Idiotype-specific CD4^+ T cells chronically stimulate autoreactive B cells to develop into B lymphomas in mice

Domain: chemistry
Published: 2026-02-25
Editorial outcome: major_revision

Abstract
Autoimmunity increases the risk of developing B lymphoma in humans; however, the molecular mechanism(s) underlying this link remain(s) unexplained. Here, we develop a mouse model to dissect the contribution of Id-driven T-B collaboration and show that chronic interaction between B cells and CD4+ T cells first leads to autoimmunity and later to the development of B (and T) cell lymphomas. We find that serum autoantibodies and lymphoma B cell receptor (BCR) are related and have the same specificity for ubiquitous self-antigens (histone and nucleosome) (Signal 1). Self-reactive B lymphoma cells are helped by CD4+ T cells that recognize a lymphoma neoantigen, an MHC class II-presented Idiotypic (Id) peptide (Signal 2). The mechanism, called Id-driven T-B collaboration, results in relentless mutual stimulation of B and T cells with germinal center markers, autoimmunity, and finally, malignant transformation of either B or T cells. Our results thus indicate Id-driven T-B collaboration as a potential mechanism linking autoimmunity and the development of lymphomas.

Introduction
Several different mechanisms have been described that contribute to the development of autoimmune diseases1,2 and B cell lymphomas3,4. However, in most cases, the etiologies of these heterogeneous diseases are unknown. Autoimmune diseases and B lymphomas are linked disorders in humans5-7. However, the mechanism(s) underling the joint pathogenesis of these diseases have not been fully elucidated.

Due to somatic hypermutation in germinal centers8, B cells often express mutated sequences [neoantigens, traditionally called idiotypic (Id) peptides] in their B cell receptor (BCR) variable (V)-regions. Moreover, B cells have been shown to antigen-process their own BCR, resulting in Id-peptides that are presented on their MHC class II molecules to Id-specific CD4+ T cells9. This phenomenon is the basis for the concept of Id-driven T-B collaboration10 which is distinct from conventional T-B collaboration11,12. We here hypothesize that Id-driven T-B collaboration may cause the joint development of autoimmunity and B cell lymphoma (Fig. 1A). A growing body of evidence suggests that Id-driven T-B collaboration may cause the development of autoimmunity and B cell lymphoma. Studies in paired Ig and TCR-transgenic mice have demonstrated that chronic Id-driven T-B collaboration can cause the development of autoimmune disease with SLE-like features12-14. Moreover, injection of Id-specific Th2 cells into mice that are transgenic for an Id+ L chain resulted in the development of marginal zone B lymphomas15.

These previous findings in gene-modified mouse models are supported by observations in humans. Thus, Id-specific CD4+ T cells have been observed in patients with SLE16, rheumatoid arthritis17, and multiple sclerosis18. Moreover, Id-specific CD4+ T cells stimulated B cell Chronic Lymphocytic Leukemia (B-CLL) in vitro19. Finally, MHCII-presented Id-peptide neoantigens were frequently found in mantle cell lymphoma20, diffuse large B cell lymphoma (DLBCL), follicular B cell lymphoma (FBL) and B-CLL21.

Evidently, Id-driven T-B collaboration must be tightly controlled by a number of mechanisms to prevent rampant lymphocyte activation and immunological havoc in normal individuals. First, T cells are tolerant to germline-encoded V region sequences and only respond to Id-peptide neoepitopes expressing somatic mutations22-24. Second, resting normal B cells display only low levels of pId:MHCII complexes on their cell surface25,26. Third, Id-driven T-B collaboration is likely to be suppressed by mechanisms that remain to be elucidated.

In previous studies on Id-driven T-B collaboration, we used 2315 Ig L chain transgenic mice in which most B cells present a mutated Id315 peptide on their MHC class II molecules13,15. To obtain a more physiological model, we recently developed a V gene segment-modified mouse (V2315m) where a V2315m-J2 rearrangement in infrequent B cells generates a complete Id-sequence spanning the V-J junction26. Importantly, ligation of the BCR by antigen resulted in increased display of pId:MHCII on the B cell surface and hence elicitation of Id-driven T-B collaboration26.

Here, we hypothesize that chronic Id-driven T-B collaboration may explain the known link between autoimmunity and B lymphomas (Fig. 1A). We show that BCR ligation by self-antigen (Signal 1), and chronic help from Id-specific CD4+ T cells (Signal 2), result in autoimmunity and later development of B cell lymphomas. These findings may have implications for the treatment of B lymphomas in humans.

Results
Incomplete T cell tolerance in mice that co-expressed the Id-modified V gene segment and the Id-specific TCR

In the V2315m mouse, germline codons in the V2 gene segment were replaced by the corresponding mutated codons found in the MOPCL315 (IgA2) multiple myeloma cell line, resulting in a Tyr94Ser95Thr96→ Phe94Arg95Asn96 exchange. In heterozygous V2315m+/- mice, about 0.5% of B cells rearrange the V2315m to J2; these B cells express a 2 CDR3 Id-peptide (residues 91-101) that spans the V2315mJ2 junction. This Id-peptide neoantigen, when bound to the MHC class II molecule I-Ed of BALB/c mice, stimulates Id-specific CD4+ T cells26. The Vλ2315m mice are close to physiological since their B cell development appears normal and since the Id-sequence is expressed at physiological levels in a small population of B cells26.

Heterozygous Vλ2315m mice were crossed with heterozygous Id-specific TCR transgenic (Tg46) mice27, yielding four types of offspring on a BALB/c background (Fig. 1B). According to the hypothesis (Fig. 1A), the Vλ2315m+/-TCR-TG+/- offspring could develop autoimmunity and perhaps even B cell lymphomas. The other three types of offspring, singly V gene-modified, singly TCR-transgenic and non-transgenic, served as experimental controls.

We first tested if T cell tolerance developed in Vλ2315m+/- TCR-TG+/- offspring. In 4-week-old mice, frequencies of double positive (DP) and single positive (SP) thymocyte subsets (gated as shown in Suppl. Fig. 1A) were unaffected (Suppl. Fig. 2A). Nevertheless, Vλ2315m+/- TCR-TG+/- offspring had a reduction in the frequency of Id-specific T cells among CD4+ SP thymocytes compared to TCR-TG mice (Fig. 1C, Suppl. Fig. 2B). Similarly, Vλ2315m+/- TCR-TG+/- offspring had a reduction of Id-specific cells among CD4+ T cells in the spleen (Fig. 1D, Suppl. Fig. 1B, 2C). The splenic Id-specific CD4+ T cells that remained had an increased frequency of FoxP3+ cells, suggestive of increased differentiation towards a regulatory phenotype (Fig. 1E, Suppl. Fig. 2D). Despite these signs of partial tolerance, the proliferative responses of Id-specific CD4+ T cells isolated from Vλ2315m+/- TCR-TG+/- mice to synthetic Id-peptide in vitro were undiminished (Fig. 1F). These results indicate that Id-specific CD4+ T cells became partly but not completely tolerized in the presence of about 0.5% of Id+ B cells. B cell subsets in bone marrow (BM) and spleen were similar in Vλ2315m+/- TCR-TG+/- and Vλ2315m+/- (Suppl. Fig. 1C, D and Suppl. Fig. 2E, F).

Development of autoimmunity in mice that co-expressed the Id-modified V gene segment and the Id-specific TCR

Vλ2315m+/-TCR-TG+/- offspring had a slightly reduced gain of body weight compared to littermates. Growth retardation was slightly more prominent in females than males (Fig. 1G, Suppl. Fig. 2G). Some mice developed inflamed eyelids and moderate hair loss but appeared otherwise healthy.

With age, ~ 70% Vλ2315m+/-TCR-TG+/- offspring developed autoantibodies detected by staining of HEp-2 cells. Autoantibodies were predominantly antinuclear antibodies (ANA) that gave a homogeneous staining pattern (Fig. 1H). Autoantibody titers were low on day 100 but increased by day 300 (Fig. 1I, Suppl. Fig. 2H). IgG2a autoantibodies were more prominent than IgG1 (Fig. 1I). Surprisingly, the autoantibodies expressed  but not  L chains (Fig. 1J). Autoantibody levels were higher in females than in males (Fig. 1K). Autoantibodies bound nucleosomes (Fig. 1L, M) and histones (Fig. 1N, O); in either case, IgG2a dominated. Levels of anti-histone and anti-nucleosome IgG antibodies correlated (Fig. 1P). Only 2/15 mice had anti-dsDNA antibodies of the IgG2a isotype in ELISA; these two sera were also positive in a Crithidia Luciliae assay (CLIFT assay) (Suppl. Fig. 2I, J).

Id-specific T cells and Id+ B cells proliferated in autoimmune Vλ2315m+/-TCR-TG+/- mice.

At 11 months of age, Vλ2315m+/-TCR-TG+/- mice without overt lymphoma had a population of peripheral blood B cells whose BCRs had a strikingly reduced  expression. These low B cells expressed increased amounts of MHCII, pId315:I-Ed [detected by a scFv TCRmimetic (TCRm)26] and CD86 (Suppl. Fig. 2 K-P), indicating an expansion of B cells that expressed 315m. Such lowTCRm+CD86Hi B cells were neither observed in blood of age-matched controls nor in young (2months-old) Vλ2315m+/-TCR-TG+/- mice (Suppl. Fig. 2Q).

At 12 months of age, splenic Id-specific CD4+ T cells had expanded in autoimmune Vλ2315m+/-TCR-TG+/- mice and attained the same frequency as in TCR-TG+/- mice (Suppl. Fig. 3A). To more stringently test for proliferation of Id-specific CD4+ T cells, 12-month-old mice without overt lymphoma received BrdU for 7 days before analysis of spleens. CD4+ T cells incorporated more BrdU (proliferated) in Vλ2315m+/-TCR-TG+/- mice compared to TCR-TG+/- mice (Fig. 2A). Moreover, Id-specific CD4+ T cells stained by a clonotype-specific mAb proliferated more extensively than did non-specific CD4+ T cells (which expressed endogenous TCR chains28) (Fig. 2A, Suppl. Fig. 3B). Similar findings were done for T follicular helper (Tfh) cells29 (Fig. 2B, Suppl. Fig. 3C, E) as well as peripheral helper T (Tph) cells30,31 (Suppl. Fig. 3D, trending but not significant, Suppl. Fig. 3F).

In the same experiment, B cells expressing pId315:MHCII+ proliferated to a larger extent in autoimmune Vλ2315m+/-TCR-TG+/- mice compared to Vλ2315m+/- mice (Fig. 2C, Suppl. Fig. 3G). The proliferating cells expressed BCRs with  but not  L chains (Fig . 2C), and the GC-marker CD95 (Suppl. Fig. 3H, trending but not significant). Consistent with these results, both the pId315:MHCII++ and the CD95++ B cell subsets were expanded in autoimmune mice (Suppl. Fig. 3I, J). These results suggest a mutual stimulation between Id-specific T cells and pId315:MHCII+ B cells in GCs in the spleens of autoimmune mice, consistent with ongoing Id-driven T-B collaboration.

Autoimmune mice have increased serum levels of cytokines associated with Tfh and DLBCL

We tested sera of 10-month-old mice for 23 chemokines and cytokines in a Multiplex assay. Autoimmune Vλ2315m+/-TCR-TG+/- mice expressed increased levels of IL-21 and IFN compared to controls while results with IL-10 and TNF were less pronounced (Fig. 2-D-G). However, the low levels of IL-10 and TNF were strikingly increased in 14-20-month-old mice that had developed B lymphomas (see below) while levels of IL-21 and IFN- were stationary (Fig. 2D-G). IL-21 has previously been associated with Tfh cells32 while IL-10 has been associated with DLBCL in humans33.

Autoimmune mice later developed lymphomas

Between 12 and 22 months of age, most Vλ2315m+/-TCR-TG+/- offspring were euthanized due to distended abdomens caused by lymphomas, while control mice were healthy (Fig. 2H). It did not influence disease development whether the mother of the offspring was Vλ2315m+/- or TCR-TG+/- (Suppl. Fig. 3K). Lymphomas developed more frequently in mice with high titers of IgG2a autoantibodies (Fig. 2I) and in females (Fig. 2J).

The lymphomas were investigated as outlined in Fig. 2K. The primary lymphomas predominantly developed in spleens and lymph nodes (Fig. 2L). Upon transfer to Rag1-/- mice, recipients developed secondary lymphomas. Lymphomas were classified according to the Bethesda proposals for classification of lymphoid neoplasms in mice34. About 60% of the lymphomas were of B cell origin. Of these, follicular B lymphoma (FBL), centroblastic diffuse large B cell lymphoma (CB-DLBCL) and histiocyte-associated (HA) DLBCL (HA-DLBCL) were of approximately equal frequency while there were fewer immunoblastic (IB)-DLBCL (Fig. 2M). Images of representative HES-stained sections are shown in Fig. 2N. 4/4 B lymphomas (2 FBL and 2 DLBCL) were BCL6+, suggestive of a GC-origin35 (Fig. 2O).

About 40% of the lymphomas were T cell lymphoblastic lymphomas (T-LBL) (Fig. 2M). Three types could be distinguished based on CD4, CD8 and TCR expression. In order of frequency, the T lymphomas were: CD4-8- Id-specific TCR+ > CD4+8- Id-specific TCR+ > CD4-8-Id-specific TCR- (Suppl. Fig. 3L). The lymphomas expressed the transgenic TCR , PD-1 and CD25 at levels similar to CD4+ T cells from TCR-TG mice while their CD44 expression was reduced (Suppl. Fig. 3M-P). 2/2 T-LBL (both CD4+Id-specific TCR+) were Bcl6+ suggestive of a Tfh origin36(Fig. 2O). Examples of immunostaining for CD3 (T cell marker), PAX5 (B cell marker) and IBA-1 (macrophage marker) are shown in Suppl. Fig. 3Q.

Characterization of primary B lymphomas

Primary splenic B lymphomas had increased expression of CD80, CD86, MHC class II, germinal center markers (CD95+GL-7+) and Ki-67 compared to B cells of control mice (Fig. 3A-E, Suppl. Fig. 4A-E). Secondary B lymphomas in Rag 1-/- recipients expressed increased levels of IgG2a BCR and had downregulated IgD and IgM (Fig. 3F). Surprisingly, primary B lymphoma mice had a reduced frequency of cell surface +CD19+ cells (Fig. 3G), even so, pId315:I-Ed expression was increased (Fig. 3H, I). An explanation could be that 2315m chains were only present intracellularly in B lymphoma cells but were nevertheless available for MHC class II presentation. Indeed, Western blots revealed lymphoma cell expression of 2/3 L chains in cell lysates (Fig. 3J). Further, a PCR employing V2315m-specific primers demonstrated that B lymphomas expressed V2315m mRNA (Fig. 3K), a result which was confirmed by sequencing in 5/5 cases. Lymphomas expressed lower than normal amounts of  on their cell surface (Fig. 3L, M), perhaps due to autoreactivity of the IgG2a BCR (see later).

Dual L chain expression in a B lymphoma cell line: cell surface  and intracellular 

A secondary B lymphoma was adapted to growth in vitro. The cell line, NT24, expressed  on the cell surface while  was expressed predominantly intracellularly as determined by flow cytometry (Fig. 3N). Nevertheless, about 30% of NT24 cells expressed pId315:I-Ed on the cell surface (Fig. 3O). Moreover, the intracellular  was identified as 2315m by proteomic analysis of cell lysates precipitated by a V-specific mAb (Fig. 3P). Confocal microscopy confirmed that  was expressed on the surface while  was expressed intracellularly (Fig. 3Q). Intracellular  co-localized with an ER marker (Fig. 3R).

B lymphomas were infiltrated by Id-specific CD4+ T cells that were activated and proliferated

Splenic B lymphomas were infiltrated by CD3+ T cells (Fig. 4A) and intimate contacts (synapses) between B lymphoma cells and Id-specific T cells were observed in the spleen (Fig. 4B). One third of infiltrating CD4+ T cells expressed the Id-specific TCR (Fig. 4C) were activated (CD69+) (Fig. 4D, Suppl. Fig. 4I), proliferated (Ki67+) (Fig. 4E, Suppl. Fig. 4J) and displayed increased levels of the memory marker CD44 (Fig. 4F, Suppl. Fig. 4K) and PD-1 (Fig. 4G, Suppl. Fig. 4L).

Bidirectional activation of lymphoma B cells and Id-specific CD4+ T cells

Ex vivo B lymphoma cells proliferated in response to Id-specific T cells (Fig. 4H). Conversely, B lymphomas induced proliferation of Id-specific T cells (Fig. 4I). When B lymphoma cells and infiltrating T cells were isolated from a single tumor and mixed in vitro, B lymphoma cells enhanced the proliferation of Id-specific T cells demonstrating that TILs were not fully anergic (Fig. 4J). These results were extended to a cloned B lymphoma cell line, NT34.E2 (Suppl. Fig. 4F-H), which similarly stimulated the proliferation of Id-specific CD4+ T cells in vitro (Fig. 4K).

B lymphoma growth was retarded in Id-specific TCR-transgenic mice

NT34 cells were labeled with luciferase and injected into Id-specific TCR-transgenic Rag1-/- mice and Rag1-/- controls. In vivo imaging showed that B lymphoma growth was retarded in mice with Id-specific CD4+ T cells (Fig. 4L and Suppl. Fig. 4M). Consistent with this, survival was prolonged in challenged TCR-transgenic mice although mice eventually succumbed to disease (Fig. 4M).

Characterization of lymphoma BCRs

Four independent IgG2a,  B lymphomas (NT7, NT8, NT18, NT24) were passaged in Rag1-/- mice. mRNA isolated from secondary lymphomas were used to amplify VDJ and VJ using specific primers. Each individual lymphoma expressed several related VDJ nucleotide sequences, indicating that they had undergone clonal evolution in vivo (Suppl. Fig. 5A-D).

For each lymphoma, a single VDJ nucleotide sequence was dominant. Since the lymphomas had a BALB/c background, the dominant sequences were compared with recently reported BALB/c germline V-D-J gene segments37. The analysis shows that all four lymphomas expressed somatic mutations in their dominant VDJ (Fig. 5A).

Similarly, for each lymphoma, the most frequent VJ nucleotide sequence was considered to represent the dominant lymphoma clone (Suppl. Fig. 5). Based on such a likely pairing of dominant VDJ and VJ , there was little sharing of Ig V-related gene segments between the four B lymphomas (Fig. 5B). [The closest IMGT names (based on C57BL/6) for the dominant VDJ andVJ are given in Suppl. Fig. 6A].

While lymphoma VDJ had increased positive charge, increased CDR3 lengths, and increased levels of somatic mutations; these features were largely absent in VJ (Fig. 5C-E, Suppl. Fig. 6B-D).

Lymphoma BCR H chains preferentially associate with  chains compared to  chains

The dual L chain B lymphomas expressed a + BCR while levels of intracellular  were increased (Fig. 3L-R). This could suggest that lymphoma BCR H chains preferentially associated with  chains compared to  chains. To test this idea, we transfected HEK293 cells with three plasmids encoding respectively (i) an H chain composed of the dominant lymphoma VDJ fused to a human (h)3 constant region (ii) an L chain composed of the dominant lymphoma VJ fused to hC constant region and (iii) a  L chain composed of VJ2315 fused to a hC . Presumably, in triple transfectants, an H chain might pair with either lymphoma  or  L chains in the ER, prior to secretion. To reveal any preferential association, levels of respectively H and H Ig in supernatants were analyzed by ELISA. Compared to a control VDJH315-h3 H chain derived from the BALB/c MOPC315 myeloma (IgA, 2315), the B lymphoma H chains preferred to pair with their endogenous  compared to the 2315 L chain (Fig. 5F).

To further test any preferential association, we performed immunoprecipitations of lysates of cloned NT34.E2 B lymphoma cells and analyzed precipitates by Western blot under non reducing conditions. Anti- precipitated a 2a H chain while anti- did not. The  was expressed either as a free L chain or as part of complete IgG2a. In contrast, the  was expressed only as a free L chain. (Fig. 5G, H). The results of the competition and the co-immunoprecipitation experiments agree with the flow cytometry and confocal microscopy data (Fig. 3L-R) and suggest that in the lymphomas, the H chain prefers to bind  over 2315 and that this happens in the ER where complete Ig is assembled.

Recombinant Ig corresponding to lymphoma BCRs were autoreactive

To test for autoreactivity of lymphoma BCRs, we made soluble recombinant (r) Ig composed of dominant lymphoma V (fused to h3) and V (fused to hC) for each of the four lymphomas. For three of them, NT8, NT18 and NT24, rIg stained HEp-2 cells with a nuclear staining pattern, while no staining was obtained with the NT7 rIg (Fig. 6A). The three ANAPOS rIgs bound histones and nucleosomes in ELISAs (Fig. 6B, C) and in line blot assays (Fig. 6D, E) but failed to bind dsDNA (Fig. 6F). Moreover, the ANAPOS rIg failed to bind a number of other autoantigens (RNP/Sm, Sm, SS-A, Ro52, SS-B, Scl70, PM-Scl100, Jo1, centromere B, PCNA, dsDNA, RIB, M2). The ANANEG ENT7 rIg had no reactivity in any of these assays. None of the rIg bound 2315 Ig (M315) (Suppl. Fig. 6E).

It was important to ascertain that the rIg corresponded to the lymphoma BCR. An initial experiment demonstrated that the NT34.E2 lymphoma cell line secreted (or shed) the lymphoma BCR in vitro at levels similar to the much-studied A20 B lymphoma cell line (Suppl. Fig. 7A). Based on this result, we injected NT18 (IgG2a) lymphoma cells into Ig-deficient Rag 1-/- mice. The recipients developed lymphomas, and sera contained IgG2a with ANA activity and reactivity to nucleosomes and histones (Fig. 6G), matching the specificity of rIg derived from NT18 (Fig. 6A). Moreover, proteomic analysis demonstrated that serum from a NT18 Rag 1-/- recipient shared V -derived fragments with NT18 rIg (Suppl. Fig. 7B, C).

Correlation between (i) specificity of lymphoma BCR expressed as rIg and (ii) specificity of autoantibodies present during the early autoimmune phase

Before overt lymphoma development, during the preceding autoimmune phase, sera of most mice had ANA activity and autoantibodies against histones and nucleosomes (Fig. 1H-P). We therefore tested day 300 sera from the mice that later gave rise to NT7, NT8, NT18 and NT24 lymphomas for the presence of autoantibodies. Autoimmune-phase sera from NT8, NT18 and NT24 mice had positive ANA as well as anti-histone and anti-nucleosome antibodies (Fig. 6A, H, I). Serum from the NT7 mouse contained only low levels of anti-nucleosome and anti-histone antibodies and gave no signal in the HEp-2 assay. These results suggest that B cells present during the autoimmune phase could later give rise to B lymphomas.

Mutated B lymphoma V sequences were detected in autoimmune phase serum Ig prior to development of overt lymphoma

To obtain more solid evidence that B lymphomas could be derived from B cells during the autoimmune phase, we performed proteomic analysis of lymphoma rIgs and autoimmune-phase serum Igs. Trypsin digestion of the rIgs resulted in fragments that could be readily detected by proteomic analysis. We chose to focus on identifiable V fragments that expressed somatic mutations since such fragments should be exceedingly rare among the vastly diversified serum Ig molecules. Such unique V fragments are indicated in NT7, 8 and 18 VDJ amino acid sequences (Fig. 5A); NT24 lacked such unique fragments altogether. The selected fragments and their sequences are given in Fig. 6J.

We first demonstrated that the selected fragments were unique for the rIgs from which they were derived, with no cross-reaction to the other rIgs, (Fig. 6K). Next, we tested if fragments unique to a rIg could be detected in corresponding sera (purified IgG) from the preceding autoimmune phase. This was generally the case with one exception: fragment number 4 of rIg NT18 was found in NT7 serum (Fig. 6L). One explanation could be that NT7 serum contained an expanded clone of Ig that happened to express the fragment 4 sequence. Taken together, these results indicate that lymphoma progenitor B cells are present already during the autoimmune phase before overt lymphomas developed. It is unclear whether progenitor B cells secreted (or shed) sufficient amounts of antibodies (or BCR) for detection by proteomic analysis. Alternatively, some progenitor B cells could have differentiated into autoantibody-secreting plasma cells.

Direct support of the hypothesis: A rare B lymphoma expressed a 315m+ BCR and + autoantibodies in serum

Summarizing the data presented above, 23/24 B lymphomas expressed a +BCR while 315m was only expressed intracellularly. This finding supports the hypothesis of Fig. 1A, but not exactly, since the MHCII-presented Id-peptide in these dual L-chain-expressing B lymphoma cells was derived from an intracellular 2315m rather than from the BCR (Fig. 6M).

However, we have recently discovered, in 16-month-old V2315m+/- Id-specific TCR-TG+/- mouse, a B lymphoma that expressed a +- BCR (Fig. 7A). The primary B lymphoma (NT30) was classified by immunohistochemistry as a CB-DLBCL. It had a phenotype similar to that described above, with increased expression of MHCII, pId:MHCII, CD80/86, and GC (CD95, GL-7) and proliferation (Ki67+) markers (Fig. 7B). Primary NT30 was successfully passaged in Rag1-/- mice. The secondary NT30 was MHCIIHi, CD86Hi and displayed pId:MHCII (Suppl. Fig. 8A-C). It expressed an IgG2a BCR (Fig. 7C) and an intracellular  (Fig. 7D). A Western blot demonstrated that the  was a 2/3 which was associated with a 2a H chain. In contrast, the  was present only as a free chain (Fig. 7E, Suppl. Fig. 8D). Secondary NT30 B lymphoma cells induced proliferation of Id-specific CD4+ T cells in vitro (Fig. 7F). These results show that the NT30 lymphoma express a 2+BCR, almost certainly due to a V2315m→J2 rearrangement.

At the pre-lymphoma stage, the mouse in which NT30 later developed had + antinuclear antibodies (Fig. 7G) with specificity for nucleosomes (Fig. 7H-K) and histones (Suppl. Fig. 8E-H). Proteomic analysis of late lymphoma phase NT30 serum demonstrated that -containing Ig expressed J2 but not J1 (Suppl. Fig. 8I). Interestingly, in the autoimmune phase, the NT30 mouse had not only + but also + anti-nucleosome/histone autoantibodies. However, by the time of overt lymphoma,  autoantibodies dominated over  autoantibodies (Fig. 7J-K). These results suggest that malignant transformation could have haphazardly occurred in a rare + autoreactive B cell within a pool of more numerous + autoreactive B cells.

Taken together, the data suggests that NT30 arose in a → receptor-revised B cell38 but where the  chain replacement by λ2 failed to extinguish autoreactivity. Most likely, the NT30 B lymphoma has a H chain that pairs better with 2315m L chain than the co-expressed  chain. These observations directly support the hypothesis of Fig. 1A (Fig. 7L).

Transfer of Id-specific CD4+ T cells to V2315m+/- mice resulted in B cell lymphoma development

To remove any thymic influence on T cell development, and to test if a more limited number of naïve Id-specific CD4+ T cells could induce lymphomas, we transferred a limited amount of Id-specific CD4+ T cells (1x106) into 2-month-old V2315m+/- mice and followed mice for development of autoimmunity and lymphoma. Non-transferred V2315m+/- mice served as controls (Fig. 7M). 15 months after transfer, Id-specific CD4+ T cells were found in peripheral blood (Fig. 7N). A third of transferred mice (2/6) had at this time point antinuclear antibodies in sera, both of these mice later developed B cell lymphomas (Fig. 7O, Supplementary Table 1). Between 16-20 months after transfer, two thirds of the transferred mice (4/6) became sick and had to be euthanized while the 4 non-transferred V2315m+/- control mice remained healthy (Fig. 7P, Supplementary Table 1). Upon euthanasia, 4/6 of transferred mice exhibited macroscopic pathology in lymphoid organs and extra-lymphoid sites (Suppl. Fig. 9A), these four mice were diagnosed with B cell lymphomas by immunohistochemistry (Fig. 7Q, Supplementary Table 1). In the remaining 2 mice, a lymphoma diagnosis could not be made with certainty (Supplementary Table 1). In sites affected by lymphoma, mice had significant amounts of Id-specific CD4+ Tfh cells and B cells that displayed pId:MHCII and GC markers (Suppl. Fig. 9B, C, Supplementary Table 1).

Methods
Mice, typing, and breeding

The Vλ2 gene-modified Vλ2315m mice26 and the Id-specific TCR-transgenic (TG) mice28 were maintained in a heterozygous state on a BALB/c background. For experiments, Vλ2315m+/- and Id-specific TCR-TG+/- were crossed, thus obtaining four types of offspring: Vλ2315m+/- Id-specific TCR-TG+/-, Vλ2315m+/-, Id-specific TCR-TG+/- and non-transgenic mice. The Vλ2315m V gene-modification26 was typed by PCR while the TCR-TG mice were typed by flow cytometry using the TCR clonotype-specific GB113 mAb64. The breeding was set up as (Vλ2315m+/- ♀ x Id-specific TCR-TG+/- ♂) for the first two cohorts. For the third cohort (Id-specific TCR-TG+/- ♀ x Vλ2315m+/- ♂) were used. Rag1-/- (C.129S7(B6)-Rag1tm1Mom/J, The Jackson Laboratory, (stock #002216)) and Id-specific TCR-TG +/+Rag1-/- mice were maintained on a BALB/c background and were used in transfer and tumor challenge experiments. BALB/c mice were purchased from Janvier labs (strain identifier: BALB/cAnNRj). Animals were housed in specific pathogen-free (SPF) unit at 20–23 °C and 45–65% humidity with a 12-hour dark/light cycle. Both the experimental and control animals were co-housed throughout the study. The mouse experiments were approved by the Norwegian Food Safety Authority (ID 12387, 27752). Experimental procedures followed institutional guidelines of the Department of Comparative Medicine, Oslo University Hospital.

Tissue culture medium

Cells were cultured in RPMI 1640+ GlutaMAX (Gibco) supplemented with 10% heat-inactivated fetal calf serum (Biochrom and Sigma-Aldrich) to which were added 0.1 mM NEAA (Invitrogen), 1 mM sodiumpyruvate (Invitrogen), 0.5 mM 1-thioglycerol (Sigma-Aldrich) and 20 µg/ml Gensumycin (Sanofi-Aventis Norway AS).

Transfer of Id-specific CD4+ T cells into Vλ2315m+/- mice

Id-specific CD4+ T cells were isolated from young TCR-TG+/- by negative selection using CD4+ T cell Isolation Kit, mouse (Miltenyi Biotec, #130-104-454). Among the purified CD4+ T cells, ~50% were Id-specific T cells. Two-month-old Vλ2315m+/- mice were injected with 2x106 CD4+ T cells amounting to ~1x106 Id-specific T cell. Age-matched Vλ2315m+/- control mice did not receive any T cells.

Follow-up of mice, dissection and tissue processing

Weekly follow-up of mice consisted of weighing, physical examination including abdominal palpation, and assessment of fur condition and motility. The humane endpoint was defined based on several criteria such as mobility, posture, weight loss and increase in abdominal size. Upon reaching the endpoint, mice were euthanized by intraperitoneal administration of ZRF (0.1 ml/10g body weight; Zolazepam 3.29 mg, Tiletamine 3.29 mg, Xylazine 0.45 mg, Fentanyl 2.6 mg) followed by a secondary methods of euthanasia, such as exsanguination or cervical dislocation, dissected and evaluated for enlargement of the spleen, the thymus and the lymph nodes (LNs). Spleens were collected into GentleMACS C tubes (Miltenyi Biotec) with RPMI tissue culture medium supplemented with 1 mg/ml of collagenase type 4 (Worthington, #LS004188) and 0.3 mg/ml DNaseI (Sigma-Aldrich, #D5025). The spleens were dissociated with a GentleMACS Dissociator (Miltenyi Biotec) and incubated with collagenase/DNase for 10 minutes at 37°C. Single cell suspensions were washed in complete medium and incubated with Tris-buffered ammonium chloride (ACT) for 7 minutes on ice to lyse erythrocytes. Following ACT treatment, the cells were washed again in complete medium and viable cells were counted using a cell counter (Countess, Invitrogen).

LNs and thymi were collected in tubes containing tissue culture medium and pressed with a syringe piston through a cell strainer. The cells were washed in complete medium and viable cells were counted.

Tissue pieces were fixed in 5% neutral-buffered formalin or frozen in isopentane chilled on dry ice.

Histopathology and immunohistochemistry of lymphomas

Histopathological examination was done on formalin-fixed, paraffin-embedded sections (FFPE) stained with hematoxylin and eosin. Guidelines for evaluation were as published34. Frozen sections were used for co-staining of T and B cells using anti-CD3 mAb (clone CD3-12, Bio-Rad, #MCA1477T, 1:100) and anti-B220 mAb (clone RA3-6B2, BD Biosciences, #553090, 1:200). For staining of Id-specific TCR+ cells, frozen lymphomatous spleen sections were cut at 5 µm, air-dried, fixed in acetone, washed, and blocked with 30% normal rat serum and 30 µg/ml of FcRγ-blocking mAb (HB197, ATCC) in PBS/BSA (30 minutes at 25°C). Sections were then incubated with anti-B220-FITC mAb (RA3-6B2, TONBO Biosciences, #SKU 35-0452-U025, 1:200), anti-Bcl-6 pAb (MBS9215821, MyBioSource, #MBS9215821, 1:25) and GB113-PE (clonotype-specific for the Id-sp TCR64, Diatec Monoclonals, 1:100) for one hour at 25°C. Secondary detection reagents were rabbit AF488-coupled anti-fluorescein (#A-11090, Thermo Fisher Scientific, 1:200) and goat anti-PE-Texas Red (#ab34734, Abcam, 1:1000), applied for 45 minutes at 25°C. FFPE sections were used to stain Pax-5, IBA-1 and CD3 using the following antibodies: anti-Pax-5 pAb (#LS-C88806-01, LSBio, 1:100), anti-IBA-1 pAb (#hs-234 013, Synaptic Systems GmbH, 1:200) and anti-CD3 pAb (#sc-1127, Santa Cruz Biotechnology, 1:100). Images were acquired using a DS-Ri1 camera and captured using NIS-Elements F4.30.01 software.

Staining of cells and flow cytometry

Single cell suspensions were stained by a number of different fluorochrome-labeled mAbs: Anti-BrdU APC from kit (#552598, BD Biosciences, 1:100), Anti-BrdU APC (clone Bu20a, Biolegend, catalogue #339808, 1:20) anti-CD19 PE-Cy7 (6D5, Biolegend, #115520, 2.5 µg/ml), anti-κ-APC-Cy7 (RMK-45, Biolegend, # 409504, 2.5 µg/ml), anti-κ-FITC (187.1, Thermo Fisher Scientific, #OB1170-02, 8 µg/ml), anti-λ-APC (RML-42, Biolegend, #407306, 4 µg/ml), anti-CD80-APC (16-10A1, Biolegend, #104714, 8 µg/ml), anti-CD86-PE (GL1, Southern Biotech, # 735-09L, 4 µg/ml), anti-mouse I-A/I-E-FITC (2G9, BD Biosciences, #553623, 8 µg/ml), anti-Ki-67-PerCP-Vio700 (REA 183, Miltenyi Biotec, #130-120-418, 2 µl/test), anti-CD95-FITC (Jo2, BD Biosciences, #554257, 8 µg/ml), anti-CD95-BV605 (SA367H8, Biolegend, #152612, 2.5 µg/ml), anti-GL7-FITC (GL7, Biolegend, #144604, 8 µg/ml), TCRm-biotin (anti-pId:MHCII scFv TCR mimetic,)26 (homemade, 1.5ug/ml), anti-CD4-APC (GK1.5, Southern Biotech, #1540-11, 4 µg/ml), anti-CD4-AF405 (RM4-5, Thermo Fischer Scientific, #MCD0426, 4 µg/ml), anti-CD69-PerCP/Cy5.5 (H1.2F3, Biolegend, #104520, 3 µg/ml), anti-mouse TCRvβ 8.1-3-FITC (F23.1, Santa Cruz Biotechnology, #sc-33648, 1:5), anti-Id-specific TCR mAb GB11328 (homemade, 8µg/ml), anti-CD93-PE (AA4.1, Biolegend, #136504, 3 µg/ml), anti-CD21/CD35-FITC (7G6, BD Biosciences, #553818, 8 µg/ml), anti-FoxP3-APC (FJK-16s, Thermo Fisher Scientific, #17-5773-82, 8 µg/ml) anti-IgM-APC (RMM-1, Biolegend, #406509, 8 µg/ml), anti-CD23-PE-Cy7 (B3B4, Thermo Fisher Scientific, #25-0232-82, 2 µg/ml), anti-CD44-PE-Cy7 (IM7, Biolegend, #103030, 3 µg/ml), anti-PD-1-APC-Cy7 (29F.1A12, Biolegend, #135224, 1.5 µg/ml), anti-CXCR5-BV421 (L138D7, Biolegend, #145512, 6 µg/ml), goat anti mouse-IgG1-AF488 (polyclonal, Thermo Fisher Scientific, #A-21121, 2 µg/ml), goat anti mouse-IgG2a (polyclonal, Thermo Fischer Scientific, #A-21134, 2 µg/ml), anti-IgG2b (G15-337, BD Biosciences, #553884, 2 µg/ml), anti-CD45R-PerCP/Cy5.5 (RA3-6B2, BD Biosciences, #552771, 4 µg/ml), anti-λ2/3-biotin (2B6, homemade, 6µg/ml)65, anti-κ-biotin (187.1, homemade, 3µg/ml). The following secondary reagents were used in combination with biotinylated antibodies: streptavidin-APC/Cy7 (#405208, Biolegend, 1:300), streptavidin-PerCP (#554064, BD Biosciences, 1:500) and streptavidin-PE (#554061, BD Biosciences, 1:1000). Labeled mAbs were diluted in PBS (#D8537, Sigma-Aldrich) containing 0.5% BSA (#805095, Bio-Rad) with 30% normal rat serum (#GTX73226, GeneTex) and FcRγ blocker mAb. Stainings were done as described in26. For intracellular stainings, BD Cytofix/Cytoperm (#554714, BD Biosciences) kit was used. For FoxP3 staining, BD Pharmingen™ Transcription Factor Buffer Set (#562574) was used. In vivo proliferation was assessed using the Ki-67 marker (mAb clone REA183, #130-100-339, Miltenyi Biotec). Ki-67 staining was done as described in66. Stained cells were run on a BD LSR II instrument and Attune NxT (from Thermo Fisher Scientific). Compensation was performed using OneComp eBeads (Thermo Fisher Scientific, #01-1111-42) stained individually with each antibody used in the panel. The data were analyzed using FlowJo_V10.6.1 (TreeStar, Ashland, OR) software.

Transfer of lymphoma cells to Rag1-/- mice

Rag1-/- mice were injected i.v. with 1x106 cells isolated from the spleens of mice with suspected primary lymphomas. The cancerous state of suspected primary lymphomas was always confirmed by establishing secondary lymphomas in Rag1-/- mice. Upon reaching the humane endpoint, the Rag1-/- recipients were dissected and lymphomatous tissues collected for IHC and for FACS staining.

In vitro BrdU proliferation assays

To study the proliferative T cell responses to synthetic peptides, Splenocytes from BALB/c were irradiated with 20Gy. CD4+ T cells were isolated from 4 weeks old Vλ2315m+/- Id-specific TCR-TG+/- and Id-specific TCR-TG+/- mice by negative selection using immunomagnetic depletion Dynabeads Mouse CD43 (Untouched B cell Kit, #11422D, Thermo Fisher Scientific) and Dynabeads Untouched™ Mouse CD4 Cells Kit, #11415D, Thermo Fisher Scientific). 1x106 splenocytes and 1x105 Id-specific T cells were added to a total volume of 1 ml complete medium in 48 well plates and stimulated with 10 µg/ml synthetic λ2 peptides [FAALWFRNHFVFGGGTK (λ2315) or FAALWYSTHYVFGGGTK (germline λ2), Genscript] for 48 hours. BrdU was then added to the culture to obtain a final concentration of 1mM. After 16 hours cells were harvested, stained and analyzed by FACS.

To study the proliferative responses of ex vivo B and T cells obtained from tumor tissues, B cells and CD4 T cells were sorted from lymphomatous spleens or BALB/c spleens by negative selection using immunomagnetic depletion. B and T cells (5x105/of each in 1 ml, using 48 well plates) were co-cultured in complete culture medium for 72 hours; the last 16 hours in the presence of BrdU (1mM). Incorporation of BrdU was detected using a BrdU Flow Kit (#552598, BD Biosciences). Briefly, 16 hours after pulsing with BrdU, the cells were harvested, washed and surface stained. Then the cells were fixed with BD cytofix/cytoperm for 15 minutes on ice. The cells were washed with BD perm/wash and incubated with BD Cytoperm Permeabilization Buffer for 10 minutes on ice. The cells were washed again and refixed with BD cytofix/cytoperm. The cells were treated with DNase I for 1hour at 37oC to expose BrdU. The cells were washed and stained with anti-BrdU APC diluted in BD perm/wash and washed. The cells were then analyzed by flow cytometry.

In vivo BrdU incorporation experiments

Age matched 14 months old Vλ2315m+/-, Vλ2315m+/- Id-specific TCR+/- and Id-specific TCR+/- mice were injected with 1 mg of BrdU (#B5002, Sigma-Aldrich) i.p. on day 1. Thereafter, mice received BrdU (0.6 mg/ml) in the drinking water for 7 days before euthanasia and analysis. Single cells from spleen and LN were surface stained and fixed with BD cytofix/cytoperm for 15 minutes on ice. The cells were then treated with BD Cytoperm™ Permeabilization Buffer Plus for 5 minutes (#561651, BD Biosciences) followed by incubation with DNase I at 37oC for 1hour. Finally, the cells were stained with anti-BrdU-APC mAb (clone Bu20a, Biolegend) at 25oC for 30 minutes and analyzed by flow cytometry.

Lymphoma cell lines and clones

Single cells from spleens of Rag1-/- mice transferred with primary B lymphoma cells were cultured in vitro in RPMI supplemented with an increased amount of FCS (20%). Generally, the cells grew only for a few days before dying. The cells from mouse #24 (NT24), however, grew for a few passages and were used for confocal microscopy and flow cytometry. Only cells from lymphoma NT34 proliferated continuously to give rise to the cell line NT34. The cells which expressed the highest amount of  L chains intracellularly were selected by limiting dilution to obtain a cell line called NT34.E2. The FCS level could be reduced to 10% with maintenance of cell growth. NT34 cells were transiently transfected with lentiviral transduction, using the bicistronic expression vector pHIV-Luc-ZsGreen, encoding firefly luciferase and the green fluorescent protein ZsGreen. Then the GFP+ cells were sorted on a BD FACS Aria. Sorted cells were called NT34.Effluc-GFP and were used in tumor challenge experiments.

The T cell lymphomas were easier to grow compared to the B lymphomas. The spleen cells of secondary lymphomas in Rag1-/- mice were cultured with 100 IU/ml IL-2. After few passages, the T cells started to grow without IL-2 supplement.

Tumour challenge and IVIS

0.5x106 NT34.Effluc-GFP cells were injected i.p. into Rag1-/- and Id-specific TCR-TG+/+ Rag1-/-. The mice were injected i.p. with D-Luciferin (150 mg/kg body weight, Sigma-Aldrich) and anesthetized with isoflurane. 10 minutes later, images reflecting tumor load were acquired on days 7, 14, 18, 21, 28 and 35 using an IVIS Spectrum imaging system (Caliper Life Sciences,). Data were analyzed using the LivingImage software (Caliper Life Sciences).

Western blot

Cells were lysed with NP-40 lysis buffer containing a protease inhibitor cocktail (P8340, Sigma-Aldrich). Samples were denatured with sample buffer containing 2-Mercaptoethanol (#M3148, Sigma-Aldrich) and Dithiothreitol (#D0632, Sigma-Aldrich) at 95oC for 5 minutes, run on a 4-12% Tris-bis gel (NW04125BOX, Novex, Carlsbad, CA, USA), and transferred to a PVDF membrane (#IB24001, Invitrogen). The membrane was blocked with 3% milk powder (#A0830, AppliChem, Darmstadt, Germany) in PBS containing 0.05% Tween. The λ Ig L chain was detected with following antibodies: Cλ2/Cλ3 L chain cross-reactive rat mAb 2B665 (1µg/ml) and Vλ1/Vλ2 cross-reactive rat mAb 9A865 (1µg/ml). Primary antibodies were detected with goat anti-rat IgG Horseradish Peroxidase (HRP) (#31470, Thermo Fisher Scientific, 1:5000) and developed with ChemiGlow West Chemiluminescence Substrate Kit (#60- 12596-00, ProteinSimple). In co-precipitation experiments, the cell lines were lysed with NP-40 lysis buffer containing 0.1% CHAPS (#C9426, Sigma-Aldrich). 30µl of protein G beads were coated with 1µg of anti- λ2315 R/A (Rabbit pAb)54 or anti-κ mAb (H139-52.1, Sothern Biotech, #1180-01, 1:2000) and blocked with rat serum. Then λ and κ Ig were precipitated from lysates and analyzed by Western blots under non-reducing condition using the biotin labelled mAb: anti-Cλ2/cλ3 (2B6, 1µg/ml), anti-κ (187.1, 1µg/ml), anti-IgG2aa (8.3, BD Biosciences, #553502, 1:1000), followed by streptavidin-HRP conjugate (#7100-05, Southern Biotech, 1:5000). Blots were captured using G:box Chemi XX6 device (SYNGENE).

Cytokine Multiplex and IL-21 ELISA

23 different cytokines in sera were analyzed using Bio-Plex Pro Mouse Cytokine 23-plex Assay (M60009RDPD, Bio-Rad). The plates were read on Bio-Plex MAGPIX. IL-21 ELISA was performed using IL-21 Mouse Uncoated ELISA Kit (8-8210-88, Invitrogen).

Mass spectrometry

For immunoprecipitation, 30 µl of Dynabeads Protein G for Immunoprecipitation (10003D, Thermo Fisher Scientific) were washed and coated with 1 ug of anti-VV mAb (9A8) at 25oC. To precipitate serum IgG, uncoated protein G beads were used. Precipitated proteins on beads were dissolved in 50 mM ammonium bicarbonate, reduced, alkylated and digested with 1 ug AspN (Promega) for 1 hour and then 1 ug trypsin (Promega) at 37oC overnight. Digested peptides were acidified and desalted by homemade C18 stage tips. LC-MS/MS analysis was carried out using a nanoElute nanoflow ultrahigh pressure LC system (Bruker Daltonics, Bremen, Germany) coupled to the timsTOF fleX mass spectrometer (Bruker Daltonics), using a CaptivAeSpray nanoelectrospray ion source (Bruker Daltonics). Raw files from LC-MS/MS analyses were submitted to MaxQuant 2.4.7.0 software for protein identification and label-free quantification. A custom database having lymphoma BCR sequences was used.

Super-resolution Immunofluorescence Microscopy

Coverslips were sterilized with 95% EtOH, flamed and placed in 6 well plates. The coverslips were treated with poly-L-lysine for 10 minutes at 37oC. The coverslips were then washed. Lymphoma B cells were cultured in RPMI w/ 20% FCS media for 2 days. Before staining, the wells were washed with PBS and the cells were fixed with 2% formaldehyde (F8775, Sigma-Aldrich). Surface  staining was done by incubating with a rat anti-mouse − FITCmAb (#MKAPPA01, Thermo Fisher Scientific, 10µg/ml) in 30% rat serum at 25oC for 2 hours. The cells were then washed and stained with Fluorescein/Oregon Green Polyclonal Antibody, Alexa Fluor 488 (#A-11090, Thermo Fisher Scientific) for 30 minutes at 25oC. For intracellular staining, the cells were then permeabilized with BD Cytofix/Cytoperm prior to incubation with goat anti-mouse λ -TRITC pAb (#1060-03, Southern Biotech, 4µg/ml) in BD perm/wash solution containing 30% rat serum. After 2 hours at 25oC, the cells were washed and mounted on a slide with Fluoromount Aqueous Mounting Medium (#F4680, Sigma-Aldrich).

For the combined ER and λ staining, the cells were fixed and intra-cellular λ staining was done. Next, ER staining was performed using the ER Staining Kit - Green Fluorescence–Cytopainter (#ab139481, Abcam). The mounted slides were imaged with Zeiss LSM880 (60X/1.42 Oil objective) microscope operating in the Airyscan mode and processed with the Zeiss Zen non-iterative deconvolution algorithm for Airyscan images. The images were cropped, and contrast enhanced for presentation purpose with Fiji (Image J).

Primers

All primer sequences are in 5’→ 3’. For gene specific RT PCER, the primers described in67 were used: ACAGTCACTGAGCTGC (IgG) and AGTGGCCTCACAGGTAR () to amplify mRNA. To amplify the H chain, forward primer (FP) MsV H (GGGAATT CGAGGTGCAGCTGCAGGAGTCTGG), which binds all mouse V 68 was used in combination with reverse primers (RP) which bind the C region of the following isotypes: ATAGACAGATGGGGGTGTCGTTTTGGC (IgG1), CTTGACCAGGCATCCTAGAGTCA (IgG2a), AGGGGCCAGTGGATAGACTGATGG (IgG2b), AGGGACCAAGGGATAGACAGATGG (IgG3). For  mCκ (RP) GATGGTGGGAAGATGGATACAGTT that binds constant domain of all κ families was used together with mixture of FP that bind different Vκ families: GGTCAGACAGTCAGCAGT (Vκ1), GTGCTCTGGATTCGGGAA (Vκ2), TGCTGCTGCTCTGGGTTCCAG (Vκ3), ATTWTCAGCTTCCTGCTAATC (Vκ4) ,TTTTGCTTTTCTGGATTYCAG (Vκ5), TCGTGTTKCTSTGGTTGTCTG (Vκ6), ATGGAATCACAGRCYCWGGT (Vκ6,8,9), GTTGTAATGTCCAGAGGA (Vκ11), GCTTACAGGTGCCAGATGT (Vκ12/13), TCTTGTTGCTCTGGTTYCCAG (Vκ14), CAGTTCCTGGGGCTCTTGTTGTTC (Vκ19), CTSTGGTTGTCTGGTGTTGA (Vκ19/28), CTCACTAGCTCTTCTCCTC (Vκ20)69, TGCTKCKCTGGGTTCCAG (Vκ21), TGTCTGGTGCCTGTGCA (Vκ22), TGGAYTYCAGCCTCCAGA (Vκ23), WTCTCTRGAGTCAGTGGG (Vκ24/25), GCTTCATGGTCAGTG (Vκ31/38), TGTTCTGCTTTTTAGGTGTG (Vκ32), GAATCCCAGGCATGATATGT (Vκ33/34), and GATATCAGGTGCCCAGTG (VκRF)70. For λ2315 PCR, the FP (CAGGCTGTTGTGACTCAG) that bind V region of λ1/2 and RP (GCCGAAAACAAAATGGTTTCTGAACC) that bind specifically to F94R95N96 in λ2315 and not germline λ2) was used.

Preparation of cDNA library

Spleen cells from terminal Vλ2315m+/- Id-specific TCR-TG+/- mice were injected into Rag1-/- mice. Once the Rag1-/- recipients reached the humane end point, mice were culled and single spleen cells were prepared. Approximately 5x106 cells were used to isolate RNA using Qiagen RNeasy kit. Subsequently, gene-specific cDNAs were made for Ig H and L chains with specific primers described above (for IgG, ) using First strand cDNA Kit (#K1612, Thermo Fisher Scientific) and then PCRs using gene specific primers and Phusion Flash polymerase (#F548S, Thermo Fisher Scientific). The PCR products were purified using AMPURE beads. The samples were sequenced using MiSEQ Nano at the The Norwegian Sequencing Centre, Oslo University hospital, Oslo.

Data Processing

Raw sequencing files (.fastq.gz files, paired-end reads) were obtained from an Illumina Nano sequencing run. Reads were processed with the MiXCR software package71 (version 4.3.20) including sequencing read alignment to a BALB/c mouse-specific germline gene database from OGRDB 37 followed by clonotype assembly using the V(D)J region sequence. Clonotypes were exported as tsv files for further analysis. SHM analysis was performed using MIXCR, which reported the number of mutations. Functional clonotypes (referring to unique sequences of VDJRegion(nt) after excluding out-of-frames and sequences containing stop codons) were included in the analysis only if they had a minimal read count of 2 72. Charge was calculated using the R package peptides73. The alignment of the dominant sequence and the phylogenetic tree were made with CLC Main workbench (Version 8.1.3, QIAGEN Aarhus A/S). The aligned sequences were submitted to Genbank: accession numbers: PV068692 - PV068753 and PV068754 - PV068825.

BCR recombinant immunoglobulin cloning and expression

The dominant VDJ and VJ sequences from four different lymphomas (NT7, NT8, NT18, NT24) were ordered from Genscript. VDJ and VJ were cloned into pLNOH2 (h3) and pLNOK (hC ) vectors, respectively, using the signal peptide sequence from the V gene of B1-8 mAb74. HEK293E (ATCC, Manassas, USA) cells cultured in multilayer cell culture flask (#353144, Falcon), were transiently transfected with combinations of H chain and κ L chain plasmids in a 1:1 ratio in the presence of Polyethylenimine (Polysciences). Supernatants were collected on days 4, 7 and 10. The recombinant immunoglobulins (rIgs) were purified on CaptureSelect FcXL affinity (#194328005, Life technologies) using AKTA prime plus (GE HealthCare) equipment. The eluted Igs were dialysed against PBS and used in various assays. rIgs were detected by sandwhich ELISA using mouse anti-human IgG (C 3 domain, H MA5-16557, Thermo Fisher Scientific, 1µg/ml) and biotin anti-human L chain κ mAb (#316504, Biolegend, 1µg/ml). The concentration of rIgs was measured on Nanodrop Lite Plus (Thermo Fisher Scientific). Anti-mouse CD40 rIg was made by cloning VDJ and VJ from anti-CD40 mAb (clone FGK45) intopLNOH2 (h3) and pLNOK (hC ) plasmids. rIg purified from supernatants of transfectants was used as a negative control,

Competition assay for H-L chain assembly

HEK293E cells cultured in 6 well plates were transfected with three plasmids: an H chain plasmid containing V H from the various lymphoma clones were fused to a h3 constant regions along with two L chain plasmids (lymphoma Vκ from the corresponding clone fused to a hCκ constant region and Vλ2315 fused to a hCλ constant region from the M315) in 1:1:1 ratio. The supernatants were collected on day 4 and 7. The expression of κIg and λIg were detected using sandwich ELISA using mouse anti-human IgG (clone R10Z8E9, Invitrogen, #MA5-16929, 1µ g/ml) and biotin anti-human L chain κ mAb (clone MHK-49, Biolegend, #316504, 1µg/ml) or biotin anti-human L chain λ mAb (clone MHL-38, Biolegend, #316604, 1µg/ml). Biotinylated mAb was detected with streptavidin-Alkaline Phosphatase (ALP) conjugate (#RPN1234, Southern Biotech, 1:3000). Signals were developed with phosphatase substrate (#P4744, Sigma-Aldrich, 1 mg/ml) dissolved in diethanolamine substrate buffer. OD was measured 405nm using EnVision 2104 Multilabel Reader with EnVision Manager 1.12 software (PerkinElmer).

Assays to detect serum autoantibodies autoreactivity of rIg from lymphoma BCR

ANA HEp-2 Fluorescent Test System (#2040, Immunoconcepts) was used to detect autoantibodies as described75. Detection of serum antibodies by indirect immunofluorescence: Goat anti-mouse IgG (H+L) Alexa Fluor™ 488 (#A28175, Invitrogen, 1:1000), anti-mouse IgG1 (γ1) PE (#P21129, Invitrogen, 1:2000), mouse IgG2a (γ2a) AF568 (#A21134, Invitrogen, 1:2000), anti-mouse IgG1 (γ1) AF488 (#A21121, Invitrogen, 1:1000), mouse IgG2a (γ2b) AF546 (#A21143, Invitrogen, 1:2000) and anti-mouse IgG1 (γ3) AF488 (#A21151, Invitrogen, 1:1000). Detection of recombinant Igs: FITC labelled anti-human IgG (from ANA kit) was used.

Crithidia luciliae immunofluorescence test (CLIFT) screening was done using nDNA Fluorescent Test System (#3040G, Immunoconcepts). After staining, the slides were mounted on coverslips using mounting media (from kit). The slides were stored at 4oC protected from light overnight, before imaging with a Nikon Eclipse Ti fluorescent microscope (Nikon Instruments).

For histone ELISA, the 96 well high binding EIA/RIA plates (Corning) were coated with 2ug/ml whole histones (#HIS-1010, Immunovision Inc,) diluted in PBS overnight at 4oC. The mice serum samples and recombinant IgG were serially diluted in ELISA buffer (starting at respectively 1/200 and ¼) and incubated for 2 hours at 37oC. Anti-histone autoantibodies were detected with 1 μg/ml anti-mouse IgG HRP (#405306, Biolegend), biotinylated anti-mouse IgG1a (#553500, BD Biosciences, 1:1000), anti-mouse IgG2aa (#553502, BD Biosciences, 1:1000), anti-mouse IgG2ba (#553393, BD Biosciences, 1:1000) or anti-mouse IgMa (#553515, BD Biosciences, 1:1000). Anti-nucleosome autoantibodies were detected using Anti-nucleosome ELISA (IgG) kit from Euroimmun (#EA 1574-9601). Sera and recombinant Ig were serially diluted in ELISA buffer (as above). Anti-nucleosome autoantibodies were revealed by anti-mouse IgG HRP. For detection of antibodies against dsDNA, EliA dsDNA Well (precoated) (#14-5500-01, Phadia ABC) was used. For detection of auto-antibodies against dsDNA in mouse serum, goat anti-mouse IgG HRP (#405306, Biolegend, 1:5000) was used.

To determine the specificity of BCR-derived rIg, immunoblot strips (#DL1590640136, EUROIMMUN) coated with parallel lines of highly purified self-antigens were used. The test strip was put into the incubation channel and sample buffer was added and incubated for 5 minutes with gentle shaking. 30 µl of recombinant Ig (about 0.1 mg/ml) was diluted in 1.5 ml sample buffer. The diluted samples were incubated for 30 minutes with gentle mixing, before strips were washed and incubated with enzyme conjugate for 30 minutes. After washings, strips were incubated with substrate for 10 minutes and washed. Scanning and evaluations of the blots were performed with EUROLineScan (digital).

Statistics

Two experimental groups were compared using two-tailed unpaired Student's t-test with Welch's correction, provided the samples had a normal distribution. A non-parametric two-tailed Mann-Whitney U test was used if there was no normal distribution. Several experimental groups with normal distribution were compared using one-way ANOVA and the Tukey/Dunnet's multiple comparisons test. Two-sided Kruskal-Wallis test, Dunn’s multiple comparison was used when comparing experimental group with several control groups without normal distribution. Comparison of survival time lengths on the tumor avoidance curve was done using the Mantel-Cox test. GraphPad Prism (Version 8.4.0 and Version 10.2) was used to make plots and perform statistical analysis. All the plots were shown as scattered dot plot or violin plot, if the data set was large. Mean ± SD was used to describe the distribution of biological replicates, while mean ± SEM was applied when the objective was to convey the precision of the estimated mean. * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001.

Data availability

The aligned lymphoma BCR sequences data generated in this study have been deposited in the Genbank database under accession code PV068692 - PV068753 [https://www.ncbi.nlm.nih.gov/nuccore/?term=VLambda2%2F-+Id-sp+TCR-Tg%2B%2F-] and PV068754 - PV068825 [https://www.ncbi.nlm.nih.gov/nuccore/?term=Vlambda2315%2B%2F-+Id-sp.+TCR-TG%2B%2F-]. The mass spectrometry data from serum and recombinant Immunoglobulin data generated in this study have been deposited in Pride under Accession numberPXD063326 [https://www.ebi.ac.uk/pride/archive/projects/PXD063326]. All data are included in the supplementary section. The raw numbers for charts and graphs are available in the Source Data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, a novel mouse model is presented that may explain the unresolved question why autoimmune diseases are often linked to the development of B cell lymphomas. The model is based on an F1 backcross of two transgenic mouse strains. One strain expresses in approximately 0.5% of all B cells a hypermutated Ig lambda light chain (derived from a plasmacytoma mouse line). By pairing with any endogenous heavy chain, a few B cells might recognize autoantigens. The second mouse line expresses a transgenic T cell receptor specific for the hypermutated motif in the lambda light chain (anti-ideotype TCR). Thus, these T cells (which surprisingly don't get clonally deleted in the F1 strain) can provide help to the B cells independent of the specificity of the BCR (Id-driven T-B collaboration). F1 mice develop overt autoimmunity and also B cell lymphomas from the age of 12 months.\nThis is a very interesting and also clinically highly relevant mouse model since it could nicely explain why several autoimmune diseases, such as Sjogren's syndrome, are associated with a high risk of lymphoma development.\nSpecific comments\n1. The group of Bjarne Bogen, who has been working on the question of Id-driven T-B collaboration for more than 30 years, previously published a similar mouse model (Zangani et al., J Exp Med, 2007). In that model, all B cells expressed the transgenic light chain and T cells were adoptively transferred. Similar to the new model, mice developed autoimmunity as well as lymphomas. While the new light chain transgenic strain with only about 0.5% expressing cells is much more physiological and a clear improvement, it is not clear why the authors switched back to an F1 backcross with the TCR transgenic strain instead of adoptively transferring of a limited number of autoreactive T cells, which would be much more physiological and also avoid potential artifacts of thymic selection. It would be good at least to see whether in such a setup some of the rather unexpected observations made in this study (such as the lambda/kappa double expressing B cells) still exist.\n2. The flow cytometry results of all intranuclear stains (FoxP3, Ki-67) look really strange with no clearly separated positive population. According to the Methods section, the BD Cytofix/Cytoperm (BD #554714) kit was used. This kit is designed for cytoplasmic, not intranuclear staining! Experiments have to be repeated using the appropriate kit (BD Pharmingen™ Transcription Factor Buffer Set, # 562574).\n3. Information on number of independent experiments and animals per group is missing for all experiments.\n4. For statistical analysis, Student's t-test or one-way ANOVA were used throughout all experiments. For many experiments this is inappropriate as there is obviously no normal distribution (e.g. Fig. 1D, 2D, 2G and many more). For many experiments, also the group sizes with sometimes as few as three animals are too small (after removing one outlier, any trends for differences between groups are gone, e.g. Fig. 2 E).\n5. Figure 3 M: According to the text and Fig. 3L, kappa expression in the B lymphoma group is reduced. The graph shows the opposite.\n6. Figure 3 N: Please check compensation.\n7. Suppl. Fig. S1 B: How do you explain the reduced TCR Vb8 expression in the Id. sp TCR+/- group compared to the double transgenic mouse?\n8. Suppl. Fig. S3 K: Please check compensation; the sharp peak obviously results from cells piling up in the sum channel of another fluorophore.", "reviewer_2": "In the study by Gopalakrishnan and colleagues, a mouse model was established to study whether idiotype-specific CD4+ T cells promote the development of idiotype+ autoreactive B cells into lymphomas. Prior studies by the group of the senior author and by other groups already showed that principally, idiotype-specific CD4+ T cells can be generated for mutated B cell receptors (BCRs), and there is a known link between autoimmunity and the development of B cell lymphomas. So the rational is clear and a highly relevant question is addressed.\nThe model was meant to be more physiological than standard transgenic mouse models, because only a small number of B cells will express the idiotype (only B cells which during their development rearrange a mutated Vlambda2 gene to the Jlambda2 gene), and not nearly all B cells. The hypothesis for the model was that if a BCR using the Vlambda2 light chain is by chance autoreactive, the autoreactive B cells would be stimulated, upregulate MHC class 2 and present the light chain idiotype to the transgenic idiotype-specific CD4+ T cells, which then further stimulate the autoreactive B cells, drive their constitutive clonal expansion and finally lead to lymphomas development. The mice indeed developed autoantibodies and after about a year in some mice B cell lymphomas developed (other developed T cell lymphomas). Hence, at first glance these findings seem to nicely support the hypothesis of the study. However, a closer inspection of the mice and their B cells revealed that the autoantibodies expressed kappa light chains, and also the lymphomas expressed a kappa BCR. The lymphomas nevertheless developed from Vlambda2 idiotype-positive B cells, but the lambda light chains were only detected with in the cells, and not as part of the BCR. Thus, allelic exclusion is violated here and the B cells co-express a kappa and a lambda light chain (but only one as part of the BCR). In the lymphomas, the BCR with the kappa light chain provides the autoreactivity, whereas peptides from the lambda light chain are presented on MHC class 2 and thereby mediate stimulation of the B cells through the idiotype-specific CD4+ T cells. The reason for the violation of allelic light chain exclusion is not clear. Perhaps, the mutated Vlambda 2 gene has problems to pair stably with most Ig heavy chains, so that additional light chain rearrangements are performed, leading to allelic inclusion. Dual light chain producing B cells have been identified, but this is clearly not the general situation in autoimmune diseases and B cell lymphomas.\nThe experiments are principally well done and nicely presented.\nSo in my opinion the study presents an unexpected and curious observation, but neither validates nor falsifies the hypothesis of the study. The original idea that an idiotypic peptide of an autoreactive BCR is presented on MHC class 2 and causes chronic stimulation of the B cells through idiotype-specific CD4+ T cells, finally leading to lymphoma development, is not tested here. This is because in the present model, unexpectedly not an idiotypic peptide from the autoreactive BCR is presented to the CD4+ T cells, but a peptide from another protein, that is not part of the BCR (i.e., the lambda light chain).\nMinor point\na) From line 106 onwards there are multiple instances where the mouse line designation is wrongly written. It should be Vl2 and then \"315m+/-\" in superscript, and not Vl with \"2315m+/-\" in superscript.\nb) Is there a mistake in figure 3M? According to the text the double transgenic mice (red symbols) have lower kappa light chain expression than the controls, but here the controls all have apparently no kappa light chain expression, but the double transgenic B cells show variable expression. Please clarify.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "Gopalakrishnan et al. have attempted to pursue the links between autoimmunity and lymphomagenesis. The created a model wherein they generated mice with a modified l light chain creating an epitope that binds to I-Ad. These mice were crossed into TCR transgenic mice the recognize the I-Ad peptide combination, generating mice heterozygous both for the light chain epitope and for the TCR transgene. These mice develop some anti-nuclear antibodies and eventually develop lymphomas. The lymphomas are kappa light chain expressors and H-chain sequences were shared between earlier autoantibody BCRs and lymphoma BCRs.\nWhie the approach has potential, and this work has some interesting aspects the main issue in the manuscript appears to be an apparent disregard for some fundamental immunological principles and the authors’ apparent decision to ignore their own data and not try to interpret it. The fundamental premise in the schematic in Figures 1A and B is not based on a sound understanding of the principles of T-B collaboration and is fundamentally incorrect, based on the data that the authors present, but inexplicably choose not to interpret and end up ignoring. A key experiment to test the likely flaws in the model in the schematic was ignored. This will be pointed out below\n1. Surprise is repeatedly expressed that the lymphomas were not lambda light chain positive! The authors show in Figures I panels H, I and J that the anti-nuclear antibodies they observe are almost exclusively kappa light chain positive. 90% of all Igs in mice are kappa positive and in the obvious scenario painted below it would be likely that most responding B cells would be kappa positive.\n2. The likely simple explanation of the data is that some IgHlambda2315 immunoglobulin molecule/s which are essentially provided in relatively high doses in these mice, are recognized by a self-reactive BCR or BCRs (at least 60-70% of all BCRs are self -reactive in practical terms) on one or a few B cell clones all of which are kappa expressors (not surprising - there is a 90% chance that a BCR would be kappa positive). Some (possibly a small fraction) of these BCRs in these mice are also capable of recognizing some epitope on a nuclear antigen.\n3. These kappa+ self-reactive BCRs internalize the IgHlambda2315 immunoglobulin self-antigen, process it and present the lambda peptide to the relatively large numbers of CD4+ T cells that are specific for the peptide. The kappa + self-reactive B cell and the CD4+ T cell collaborate at the T-B boundary generate an extrafollicular B cell response that generates plasmablasts secreting anti-nuclear antibodies. No autoimmune disease necessarily, just an autoimmune phenomenon which is not such a big deal\n4. As these kappa+ self-reactive B cell clones are repeatedly stimulated by T cell help since antigen is always available, and therefore expand, occasionally additional hits accumulate and lymphomas evolve. The data says most of this!!!\n5. The authors should do the following:\na) use the Wardemann and Nussenzweig approach and clone V regions of Ig H and kappa light chains from some lymphomas, clone into the already widely used Ig vectors, and then express in 293T cells\nb) show that these clonal Igs are self-reactive using the assays they have used in the manuscript\nc) delete and correct the old schematic in Figure 1\nd) In the abstract and manuscript stick to the use of “autoantibodies” or “autoimmune phenomena” and delete “autoimmunity” in the manuscript. There is no good evidence for an autoimmune disease in this manuscript. We all have autoantibodies! Self-reactivity is a normal phenomenon – some extreme self-reactivity is eliminated by central tolerance mechanisms."}, "author_response": "Our response: We would like to thank the Reviewer for highlighting the novelty of our work and finding our mouse model to be interesting and clinically highly relevant.\nSpecific comments\nComment 1. The group of Bjarne Bogen, who has been working on the question of Id-driven T-B collaboration for more than 30 years, previously published a similar mouse model (Zangani et al., J Exp Med, 2007). In that model, all B cells expressed the transgenic light chain and T cells were adoptively transferred. Similar to the new model, mice developed autoimmunity as well as lymphomas. While the new light chain transgenic strain with only about 0.5% expressing cells is much more physiological and a clear improvement, it is not clear why the authors switched back to an F1 backcross with the TCR transgenic strain instead of adoptively transferring of a limited number of autoreactive T cells, which would be much more physiological and also avoid potential artifacts of thymic selection. It would be good at least to see whether in such a setup some of the rather unexpected observations made in this study (such as the lambda/kappa double expressing B cells) still exist.\nOur response:\nThe Reviewer is right in stating that: “….the new light chain transgenic strain with only about 0.5% expressing cells is much more physiological and a clear improvement,…” . In a further comment to the Zangani paper, the Reviewer writes: “ Similar to the new model, mice developed autoimmunity as well as lymphomas.” . However, the Zangani et al. paper did not study autoimmunity at all. Thus, the link between autoimmunity and later B lymphoma development is novel to the present study and represents a major advance, as indicated by the title of the present paper.\nThe Reviewer wonders why we in the present study use F1 backcross mice rather than transfer of T cells. In our opinion, it is more physiological not to transfer T cells (which had to be done multiple times in the Zangani paper, in addition, that study used unphysiological in vitro-polarized Th2 cells). In contrast, in the present study, naïve Id-specific T cells develop along normal pathways, including thymic education, which we feel is more physiological.\nThe Reviewer asks why we have not transferred limited numbers of autoreactive (idiotype-reactive?- our comment) T cells into V gene-modified mice. This is a relevant question since Id-specific TCR-transgenic mice have an unphysiologically high frequency of Id-specific CD4+ T cells.We already did the experiment requested by the reviewer, but did not include the results in the submitted manuscript. The data have now been included in the revised manuscript. In brief, 6 V2315m+/- mice (2-month-old) were transferred with 1 million naïve Id-specific CD4+ T cells. Controls were 4 age-matched V2315m+/- mice that did not receive any T cells. Within 13 months, 2/6 of transferred mice developed autoantibodies. Later these two mice developed B cell lymphomas. Another 2 transferred mice developed B cell lymphomas without preceding autoimmunity. Thus, altogether 4 out of 6 transferred mice developed B cell lymphomas within 20 months. In lymphoma mice, there was expansion of Id-specific T cells and pId:MHCII+ B cells. None of the control mice (0/4) developed autoantibodies or lymphomas. This experiment shows that even when strongly reducing the number of Id-specific T cells, and circumventing T cell development in the thymus, autoimmunity and B lymphomas were induced albeit at lower levels. The data obtained in this transfer experiment has not allow us to address the issue of lambda/kappa double-expressing B cells. A difficulty has been that we don’t have adequately frozen cells to establish secondary lymphomas in Rag1-/- recipients.\nChanges done in the revised manuscript: The transfer experiment asked for by the reviewer is presented in Fig. 7M-Q, Supplementary Fig. S8 and Supplementary Table-1. The experiments are described in the Methods (line 502-507), Results (line 344-361) and in the Discussion (lines 446-451).\nComment 2. The flow cytometry results of all intranuclear stains (FoxP3, Ki-67) look really strange with no clearly separated positive population. According to the Methods section, the BD Cytofix/Cytoperm (BD #554714) kit was used. This kit is designed for cytoplasmic, not intranuclear staining! Experiments have to be repeated using the appropriate kit (BD Pharmingen™ Transcription Factor Buffer Set, # 562574).\nOur response: We have repeated the FOXP3 staining (Fig . 1E) with BD Pharmingen™ Transcription Factor Buffer Set, # 562574 and have updated the figure and Materials and Methods. Regarding the nuclear antigen Ki-67 staining (Fig. 3E and 4E), it was done as described in Graefe C et al.,Optimized Ki-67 staining in murine cells: a tool to determine cell proliferation. Mol Biol Rep. 2019.\nChanges done in the revised manuscript: We have updated the FoxP3 staining (Fig 1E). We also updated the Materials and Methods section to explain the Ki-67 staining in more detail, and also referred to the Graefe et al. paper mentioned above (lines 562-565).\nComment 3. Information on number of independent experiments and animals per group is missing for all experiments.\nOur response: We thank the reviewer for pointing this out.\nChanges done in a revised manuscript: Information on number of independent experiments, and mice per group is now stated in the legends of Figs. and Supplementary Figs. These changes are too many to be indicated individually but changes are clearly marked in the revised manuscript.\nComment 4. For statistical analysis, Student's t-test or one-way ANOVA were used throughout all experiments. For many experiments this is inappropriate as there is obviously no normal distribution (e.g. Fig. 1D, 2D, 2G and many more). For many experiments, also the group sizes with sometimes as few as three animals are too small (after removing one outlier, any trends for differences between groups are gone, e.g. Fig. 2 E).\nOur response: We thank the reviewer for pointing this out.\nConcerning statistics: We have repeated the statistics according to the Reviewers’ suggestion. Two experimental groups were compared using unpaired Student's T-test with Welch's correction provided the samples had a normal distribution. A non-parametric Mann-Whitney U test was used, if there was no normal distribution. Several experimental groups, with normal distribution, were compared using one-way ANOVA or the Tukey's multiple comparisons test. Kruskal-Wallis test, Dunn’s multiple comparison was used when comparing several experimental groups not having normal distribution. Survival curves were compared using the Mantel-Cox test.\nWhen we repeated the statistical analysis as described above, most of the significant p values were retained. However, in two cases, Fig. 2C and 2E, the significance was lost. These two Fig. items have been transferred to Supplementary Figs. as new Fig. S2D and Fig. S2H. The Results text has been correspondingly changed, emphasizing that the results in Fig. S2D and Fig. S2H are trending but not statistically significant.\nConcerning group sizes: The group sizes in the various experiments of the originally submitted paper are given below.\nFig 1: C,D: 3-4 mice/group; E: 3-4/group; G: 7/group; I: 6–15/group; J: 6; K: 6–12/group; L: 4–14/group; M: 14; N: 4–11/group; O: 12, P: 11. Fig. 2: A-C: 4 /exp. group, 3/ control group; D, E, F, G: 4–5/group; H: 15–40/group; I: 4–14/group; J: 7–12/group; M: 40 (total number of classified lymphomas). Fig. 3: A-E: 10–12/group; F: 10/group; G: 12–14/group; I: 6/group; M: 8–10/group. Fig. 4: C:11–14/group; D-G: 6–13/group; L, M: 4/group. Fig. 7: F: 4/group, H- K: 4/group; M-Q: 6/exp. group and 4/control group.Fig. S1: A: 4–5/group; F: 4–5/group; G: 6/group; H, I: 6–15/group; Q: 14/ exp. group and 4–5/ controls groups. Fig. S2: A: 3–4/group; E-F: 3–4/group; I, J: 3–4/group; S2K: 12–14/cohort; L: 8; M-P: 3–8/group. Fig. S3: F-H, M: 4/group. Fig. S8: B-C: 6/ exp. group and 4/ control group.\nAs can be seen, we have generally used a sufficient number of mice/group for statistical comparisons except in Fig. 1C, D, E where only 3-4 mice/group were used. To remedy this, we have for Fig. 1C,D combined three similar experiments, each giving similar results, thereby increasing group sizes to 6-10(11) mice/group. For Fig. 1E, we pooled data from two novel and independent experiments performed with BD Pharmingen™ Transcription Factor Buffer Set, # 562574 as recommended by the Reviewer (see Comment 2, above). Group sizes are now n=9/group.\nThe cytokine measurements in sera of 10-month-old mice (Fig 2F-G in submitted version) are in the cases of IL-10 and TNF-somewhat weak (while the results for IL-21 and IFN are clear-cut). These data have now been bolstered by including cytokine levels in sera of 14-20 months old mice with B cell lymphomas (Fig. 2D-G in revised version). The novel data show that the two cytokines which were only weakly upregulated at 10 months (IL-10, TNF-) were strongly upregulated with lymphoma development.\nChanges done in the revised manuscript:\nThe statistics section in the Materials and Methods have been updated according to the Reviewer’s suggestion (lines 804-812).\nThe statistical methods employed are described in the Fig. legends. Specifically, the statistical analysis has been updated for Fig. 3A, 3D, 3G, 4D, and 4G with maintenance of significance.\nFig. 1M, S2E, S2I and S2J lacked description of statistics in the submitted version were updated now.\nData of Fig. 2C and 2E became non-significant upon reanalysis, these two Fig items have been transferred to Supplementary as Figs. S2D and S2H, respectively.\nThe Results section have been altered to accommodate the changes (lines 138-143).\nThe number of mice in Fig 1C, D, E has been increased to 6-11 mice.\nThe cytokine data in Fig. 2D-G have been updated in the revised manuscript by including data from 14–20-month-old lymphoma mice. The corresponding text in the Results section have been updated (lines150-155).\nComment 5. Figure 3 M: According to the text and Fig. 3L, kappa expression in the B lymphoma group is reduced. The graph shows the opposite.\nOur response: Thanks for pointing this out. We have mistakenly copied graph 3I onto 3M (i.e., duplicated 3I). How this inadvertently happened we do not know.\nChanges done in the revised manuscript: The correct graph has been included in the revised manuscript. The new graph clearly illustrates the reduced k expression on B lymphomas compared to controls.\nComment 6. Figure 3 N: Please check compensation.\nOur response: We reanalyzed the data and checked the compensation. The compensation appears correct. We think the reason for the unusual appearance might be due to stretching of the axes of the contour plot which results in an impression of potential compensation issues.\nChanges done in the revised manuscript: We have updated the contour plot without having stretched the axes.\nComment 7. Suppl. Fig. S1 B: How do you explain the reduced TCR Vb8 expression in the Id. sp TCR+/- group compared to the double transgenic mouse?\nOur response: Thank you for making this keen observation. Fig. S1B is related to Fig. 1C. As stated in our response to Comment 4 of the Reviewer, above), we have combined 3 similar experiments in the new Fig. 1 C. Upon closer examination of the stainings from these three experiments, we don’t think that there is a much-reduced TCR Vb8 expression in the Id-specific TCR+/- group.\nChanges done in the revised manuscript: We have replaced Fig. S1 B with a more representative histogram for the Id-specific TCR+/- group.\nComment 8. Suppl. Fig. S3 K: Please check compensation; the sharp peak obviously results from cells piling up in the sum channel of another fluorophore.\nOur response: Other histograms representing the same staining do not show this sharp peak.\nChanges done in the revised manuscript: We replaced Fig. S3K with another representative histogram (without a sharp peak).\nReviewer #2 (Remarks to the Author):\nIn the study by Gopalakrishnan and colleagues, a mouse model was established to study whether idiotype-specific CD4+ T cells promote the development of idiotype+ autoreactive B cells into lymphomas. Prior studies by the group of the senior author and by other groups already showed that principally, idiotype-specific CD4+ T cells can be generated for mutated B cell receptors (BCRs), and there is a known link between autoimmunity and the development of B cell lymphomas. So, the rational is clear and a highly relevant question is addressed.\nThe model was meant to be more physiological than standard transgenic mouse models, because only a small number of B cells will express the idiotype (only B cells which during their development rearrange a mutated Vlambda2 gene to the Jlambda2 gene), and not nearly all B cells. The hypothesis for the model was that if a BCR using the Vlambda2 light chain is by chance autoreactive, the autoreactive B cells would be stimulated, upregulate MHC class 2 and present the light chain idiotype to the transgenic idiotype-specific CD4+ T cells, which then further stimulate the autoreactive B cells, drive their constitutive clonal expansion and finally lead to lymphomas development. The mice indeed developed autoantibodies and after about a year in some mice B cell lymphomas developed (other developed T cell lymphomas). Hence, at first glance these findings seem to nicely support the hypothesis of the study. However, a closer inspection of the mice and their B cells revealed that the autoantibodies expressed kappa light chains, and also the lymphomas expressed a kappa BCR. The lymphomas nevertheless developed from Vlambda2 idiotype-positive B cells, but the lambda light chains were only detected with in the cells, and not as part of the BCR. Thus, allelic exclusion is violated here and the B cells co-express a kappa and a lambda light chain (but only one as part of the BCR). In the lymphomas, the BCR with the kappa light chain provides the autoreactivity, whereas peptides from the lambda light chain are presented on MHC class 2 and thereby mediate stimulation of the B cells through the idiotype-specific CD4+ T cells. The reason for the violation of allelic light chain exclusion is not clear. Perhaps, the mutated Vlambda 2 gene has problems to pair stably with most Ig heavy chains, so that additional light chain rearrangements are performed, leading to allelic inclusion. Dual light chain producing B cells have been identified, but this is clearly not the general situation in autoimmune diseases and B cell lymphomas.\nOur response:\nThe reviewer nicely summarizes the results.\nConcerning allelic inclusion, we agree with the possible explanation suggested by the Reviewer:“Perhaps, the mutated Vlambda 2 gene has problems to pair stably with most Ig heavy chains, so that additional light chain rearrangements are performed, leading to allelic inclusion”. Supporting the Reviewer’s view, we do present evidence that the various lymphoma BCR H chains poorly associated with λ2315 compared with the B lymphoma (Fig. 5F). In contrast, the H chain of the MOPC315 myeloma pairs well with λ2315 (Fig. 5F). We comment upon these datain Results, lines 243-254.\nThe Reviewer writes: “Dual light chain producing B cells have been identified, but this is clearly not the general situation in autoimmune diseases and B cell lymphomas.” Dual L chain B cells have been described in autoimmune diseases in bothmouse and humans (references 50-53 in the manuscript). Similarly, human B cell malignancies with dual L chains have also been reported previously, mostly in case reports (Aggressive B lymphoma, Fujiwara T et al., Intern Med. 2007; Mantle cell lymphoma, Wang et al.,Int J Lab Hematol. 2023; B-CLL, del Senno et al., Leuk Res. 1987 and Xu et al, Arch Pathol Lab Med. 2006). In a larger study on B-CLL patients, 1 out of 56 patients expressed dual L chains (Peltomaki et al., Eur J Cancer Clin Oncol. 1988). In all these studies, dual L chains were expressed in the cell membrane of malignant B cells as detected by flow cytometry. The frequency of dual L chain expression in human malignancies has not been exactly defined, but it is likely to be low, as the Reviewer suggests (e.g. in the 1-3% range?). However, and importantly, it could well be that the prevalence of dual L chain expression in B cell cancers could turn out to be higher if surface and intracellular staining is combined, similar to our findings in the current mouse model. This should be explored in future studies.\nChanges done in the revised manuscript: We already have 4 references (refs 50-53) related to dual L chain B cells in autoimmune diseases in mice and humans. We are hesitant to include more refs (see above) since the paper is already long. However, we include two new paragraphs on dual L-chain expression and receptor revision in the Discussion. This is described in more detail below.\nThe experiments are principally well done and nicely presented.\nOur response: We thank the reviewer for pointing this out.\nSo in my opinion the study presents an unexpected and curious observation, but neither validates nor falsifies the hypothesis of the study. The original idea that an idiotypic peptide of an autoreactive BCR is presented on MHC class 2 and causes chronic stimulation of the B cells through idiotype-specific CD4+ T cells, finally leading to lymphoma development, is not tested here. This is because in the present model, unexpectedly not an idiotypic peptide from the autoreactive BCR is presented to the CD4+ T cells, but a peptide from another protein, that is not part of the BCR (i.e., the lambda light chain).\nOur response: We appreciate the Reviewer’s comment.We started with a hypothesis (Fig 1A) but we found something “unexpected and curious” as pointed by the Reviewer. In our opinion this makes the present work even more interesting. Clearly, the statement that “Id-driven collaboration can drive development of autoimmunity and B lymphoma” is still true. It is just that the Id-peptide is derived from an intracellular L chain, rather than a BCR L chain.\nImportantly, directly addressing the Reviewer’s concern, we have recently discovered, in an aged V2315m+/- Id-specific TCR-TG+/- mouse, a B lymphoma that expressed a m+- BCR and that stimulates Id-specific CD4+ T cells. In the pre-lymphoma phase, the mouse had autoantibodies that bound nucleosome/histone. These novel findings directly supports the hypothesis of Fig. 1A. Interestingly, this B lymphoma has an intracellular k and is likely to have undergone a κ→2315mrevision.\nBased on the present results, and the previous findings that (i) BCR-ligation by antigen results in pId:MHCII presentation by normal B cells (Huszthy et al., PNAS 2019) and (ii) that nascent 2 L chains in the ER are antigen-processed and MHCII-presented (Weiss and Bogen Cell 1991), we consider it likely that the Id-peptide in the current B lymphomas can be derived from a 2 L chain that is either part of cell surface BCR or nascent 2 L chain in the ER.\nIt is striking that 23/24 (96%) of B lymphomas probably have undergone a 2315m→k revision while 1/24 (4%) is likely to have undergone the reverse revision, k→2315m. These frequencies correspond roughly to k/ ratios in mouse serum Ig. The H chain that happens to be expressed early in a B cell’s development could influence this result by preferentially pairing with either  or  L chains. It is also striking that we did not find any B lymphomas that only expressed one L-chain, i.e. 2315m. We speculate that this could be related to the reinstated Rag expression in dual L chain B cells that somehow could be lymphomagenic.\nChanges done in the revised manuscript: For increased clarity, we added a new Fig 6M to the revised manuscript illustrating that the Id-peptide in the great majority of the present B cell lymphomas is derived from an intracellular source. The novel data on the 2315m+ B lymphoma are presented in Fig. 7 A-L, Supplementary Fig. 7, and Results lines 313-343. Discussion lines 415-437 have been modified to encompass the novel data and a possible explanation for the results (see above). The new Fig. 7L illustrates that the 2315m+ B lymphoma (and 2+ autoantibodies) directly supports the hypothesis of Fig. 1A.\nMinor point a)\nFrom line 106 onwards there are multiple instances where the mouse line designation is wrongly written. It should be Vl2 and then \"315m+/-\" in superscript, and not Vl with \"2315m+/-\" in superscript.\nOur response: Thanks for pointing this out.\nChanges done in the revised manuscript: This has been changed from line 106-160.\nMinor point b)\nIs there a mistake in figure 3M? According to the text the double transgenic mice (red symbols) have lower kappa light chain expression than the controls, but here the controls all have apparently no kappa light chain expression, but the double transgenic B cells show variable expression. Please clarify.\nOur response: Thanks for pointing this out. Reviewer 1 made the same observation. We have mistakenly copied graph 3I onto 3M (i.e., duplicated 3I). How this inadvertently happened we do not know.\nChanges done in the revised manuscript: The correct graph has been included in the revised manuscript. The new graph clearly illustrates the reduced k expression on B lymphoma cells compared to controls.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nOur response: We would like to thank Reviewer #3 for reviewing this manuscript.\nReviewer #4 (Remarks to the Author):\nGopalakrishnan et al. have attempted to pursue the links between autoimmunity and lymphomagenesis. The created a model wherein they generated mice with a modified l light chain creating an epitope that binds to I-Ad. These mice were crossed into TCR transgenic mice the recognize the I-Ad peptide combination, generating mice heterozygous both for the light chain epitope and for the TCR transgene. These mice develop some anti-nuclear antibodies and eventually develop lymphomas. The lymphomas are kappa light chain expressors and H-chain sequences were shared between earlier autoantibody BCRs and lymphoma BCRs.\nOur response: Just a minor point: the Id epitope binds I-Ed, not I-Ad.\nWhie the approach has potential, and this work has some interesting aspects the main issue in the manuscript appears to be an apparent disregard for some fundamental immunological principles and the authors’ apparent decision to ignore their own data and not try to interpret it. The fundamental premise in the schematic in Figures 1A and B is not based on a sound understanding of the principles of T-B collaboration and is fundamentally incorrect, based on the data that the authors present, but inexplicably choose not to interpret and end up ignoring. A key experiment to test the likely flaws in the model in the schematic was ignored. This will be pointed out below\nOur response: With all due respect, the Reviewer appears to have overlooked crucial information in the manuscript which has led to a misunderstanding on part of the Reviewer. The paper is not about conventional T-B collaboration (Lanzavecchia Nature 1985), as reviewer 4 appears to think, but rather about Id-driven T-B. The concept of Id-driven T-B collaboration is described in detail in the Introduction (lines 37-69, refs 9-25) and in Fig. 1A, the concept has been established by us and others over the last 30 years. In contrast to Reviewer 4, Reviewers 1-3 have perfectly well understood Id-driven T-B collaboration and the hypothesis of Fig. 1A. Thus, it should not be anything wrong with the description of Id-driven T-B collaboration in the Introduction, and the hypothesis in Fig 1A. However, Id-driven T-B collaboration is clearly less well known compared to conventional T-B collaboration which probably contributes to the misunderstanding.\nThis having been said, soluble Id+Ig can induce conventional T-B collaboration where B cells express an anti-Id BCR that bind Id+ Ig and where pId:MHCII is recognized by Id-specific CD4+ T cells. This was shown by senior author Bogen and colleagues by generating and employing an Ig knock-in mouse that expresses an anti-Id BCR (Jacobsen et al. Naive idiotope-specific B and T cells collaborate efficiently in the absence of dendritic cells. J Immunol. 2014). Reviewer 4 is obviously thinking along these lines as becomes clear from the Reviewer’s comment further below. But Id in conventional T-B collaboration is not the topic of the present paper; rather, the topic is Id-driven T-B collaboration as outlined in the Introduction and Fig. 1A.\nChanges to be done in the revised manuscript: Since Reviewer 4 has misunderstood crucial aspects of the paper, it is important to prevent other readers from also misunderstanding. To this end, a sentence in the Introduction (line 42) has been modified. It now reads: “This phenomenon is the basis for the concept of Id-driven T-B collaboration, which is distinct from conventional T-B collaboration11,12”. Ref 11 (Lanzavecchia Nature 1985) is new, ref 12 (Muthe et al. J Immunol 2004) was included in the submitted version but has now been moved and got a new number.\nComments 1 and 2.\n1. Surprise is repeatedly expressed that the lymphomas were not lambda light chain positive! The authors show in Figures I panels H, I and J that the anti-nuclear antibodies they observe are almost exclusively kappa light chain positive. 90% of all Igs in mice are kappa positive and in the obvious scenario painted below it would be likely that most responding B cells would be kappa positive.\n2. The likely simple explanation of the data is that some IgHlambda2315 immunoglobulin molecule/s which are essentially provided in relatively high doses in these mice, are recognized by a self-reactive BCR or BCRs (at least 60-70% of all BCRs are self -reactive in practical terms) on one or a few B cell clones all of which are kappa expressors (not surprising - there is a 90% chance that a BCR would be kappa positive).\nOur response: The interpretation of the Reviewer is seemingly clever but erroneously based on conventional T-B collaboration (see above). There are several strong arguments against the Reviewer’s interpretation.\n• First, the new Vλ2315m mice used herein express λ2+ B cells in physiological amounts, and only low levels of λ2+ Ig (~500 ng/ml, Huszthy et al. PNAS 2019). Thus, the Reviewer’s statement: “The likely simple explanation of the data is that some IgHlambda2315 immunoglobulin molecule/s which are essentially provided in relatively high doses in these mice” is not consistent with previous data.\n• Second, we have extensively tested the specificity of serum autoantibodies (Fig. 1H, I, L, N and S1I, lines 104-120). We only found reactivity against nucleosomes and histones and occasional reactivity to dsDNA (Figs. S1I and J, lines 118-120). Thus, the autoantibodies in this model are not broadly cross-reactive (e.g to 2315Ig, like the Reviewer suggests).\n• Third, and most importantly, we have now tested if four different recombinant lymphoma BCR with specificity for histones/nucleosomes can bind λ2315 Ig (myeloma protein M315) in ELISA. None of them did. This speaks strongly against the Reviewer 4’s suggestion that autoreactive BCR could cross-react with λ2315 Ig, thus initiating conventional T-B collaboration.\nChanges done in the revised manuscript: The novel data on lack of BCR cross-reactivity to 2315Ig is included in a Supplementary Fig.S5E and commented upon in the text ( Line 272).\nComment 3.\nThese kappa+ self-reactive BCRs internalize the IgHlambda2315 immunoglobulin self-antigen, process it and present the lambda peptide to the relatively large numbers of CD4+ T cells that are specific for the peptide. The kappa + self-reactive B cell and the CD4+ T cell collaborate at the T-B boundary generate an extrafollicular B cell response that generates plasmablasts secreting anti-nuclear antibodies. No autoimmune disease necessarily, just an autoimmune phenomenon which is not such a big deal\nOur response: There are several strong arguments against the Reviewer’s suggestion, some of which are conclusive.\n• First, if λ2315 Ig is internalized via the BCR, it should be found in endosomes. However, it is not. Rather, the λ2315 L chain is localized to the ER (Fig. 3Q and R), which is inconsistent with endocytosis of λ2315 Ig.\n• Second, an ER localization of λ2315 was previously found in plasmid-transfected B lymphoma cells, a finding that is consistent with an endogenous origin (Weiss and Bogen Cell 1991). This supports that the ER-localized λ2315 in the present lymphomas is endogenously produced.\n• Third, and most conclusive, we show that lymphoma cells grown in vitro for prolonged periods of time express λ2315 in the ER, and display Id peptide on their MHCII molecules (Fig. 3O). Since there is no λ2315 Ig in the culture medium, this excludes the reviewer’s suggestion of endocytosis of λ2315 Ig.\n• Fourth, and most conclusive, we show that B lymphoma cells express mRNA for λ2315 L chains and thus produces λ2315 themselves (Fig. 3K).\nChanges to be done in the revised manuscript: We have added a few words in Discussion to emphasize that λ2315is endogenously produced in the B lymphoma cells.(lines 425-430).\nComment 4.\nAs these kappa+ self-reactive B cell clones are repeatedly stimulated by T cell help since antigen is always available, and therefore expand, occasionally additional hits accumulate and lymphomas evolve. The data says most of this!!!\nOur response: As should be clear from the data in the paper and our comments above, + self-reactive B cells derive the MHCII-presented Id-peptide not from endocytosed λ2315 Ig but rather from endogenously produced λ231. This said, we agree with the latter part of the Reviewer’s statement: chronic mutual stimulation between autoreactive, Id-presenting B cells and Id-specific CD4+ T cells results in chronic proliferation, acquisition of mutations, and lymphomagenesis. This idea is already expressed in the Discussion lines 390-397, hence, there should no need for changes to the manuscript.\nComment 5 (a-d).\nThe authors should do the following:\na) use the Wardemann and Nussenzweig approach and clone V regions of Ig H and kappa light chains from some lymphomas, clone into the already widely used Ig vectors, and then express in 293T cells\nb) show that these clonal Igs are self-reactive using the assays they have used in the manuscript\nOur response:We already did the experiment suggested by the Reviewer. We generated recombinant Ig corresponding to BCRs from 4 independent B lymphomas. The following results were obtained:\n• First, recombinant lymphoma BCRs are indeed specific for histones/nucleosomes, like serum autoantibodies. Importantly, they do not cross-react with λ2315 Ig (new experiment, see above). The data are presented in Figure 6 A, B, C and F and Supplementary Figs S4 and S5. The data are commented upon in Results lines 263-290 and in the Discussion lines 398-404.\n• Second, the recombinant lymphoma BCRs were crucial to demonstrate by proteomic analysis that autoantibodies and lymphoma BCRs share unique sequences. Hence, B lymphomas appear to be derived from autoreactive B cells which is an important conclusion of the paper (Fig. 6 J-L, Results lines 291-3118. Discussion lines 398-404).\nIt is somewhat surprising that Reviewer 4 has missed this large body of data so important for the conclusions of the paper. No changes to the paper should be needed.\nc) delete and correct the old schematic in Figure 1\nOur response: As explained above, the Fig. 1A is correct and should be kept.\nChanges done to the manuscript:\nAs mentioned above, we have modified a sentence in the Introduction, line 42 to emphasize that Id-driven T-B collaboration is distinct from conventional T-B collaboration. We have introduced Fig. 6M and Fig. 7L that further explains the concept of Id-driven T-B collaboration and that the Id peptide can be derived from either the BCR or nascent L-chain in the ER (see also our comments to reviewer 2).\nd) In the abstract and manuscript stick to the use of “autoantibodies” or “autoimmune phenomena” and delete “autoimmunity” in the manuscript. There is no good evidence for an autoimmune disease in this manuscript. We all have autoantibodies! Self-reactivity is a normal phenomenon – some extreme self-reactivity is eliminated by central tolerance mechanisms.\nOur response: We agree with the reviewer that autoimmune disease in mice was mild [slightly reduced gain of weight, inflamed eye lids (blepharitis), moderate hair loss, as described in Results lines 106-109 and Fig. 1G, Fig. S1G]. This said, a more fulminant expression of autoimmune disease could have been disguised by lymphoma development.\nWe agree that the term autoantibodies and autoreactive B cells should be preferred, and we have tried to do so throughout the paper. We have avoided the term autoimmune phenomena since that implies autoimmune disease manifestations, which are mild. However, according to textbook immunology, it is justified to use the term autoimmunity if autoantibodies are detected, which is the case in our model. We do use the term autoimmunity several places in the manuscript and do not wish to change that.\nMinor changes to the manuscript (typos and edits)\nThe authors have made a few changes to the manuscript for reasons of correctness and clarity. None of the changes influence the scientific content of the paper. The changes are marked in the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised version of the manuscript satisfactorily answered most of the open questions. Only the intranuclear stainings shown in the manuscript require further attention.\n1. Please compare the FoxP3 staining shown in Suppl. Fig. S1D to a typical outcome shown on the manufacturer's homepage (clone FJK-16s from Thermo Fisher Scientific; not \"FJK-162 by Thermofischer\" - see below). What you expect is a clearly separated positive population, not a shift of the entire population and a high background over the whole fluorescence range. To demonstrate specificity of the staining, the authors should plot FoxP3 against CD4 (gated on CD3+ or live) and FoxP3 against TCRb8 or GB113 (gated on CD4+). This would be a better way than the isotype control to judge signal versus background.\n2. This manuscript requires careful reading by the senior corresponding author - there are so many inconsistencies and small errors! Please check clone names (see above), spelling of fluorophores (e.g. it is not \"PecP-vio700\" but \"PerCP-Vio700\") and manufacturer's names (e.g. it is not \"Miltney BioTech\" but \"Miltenyi Biotec\").", "reviewer_2": "My criticism to the first version of the manuscript has been adequately addressed. The manuscript is further improved in response to issues raised by the other referees.\nThere is one minor point:\nIn the legend to figure 7, there is a mixup of letters for the subfigures O -Q, and N is missing.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "All issues have been addressed."}, "author_response": "Point-to point Rebuttal letter\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe revised version of the manuscript satisfactorily answered most of the open questions.\nOnly the intranuclear stainings shown in the manuscript require further attention.\n1. Please compare the FoxP3 staining shown in Suppl. Fig. S1D to a typical outcome shown\non the manufacturer's homepage (clone FJK-16s from Thermo Fisher Scientific; not \"FJK-162\nby Thermofischer\" - see below). What you expect is a clearly separated positive population,\nnot a shift of the entire population and a high background over the whole fluorescence range.\nTo demonstrate specificity of the staining, the authors should plot FoxP3 against CD4 (gated\non CD3+ or live) and FoxP3 against TCRb8 or GB113 (gated on CD4+). This would be a\nbetter way than the isotype control to judge signal versus background.\nOur Response: We thank the reviewer for pointing this out. We have now done as the\nreviewer suggests. We plotted FoxP3 against CD4 (gated on live cells). CD4+ cells were\ngated for further analysis in a FoxP3 versus TCRVβ8 plot (the transgenic TCR is TCRVβ8.2).\nThe plots are shown in Suppl. Fig.S2D (previously Fig. S1D) where we compare results with\nV2315m+/- Id-spTCR-TG+/- and Id-spTCR-TG+/- mice by overlays. By this approach a clear\npopulation of V8+FoxP3+ CD4 T cells is seen. Based on this staining strategy, we\nreanalyzed all mice in the group and recalculated data of Fig. 1E so that FoxP3+/splenic\nCD4+ TCRVβ8+ (%) is now displayed on the y-axis. We did a similar analysis using the\nclonotype-specific GB113 mAb instead of the anti-Vβ8 mAb. Results were similar but data\nwere clearer with the anti-Vβ8 mAb – which are therefore presented.\nChanges in the manuscript: Updated Fig. S2D (previously Fig. S1D) and the corresponding\nFig. legend. Updated Fig.1E and the corresponding Fig. legend.\n2. This manuscript requires careful reading by the senior corresponding author - there are so\nmany inconsistencies and small errors! Please check clone names (see above), spelling of\nfluorophores (e.g. it is not \"PecP-vio700\" but \"PerCP-Vio700\") and manufacturer's names\n(e.g. it is not \"Miltney BioTech\" but \"Miltenyi Biotec\").\nOur Response: We thank the reviewer for pointing this out. We carefully went through the\ndetails in the manuscript and made several minor changes to avoid inconsistencies and minor\nerrors. The fluorophore name PerCP-Vio700 has been updated in Suppl. Fig.S4E and J.\nChanges in the manuscript: Updated Suppl. Fig. S4E and J. Several minor changes in\nmanuscript has been made (indicated by track changes, too many to list).\nReviewer #2 (Remarks to the Author):\nMy criticism to the first version of the manuscript has been adequately addressed. The\nmanuscript is further improved in response to issues raised by the other referees.\nThere is one minor point:\nIn the legend to figure 7, there is a mixup of letters for the subfigures O -Q, and N is missing.\nOur Response: We thank the reviewer for pointing this out. The legend to Figure 7 has been\nupdated and shortened to conform to the length requirement.\nChanges in the manuscript: Updated legend to Figure 7, lines 1209-38.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nOur Response: We thank the reviewer for co- reviewing this manuscript.\nReviewer #4 (Remarks to the Author):\nAll issues have been addressed.\nOur Response: We thank the reviewer for reviewing this manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_1": "All issues have been addressed"}, "author_response": ""}]

Decision letter