti-IgM (II/41), anti-GL7 (GL7), anti-Ly6C (AL21), anti-CXCR5 (2G8) and anti-I-Ak (10-3.6; all from BD Pharmingen); anti-CD4 (GK1.5), anti-CD11c (N418), anti-CD278 (ICOS; C398.4A), anti-Gr-1 (RB6-8C5) and anti-I-Ab (KH74; all from BioLegend); anti-CD19 (6D5), anti-CD38 (90), anti-CD44 (IM7), anti-PD1 (J43) and anti-F4/80 (BM8; all from eBioscience); anti-CD11b (M1/70.15), anti-Vβ3 (KJ25) and anti-IgD (11.26; all produced in-house); antibody to HEL amino acids 48–61 in I-Ak (Aw3.18), NP (4-hydroxy 3-nitrophenylacetyl; Biosearch); and anti-HEL (Sigma). For the double-biotinylated monoclonal antibody strategy, cross-reactivity was diminished by blockade with excess free biotin (Perbio).

For intracellular staining, surface-labeled cells were made permeable and fixed with Cytofix/Cytoperm (BD Pharmingen), then labeled with the fol-lowing monoclonal antibodies: anti-IgG1 (A85-1), anti-IgG2a (R19-15), anti-IgG2b (R12-3), anti-IgG3 (R40-82), anti-IgA (R5-140), anti-IgM (R6-60.2) and anti-CD74 (In-1; all from BD Pharmingen); anti-H2-M (2C3A) and anti-H2-O (Ob1; both from L. Denzin); and antibody to I-Ab–CLIP (15G4; from A. Rudensky). Samples were analyzed on FACSVantage or FACSAria III (BD Biosciences) and data were processed with FlowJo software (TreeStar). Exclusion criteria (Dump) included anti-CD4, anti-CD8, anti-Gr-1 and anti-F4/80 (for plasma cells and naive B cells); anti-B220, anti-CD8 and anti-CD11b (for CD4+ helper T cells); and anti-CD19 and anti-CD3 (for CD11c+ DCs).

BrdU analysis. Mice received a single intraperitoneal injection of 800 μg BrdU (5-bromodeoxyuridine; BD Biosciences) and cells were prepared with a BrdU flow kit (BD Pharmingen).

Antigen-presentation assays. After being primed in vivo with PCC or adju-vant only, plasma cells were sorted as PI−Dump−IgD−CD138+IgM− from lymph nodes 10 d after immunization and were cultured for 4 d at a den-sity of 5 × 103 cells per well with 5 × 104 naive PCC-specific helper T cells (PI−Dump−CD4+CD44−CD62L+) sorted from 5C.C7 αβTCR–transgenic mice and labeled for 5 min at 24 °C with 5 μM CFSE (carboxyfluorescein diacetate succinimidyl ester; Molecular Probes). For in vivo antigen presentation, 9.0 × 105 isotype-switched plasma cells were sorted from lymph nodes 5 d after immunization with adjuvant only, then were pulsed for 1 h at 24 °C with 10 μM HEL peptide and transferred subcutaneously into B10.BR mice that had previously received the equivalent of 5 × 106 CFSE-labeled helper T cells from donor 3A9 αβTCR–transgenic spleens. T cell proliferation was analyzed 3 d later. For assay of in vitro antigen presentation to naive helper T cells,

5 × 104 CFSE-labeled naive PCC-specific helper T cells were cultured with 5 × 103 plasma cells or DCs (PI−Dump−CD11c+), sorted from lymph nodes 5 d after immunization with adjuvant only, and with 10 μM MCC peptide (amino acids 88–103; Anaspec) or 10 μM PCC protein.

For analysis of antigen presentation to activated T cells, PCC-specific naive T cells were activated either in vitro for 4 d with DCs in the presence of PCC or in vivo for 7 d after transfer of 5 × 105 5C.C7 αβTCR–transgenic helper T cells into B10.BR mice simultaneously immunized with PCC-adju-vant or sorted antigen-activated Vα11+Vβ3+ helper T cells from B10.BR mice without transfer as described8. Subsequently, activated helper T cells (PI−Dump−CD4+CD44hiCD62Llo) were sorted and cultured for 3 d or 24 h at a ratio of 10:1 with plasma cells or DCs with or without PCC. All cultures were done in RPMI medium supplemented with 10% (vol/vol) FCS, 1× penicillin–streptomycin–l-glutamine and 50 μM 2-mercaptoethanol (Invitrogen). For adoptive transfer into Blimp-1-cKO mice and littermate control mice, 1.0 × 108 unfractionated spleen and lymph node mixtures from C57BL/6 mice on day 4 after secondary immunization with NP-KLH in Ribi were distributed evenly and injected intraperitoneally and into the base of the tail of recipient mice at day 9 after initial priming of both groups of mice. For assay of antigen presentation to antigen-specific TFH cells in vivo, 3.0 × 105 isotype-switched plasma cells were sorted from lymph nodes 5 d after immunization with adjuvant only, then were pulsed for 1 h at 25 °C with 10 μM MCC peptide and were transferred subcutaneously into B10.BR mice that had been immunized 5 d earlier with PCC in adjuvant. TFH cells were sorted 2 d later for gene-expression analysis.

Enzyme-linked immunospot assay. Plasma cell function was measured as described14 with NP-BSA or IgM and IgG antibody to mouse (Southern Biotechnology Associates).

Estimation of mRNA by quantitative RT-PCR. For quantitative PCR ex vivo immediately after isolation, 1 × 106 helper T cells (PI−Dump−CD4+), DCs (PI−Dump−CD11c+), naive B cells (PI−Dump−B220+IgD+IgM+) or isotype-switched plasma cells (PI−Dump−IgD−CD138+IgM−) were sorted either from lymph nodes of HEL-adjuvant–immunized B10.BR mice 1 d after immuniza-tion (T cells and DCs) or from spleens of HEL-adjuvant–immunized B10.BR mice 5 d after secondary immunization (B cells and PCs). For gene-expression analysis after antigen-presentation assay, activated helper T cells (PI−Dump− CD4+CD44hiCFSElo) were sorted from individual wells after culture. For gene-expression analysis after transfer of plasma cells in vivo, antigen-specific TFH cells (PI−Dump−CD4+Vα11+Vβ3+CD44hiCD62LloCXCR5+) were sorted from B10.BR mice 7 d after immunization with PCC. All samples were sorted for RNA extraction, cDNA synthesis and quantitative PCR as described8. The mRNA purified after in vitro assay of antigen presentation to naive helper T cells (Fig. 4b,c) was amplified by two rounds of in vitro transcription as described8 (primers, Supplementary Table 1). Results were analyzed with Rotor-Gene 6.0 software or StepOne software version 2.0.

Confocal microscopy. Inguinal lymph nodes were embedded in Tissue-Tek optimum cutting temperature compound (Sakura) and were snap frozen on dry ice. Sections 6–8 μm in thickness were fixed in cold acetone and were stained in PBS containing 10% (vol/vol) FCS and 0.1% (vol/vol) sodium azide with the following labeled antibodies: anti-IgD (11.26) and anti-Vβ3 (KJ25; both produced and labeled in the laboratory); anti-CD90.2 (53-2.1) and anti-CD138 (281.2; both from BD Biosciences); IgG antibody to rat (Vector); and rhodamin red–streptavidin (Invitrogen). Confocal images were analyzed with an Olympus Fluoview 500 confocal microscope.

Statistical analysis. Mean values, s.e.m. values, Student’s t-test (unpaired) and Mann-Whitney test were calculated with GraphPad Prism (GraphPad Software).

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Addendum: The function of follicular helper T cells is regulated by the strength of T cell antigen receptor bindingNicolas Fazilleau, Louise J McHeyzer-Williams, Hugh Rosen & Michael G McHeyzer-WilliamsNat. Immunol. 10, 375–384 (2009); published online 1 March 2009; corrected online 8 March 2009; addendum published after print 8 March 2011

Plasma cells negatively regulate the follicular helper T cell programNadége Pelletier, Louise J McHeyzer-Williams, Kurt A Wong, Eduard Urich, Nicolas Fazilleau & Michael G McHeyzer-WilliamsNat. Immunol. 11, 1110–1118 (2010); published online 31 October 2010; addendum published after print 8 March 2011

It has been called to our attention that in a series of studies on the development of follicular helper T cells (TFH cells), the primers we used to amplify Bcl6 were specific for Bcl6b, not Bcl6. We have now traced back the results and have redone experiments that demonstrate that both Bcl6 and Bcl6b are modified in the same way and to a similar extent in antigen-specific and non–antigen-specific TFH cells induced both in vivo and in vitro.

In the original studies we inadvertently mixed up primers specific for Bcl6b (but not for Bcl6) with those specific for Bcl6. After closer inspection, we found that this error was made when the wrong set of primers was ordered and labeled “Bcl6 #2” in our laboratory but the primers were in fact specific for Bcl6b. We used both sets of primers in the original studies of gene expression on antigen-specific TFH cells analyzed immediately after isolation (Fig. 3f in Nat. Immunol. 10, 375–384 (2009), and Addendum Fig. 1). Hence, the expression of both Bcl6 and Bcl6b seemed to be three- to fourfold higher in pigeon cytochrome c (PCC)-specific TFH cells than in naive CD4+ helper T cells.

As these genes are structurally related, we were concerned that the primers for Bcl6b may have sufficiently cross-reacted with Bcl6 and perhaps amplified the latter gene in T cells. Thus, we sorted antigen-specific TFH cells immediately after isolation and amplified random hexamer–primed cDNA with both sets of primers (Addendum Fig. 2). Both gene products were amplified and produced PCR products of different sizes (the predicted sizes for the different gene products). We then sequenced these products to demonstrate they belonged to the different genes as presented. Thus, both primers amplified the expected PCR products in both naive helper T cells and antigen-specific TFH cells.

To explore this issue further, we sorted non–antigen-specific TFH cells on the basis of cell surface phenotype and amplified DNA from these cells by RT-PCR without further treatment (Addendum Fig. 3). These experiments also showed that both Bcl6 and Bcl6b had similar basal expression in naive helper T cells and a similar change of expression in CXCR5+ TFH cells.

In our more recent study on antigen presentation by plasma cells (Nat. Immunol. 11, 1110–1118 (2010)), we reported that plasma cells negatively regulate the TFH program in vitro and in vivo. Our conclusions were based on expression of the genes encoding interleukin 21 (IL-21) and Bcl-6, as measured by reverse transcription and quantitative PCR. The original primer mix-up continued in the early phase of these studies. However, the error was sporadic and these more recent studies reported on Bcl6 and not Bcl6b, as outlined below (Addendum Table 1).

Hence, we concluded that antigen presentation by plasma cells inhibits the expression of mRNA for IL-21 and Bcl-6 by in vivo–derived, endogenous, antigen-specific TFH cells (Fig. 5d, second panel, in Nat. Immunol. 11, 1110–1118 (2010)). Furthermore, the adoptive transfer of antigen-pulsed plasma cells significantly inhibited the expression of mRNA for both IL-21 and Bcl-6 on antigen-specific TFH cells (Fig. 7i, in Nat. Immunol. 11, 1110–1118 (2010)) without affecting the expression of mRNA for IL-4 or the transcription factor GATA-3 in vivo. Therefore, this provided evidence of antigen-specific negative regulation of the TFH program by plasma cells in vivo.

We have also repeated the in vitro experiments using dendritic cells (DCs) or plasma cells as antigen-presenting cells to evaluate the expression of Bcl6 and Bcl6b after in vitro induction of TFH cell activity (Addendum Fig. 4). These studies demonstrated equivalent induction of both Bcl6 and Bcl6b in vitro with DCs as antigen-presenting cells and naive helper T cells with transgenic expression of the 5C.C7 ab T cell antigen receptor (abTCR) as antigen-specific helper T cells in these cultures. Consistent with the overall conclusions of our earlier study (Nat. Immunol. 11, 1110−1118 (2010)) plasma cells with antigen did not induce Bcl6 or Bcl6b in 5C.C7 abTCR–transgenic helper T cells in vitro.

We did these new experiments without secondary in vitro transcription or specific synthesis of target cDNA and preamplification by PCR to evalu-ate the extent of signal to be expected for Bcl6 after in vitro activation of TFH cells. The tenfold induction of both Bcl6 and Bcl6b in these studies is more in line with the expected results and the results of other studies using standard culturing conditions to induce TFH cell activity. These data are also more in line with the expression of Bcl6 and Bcl6b on antigen-specific TFH cells derived in vivo we have reported in all our studies. We believe that the starting cell population, in particular the primary culture stimulus, atypical secondary culture conditions and secondary amplification of the assay all contributed to the more enhanced signals we reported before for Bcl6b (Fig. 5b,c in Nat. Immunol. 11, 1110−1118 (2010)).

In conclusion, we mixed up primers for Bcl6 amplification and in parts of our published work inadvertently reported on Bcl6b, not Bcl6. Bcl6b is a distinct gene but remains a structurally and functionally related member of the Bcl6 gene family. We have now been able to revisit this issue and have demonstrated that both Bcl6 and Bcl6b seemed to be coexpressed to a similar extent in naive helper T cells. Both gene products were similarly enhanced in TFH cells that emerged in vivo or were derived in vitro. Furthermore, both genes seemed to be inhibited similarly by antigen presenta-tion by plasma cells. Hence, we believe that this addendum will rectify the mistake made in the initial studies and clarify the coexpression of Bcl6 and Bcl6b in antigen-specific TFH cells.

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Addendum Table 1 Induction and inhibition of Bcl6 or Bcl6b in TFH cellsFigure amplification method Tm Gene amplified

4c IVT 86.88–87.32 Bcl6b

5b none 87.02–87.31 Bcl6b

5c STa 87.03–87.47 Bcl6b

5d STa 82.75–83.06 Bcl6

7i STa 82.99–83.56 Bcl6

Induction of Bcl6 or Bcl6b in TFH cells selectively by dC antigen presentation and inhibi-tion of Bcl6 by PC antigen presentation, assessed by two rounds of in vitro amplification (IVT) of Rna with a Messengeramp II aRna amplification kit (ambion), similar to what is done before microarray analysis, followed by the generation of cdna from 10 mg amplified Rna as the starting material for SYBR Green–based quantitative PCR analysis; by specific target amplification (STa) with specific primers to generate cdna, followed by 14 cycles of PCR preamplification and dilution of the product 1:5 for use as the starting material for SYBR Green–based quantitative PCR analysis; or the melting curve (Tm) peak signal after SYBR Green–based quantitative PCR analysis.

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Addendum Figure 2 Primers for Bcl6 and Bcl6b amplify the expected PCR products from naive helper T cells and TFH cells. PCR analysis of Bcl6 and Bcl6b in naive helper T cells or antigen-specific TFH cells (5 × 103) sorted (as described in Addendum Fig. 1) on day 8 of the memory response to PCC; cdna synthesized from Rna by random-hexamer priming was analyzed by 40 cycles of PCR with both sets of primers (Addendum Fig. 1), then primers were removed and PCR products were analyzed directly with a Bigdye Terminator Cycle Sequencing kit on a 3130 Genetic analyzer.

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Addendum Figure 3 equivalent upregulation of Bcl6 and Bcl6b in CXCR5+ TFH cells assessed immediately after isolation. Reverse transcription and quantitative PCR analysis of Bcl6 and Bcl6b in naive helper T cells (PI–Cd8–Cd11b–Cd4+Cd44loCd62LhiCXCR5–) or TFH cells (PI–Cd8–Cd11b–

Cd4+Cd44hiCd62LloCXCR5+) from the draining lymph nodes of mice immunized with Ribi adjuvant only and sorted, results are presented in arbitrary units relative to the expression of b-actin mRna.

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Addendum Figure 4 Selective upregulation of Bcl6 and Bcl6b in antigen-specific TFH cells with the use of dCs but not PCs for antigen presentation in vitro. Quantitative PCR analysis of Bcl6 and Bcl6b in cells derived from Cd11c+ dCs or IgM–Cd138+ plasma cells (PC) sorted ex vivo with protein antigen and cultured for 4 d in vitro together with cytosolic dye CFSe–labeled naive Cd4+ PCC-specific 5C.C7 abTCR–transgenic helper T cells, followed by sorting of Cd44hi 5C.C7 abTCR–transgenic helper T cells that diluted CFSe, for Rna extraction, cdna synthesis and SYBR Green–based quantitative PCR amplification of Bcl6 and Bcl6b (details as for Fig. 4 in the original study (Nat. Immunol. 11, 1110−1118 (2010))); results are presented in arbitrary units relative to b2-microglobulin mRna expression.

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Addendum Figure 1 Upregulation of both Bcl6 and Bcl6b in antigen-specific TFH cells analyzed immediately after isolation. Quantitative PCR analysis of Bcl6 mRna and Bcl6b mRna in naive helper T cells (Va11+Vb3+Cd44loCd62Lhi) or PCC-specific TFH cells (PI–B220– Cd11b–

Cd8–Va11+Vb3+Cd44hiCd62LloCXCR5hi) sorted from draining lymph nodes on day 7 after subcutaneous priming with PCC in Ribi adjuvant (2 × 103 cells), followed by cdna synthesis and amplification with Platinum SYBR Green (details as for Figs. 1–3 in the original study (Nat. Immunol. 10, 375−384 (2009))); results are presented in arbitrary units (aU) relative to the expression of b2-microglobulin mRna, set as 1. Other antigen-specific helper T cell subsets are sorted on the basis of differences in the expression of Cd62L and CXCR5 as described in detail in the original study (Nat. Immunol. 10, 375−384 (2009)).

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